The Second
Ministry of Agriculture,
Forestry and Fisheries,
Japan
International
Workshop on Genetic Resources

Soil Microorganisms
Tsukuba Officeof MAFF Research Council
1994. 12. 5-7
Sponsored by
Agriculture, Forestry and Fisheries Research Council Secretariat
National Institute of Agrobiological
Resources

in cooperation
National

Agricultural

withResearchCenter,National Instituteof Agro-Enviromental
Sciences,
Japan International
Research Center for Agricultural
Sciences,
National Food Research InstituteandForestry and Forest Products Research InstituteandContents
Page
Welcome address
Opening

S. Kida3

H. Fujimaki

5

address

Keynote addresses
Microbial
CultureResource Collections:Collections

Session1: Collection

Collection,

Evaluationin Japan:

Current Status
Present

Statusand Future Strategy
D. Smithand Future Plans
T. Nakaseand Preservationand Use of Microorganisms in the Philippines
R. E. dela Cruz and R. G. Monsalud
A Note on the Present State of Indonesian Microbial Diversity
M.A.Rifai
Collection,
Preservationand Use of Microbial Genetic Resources:
Activitiesof the MAFF Gene Bank
H. Kaku
Discussion
Session2: Nitrogen-Fixing

Microorganisms

Collection
and Maintenance of Germplasm of Nitrogen-Fixing
Organisms from Lowland Rice Ecosystem
J. K. Ladha, T. Ventura and R. So
Collection
and Use of Tropical Rhizobia for Increasing
LegumeYield in Thailand
B. Tangcham, P. Wadisirisukand C. Aroonsri
Genetic Variabilityof the Common Nod Gene in Soybean Rhizobia
Strains with Particular
Reference to Strains from Thailand and JapanT. Yokoyama and S. Ando
Discussion

7
17
27

29
37

47
55
59

61

71

79
95

Session

3: Plant

Pathogenic

Bacteria

97

Diversityof the Genus Agrobacterium and its Relatives
H. Sawada

99

Diversityof Pseudomonassolanacearum in China
109

Diversity

L. He
of Pseudomonassolanacearum in Japan and Southeast Asia
K.Tsuchiya and M. Horita

Discussion
SessionDiversity

4: Plant

Pathogenic

Distribution

and Toxigenic

133

Asia
B. Salleh
Pathogenic
Specializationof Fusarium oxysporumCausing Wilts of Cucurbits
F. Namiki and K. Nishi
Mating Types, Isozyme Polymorphism
and Metalaxyl Sensitivityof
Phytophthora infestans Isolated from Several Countries in Asia
A. Ogoshi
Discussion
Sessionof Pathogenic

Fungi
Fusariain Southeast

5: Mushroomsof Biological

121
13 1

135
147

157
1 67
171

Speciesof Genus Armillaria

in Japan
E. Hasegawa
Distributionof the Ectomycorrhizal
Fungus Tricholoma matsutake andthe Related Species and Some Characteristicsof their Isolates
K. Iwase
Molecular Genetic Analysis of Diversity
in Agaricus bisporus
B. A. Bunyard and D. J. Royse
Discussion

189
207

GeneralDiscussion

209

Closing

Remarks
M.Nakagahra

Listof Participants

173

179

213
217

INTRODUCTION
Preliminary
address
Keynote address

Chairperson
Takahito SuzuiWelcome Address
SHIGEKI KIDA
Research Councillor
Secretariat
Council of AFFRC, MAFF

Distinguishedguests and participants.
On behalf of the Agriculture,
Forestry and Fisheries Research Council, it is
my great pleasure to extend sincere greetings and best wishes to all participantsin this"MAFF International
Workshop on Genetic Resources.
This workshop has beenorganized and sponsored by the Agriculture,
Forestry and Fisheries Research Council
and the National Institute of Agrobiological
Resources in collaboration
with associatedinstitutes
in Tsukuba of the Ministry of Agriculture,
Forestry and Fisheries.
Recently, Japan celebrated 100 years of agriculturalresearch. During the past
100 years genetic resources activities
have supported basic agriculturalresearch andfurnished invaluable germplasm for plant breeding and microbial research. In responseto the continuous and increasingneed for collection
and preservationof genetic
resources, the MAFF gene bank project was initiatedin 1985.
This year, we establisheda DNA bank project which supports genetic
resources research. The DNA bank project will involve the systematic preservation,
accumulation and distributionof DNA and information related to DNA.
Weare making these efforts, because we firmly beleive that genetic resources
are vital for agricultureof the future and that conservationof the global environmentis essential for mankinds survival.
Drastic changes in the global environment led to the Conservationon
Biological
Diversity
which came into effect recently. The first meeting of countries
which support the Biological
Diversity
Convention , which includes Japan, is now
being held in the Bahamas. This convention has broad aims in the area of both in situ
and exsitu conservation of biologicaldiversity.
The increased attention by the global
community to the preservationof genetic resources will result in safer world for all.
Recognizing
the importance of genetic resources and global changes, theresearch council secretariat of MAFF decided to hold this workshop in order to support
the preservationof genetic resources and promote research on genetic resources
worldwide.
This is the second of a series of genetic resources workshops. The first
focused on "Root and Tuber Crops" last March. We hope that this weeks meeting
will bring about useful cooperation and you will have valuable discussions which willlead to an improved international
genetic resources network. We look forward to
valuable contributions
over the next two days on the subject of "Microorganisms
Genetic Resources" .
Thank you.

Opening Address
HIROSHI FUJIMAKI
Director General, NIAR

It is with great pleasure that I extend a warm welcome to all participantsofthis Ministry of Agriculture,
Forestry and Fisheries workshop on Genetic Resources.
I would particularly
like to extend a special welcome to all those who have travelled
a long way to attend this workshop here in Tsukuba. We hope that the next few days
will make that journey well worthwhile.
I would like to acknowledge the contributionof several organizations.
The
workshop was organised and sponsored by the Agriculture,
Forestry and Fisheries
Research council of MAFF. Here in Tsukuba to prepare for this workshop the National
Institute of Agrobiological
Resources was helped by staff of the National Agricultural
Research Center, National
Instituteof Agro- Environmental
Sciences,
Japan
International
Research Center for Agricultural
Sciences, National Food ResearchInstitute,and Forestry and Forest Products Research Institute.
The global concern for the conservation of, fast dwindling,
genetic resources
requiresan international
approach to protect this fundamental
genetic base forhumanity. Microorganisms are one component of the worlds genetic resources which
require conservation. Japan has in recent years significantly
increased its commitment
to conservation and evaluation of genetic resources.
In 1985 MAFF embarked on an ambitious project called"The Genebank
Project". This did not just involve the buildingof a new genebank, it involves
hundreds of scientists
from all over Japan and collaboration
with scientists
from allover the world in the conservationand evaluation of a multitude of microorganism,
plant and animal genetic resources.
In addition to the microorganism, plant and animal genetic resources, a DNA
Bank was establishedlast year, and additional floor was constructed for the DNA Bankon the genebank building of our Institute. We would like to introduce you to our Gene
Bank the day after tomorrow as one of the programs of the excursion.
Microorganisms
have great potential
to improve agriculture.
For instance,
Agrobacterium tumefaciens', a soil microorganism,
is a plant pathogen. However, thisorganism plays a role in biotechnology
as a gene vector. Many soil microorganisms
produce antibiotics,
and they are used to produce medicines and agrochemicals.
Further prospects
for microorganisms
are their use in biologicalcontrol andbiofertilizers
for the development of low input sustainableagriculture.
Microorganisms are a vast but usually cryptic component of the biologicalrealm and they include bacteria, fungi, mycoplasma, viruses among other groups oforganisms. In this workshop only the tip of this fascinatinggroup of organisms will be
discussed,
mainly soil microorganisms.
It is clear that a world-widesystem toconserve and evaluationof microorganism genetic resources has yet to be fully
developed.
We hope that this seminar draws people together in a way in which willlead to a better understandingof what is being done in various countries to conserve
microorganism gemplasm. However, perhaps more important will be to determine
areas where we are not doing a good job and where more attention is needed inconserving and evaluating microorganism genetic resources.
Finally let me encourage you all to fully participate
in the meeting. Pleaseask lots of questions and make full use of your time together.
Thank you.

KEYNOTE ADDRESS I
Microbial
Strategy

Resource Collections:

Current

Statusand Future

DAVID SMITH
International
Mycological
Institute
Bakeham Lane, Egham, Surrey, TW20 9TY, UK

Introduction
Microbial resource collections provide a valuable source of microorganisms
for education,
the scientificcommunity and industry.
There are many collections
worldwide, 481 are registered with the World Data Centre for Microorganisms holding
in excess of 786,000 strains. However, this represents only 10 - 15% of those species
known and is a much smaller representationof the estimated total population.
The
potential number is best illustrated
in the fungi where there is an estimated 1.5 million
species, yet only ll,500 are held in microbial collections.
The problem of maintaining
adequate representation
becomes enormous when appropriate
numbers of strains of asingle species are taken into consideration.
Exsitu conservation of microorganisms
requires several issues to be addressed including,capturing a greater proportionoforganisms, improvement of collaborationand coordinationof the world's collections,sustained funding, integrationof ex situ collection with conservation programs andthe development of strategies for the incorporationof hitherto uncultured organisms.
The Current Position
There are 481 collectionsof microorganisms registered with the World Data
Center for Microorganisms(WDCM) holding 786,328 strains with 19,392 names(Sugawara et aL, 1993). 610,228 of the strains are bacteria and fungi. Currently there
are 72,000 known species of fungi and over 3,000 species of bacteria.
At best 26% of
the species of bacteria and fungi are held in the collectionsof the WDCM. In reality
the actual number of species held by these collections is much lower and less than 15%
is probably
a more accurate figure when synonyms, teleomorph and anamorph
relationships,
invalid names and mispellings
used by the collections
are taken into
account. Ten of the WDCMlisted collections
hold 35% of all strains held, quite animbalance in distribution.
In this paper I will attempt to answer four basic questions
in relation to
culture collections :1.

Are there sufficientcollections

to preserve adequate representatives

of microorganisms?
Are those that exist duplicating
effort?
What could be done to rationalize
existing collections?
What should be done to coordinate
the activities
of collections
worldwide?
The role of Microbial Resource Collections
is to conserve representativesofbiodiversity
to supply to users. In theory, all microorganisms have something to offerwith regard to morphologicalor biochemical properties and there can be vast differences
between propertiesof strains within one species.
Therefore the task of providing
adequate coverage of microorganisms is an enormous one. The estimated number offungi in nature is 1.5 million (Hawksworth, 1991), the majority of which are yet to bediscovered, and about ll,500 species of fungi (0.77%) were held in collectionsin 1990.
There are, on average 41, strains for each name listed in the WDCM.
Following compensation for teleomorph/anamorph
connections, spelling variations andsynonyms there is an average of 71 strains per species currently held. An example ofthe variation in a species is seen in the holdings of the Fungal Genetics Stock Centre
where in excess of 3,000 strains of geneticallymarked Neurosporacrassa are kept.
A conservative estimate to give modest representation
for a species could be as highas 50, based on morphological and physiologicalvariation within a species this would
mean75 million strains would have to be maintained by collections.
Each of the 246 fungal collections
listed in the WDCM would need to hold304,878
strains to achieve this target. This is far too large a challenge for existingcollections,particularly
with the current lack of coordination
which has led to overlap
between them. In reality, these figures are extremely optimistic
as not all fungi can begrown in culture. Although methods are available to preserve strains directly on, or
harvested from, their hosts it is unlikelythat all species could be gathered with asufficiently large amount of inocula to store in collections.
If the microorganisms cannot
be multiplied
easily then they are less likely to be suitable for wide application.
Theiruse for the production of natural products would be limited, particularly
if the host israre, economically important, an animal or man.
However, it is essential that the vast potential of microbial diversity is madeavailable to science for screening for useful properties
and research. To achieve this,
it is necessary to establish or consolidate national or regional gene banks. Several issues
that require addressing
by microbial
resource collections
were summarized in the
IUBS/IUMS workshop on biodiversity
action statement published as Microbial Diversity
21 (Hawksworth & Colwell, 1992).
One of the main objectives
should be to capture
a greater proportionof the microbial gene pool in world collections,beginning withcollectionof material from disappearing
habitats
and preserving them by long-term
cryopreservation
methods to safeguard their genetic potential.
This must be done while
following the relevant components of the Convention on Biological Diversity with regard
to conservation and national property rights.
Other recommendations outlined in Microbial Diversity 21 of relevance here
were; to improve collaborationand coordination of the world's collections
and develop
2.
3.
4.

mechanisms; to maximise complementation to reduce duplicationof effort; to consider
establishinga network of collections
charged with specific tasks such as on the linesof the base collections
establishedby the International
Plant Genetic Resources
Institute (IPGRI);
to develop sustainable funding mechanisms for the world's microbial
diversity.
There is a need to consider how to integrate microbial resource collections
into ex- situ genetic resource conservation programs and a strategy is required for theisolation, collection, growth and storage of hitherto uncultured organisms. Such tasks
must be addressed at an international
level.
It is evident that collections
are duplicating
effort, clearly seen in microbial
resource collection catalogues where acronyms of other collections
are commonplace.
The International
Mycological
Institute(IMI), U.K., collection
holds 2,255 strains(<1 1.7%) also held in the Centraalbureau voor Schimmelcultures(CBS), the Netherlands,
and 1,685 strains (8.76%) that are also held by the American Type Culture Collection(ATCC), U.S.A. In general, this overlap represents ex-type and reference strains heldin several collections
for security and as comparative standards for identification.
However, 8,900 strains are unique to IMI. Of the 88 ,981 strains of fungi and bacteria
listed in the Microbial
Information
Network Europe (MINE) database 64,592 areunique. The difference of 24,389 represents the distributionof the same 9,439 isolates
between membercollections,each one kept almost in triplicate.
Of the 19,392 names
listedin the WDCM 9,500 are held in only 1 collection
making those organisms
vulnerable. Some duplication,
particularlyof currently non-duplicatedorganisms, isuseful where extremely important strains can be held in more than one collection
ensuring they remain available if one collection
loses them. Access to cultures can beeasier if the strain can be obtained in the customer's home country. This is often thedriving force for duplication- not the security of the organism. Greater collaborationis necessary to avoid unncessary duplication
but also to ensure the securityofextremely important strains.
The UK collections
were organized in 1947 so that such duplication
was
minimized giving the responsibilityof particulargroups of organisms to particularinstitutions
or collections.
However, this type of arrangement is not common.Sucha division is difficult
to formulate on an equal basis. Some groups are more economicallyimportant than others, some are more difficult
to maintain and require more expensive
facilities
and generally costs of preservation,
handling and growth of these strains aremuch higher. The different
collections
have their own interests and policies whichcannot easily be influenced
by outside bodies. Some form of incentive to specialize
is necessary.
It is not only the holdings of collections
that are duplicated.
Collections
have
many things in common, their preservationequipment,
information
storage andprovision, culture supply, safe storage and identification
services, being some obvious
ones. Setting up collections
where equipment is under-used and that require expert
back-up is not cost effective.
Research and development of improved preservation
procedures is an area
in which many collections are interested and in which they invest at least some of their

10

resources. It would be much more cost effective if these resources were pooled toenable meaningful research projects into modern methods and improved technologies
to be undertaken.
European collections have collaborated
under various European Union (EU)programs to improve preservation
procedures(Smith et aL, 1990) and to makeavailable
strain data through the Microbial
Information
Network Europe (MINE)(Gams et al., 1989). It is only where funding is central and controlled that collaboration
has come about. There is a limited amount of support available
and collections
compete for customers to buy their product and funding to support their projects.
The task of preserving microbial diversity cannot be left to chance. It is notpossibleto predict what will be useful in the future and it is often too late to act inretrospect. Once a property is the subject of a publication
it is quite often the case thatthe original
culture displaying
the property has been lost or discarded rendering thatwork unrepeatableand unable to be confirmed thus preventing
further tests orexperiments.
Tackling

the Problem

With the task of maintaining
representative
strains of microbial diversity
incollections as vast as it is, there must be an initial focus. This could be on industriallyor economicallyimportant organisms found in geographicallocationswith little
previous exploration
and with unique habitats.
Priorities
can be set taking note ofdiversity, uniqueness and stabilityof populations.
Many organisms will be present inthese areas that will not grow in culture. The existing methodology
to collect, extractand store DNAgives the opportunity to maintain hitherto uncultured microorganisms.
Although this may be limiting the use of this germplasm, particularly
if a suitable hostorganism cannot be found to express useful properties,
it does offer the possibilityofconservation until new technologies
are developed.
This can back up frozen stocks of
diseased tissues or harvested propagules.
It is necessary to preserve strains as they are
discoveredas there is evidence to show that species have only been found once.
Returning to the same location cannot guarantee that the same organisms with the same
properties
will be found.
Preservation

It is essential that long- term methods of preservation that retain stablility
areused. Methods should be optimized for the cells being preserved, major collectionsare
set up to do this and should help those collections
who are not. Understandingthescience of low temperature chemistry and physics and the ability
to observe what
happpens to cells is essential for technique design.
Methods normally used to maintain long- term stability of organisms (Smith & Onions,1994)are:(1) Freeze-drying

ll

(2)

Cryopreservation:either in or above liquid
nitrogenor in a lowtemperature freezer.
It is extremely expensive to set up such facilities
in every collection.
Collaboration
andshared use is essential to increase cost effectiveness.
Cryogenic

light

microscopy

It is essentialthat collections
endeavour to ensure complete stabilityofpreserved microorganisms. They must be preserved in such a way that they retain theirfull potentialof biochemical
abilities.
New discoveries of uses and natural products
from microorganisms requires that collections
can provide representative
strains thatmayexpress these new properties.
If strains are not maintained well then yet unkown
properties
could be lost. It becomes imperativethat preservation
techniquesareoptimized and the use of cryomicroscopy is just one way of achieving this.
A light microscope with a conductive stage enabling accurate temperature
control of living cells and observationof their response to cooling and subsequent
warming has proved a useful tool in technique development. Cryomicroscopy (McGrath,1987) has been used for many years to observe cells to help determine optimum
preservation conditions.
This technique has been used more recently with fungi (Thomas& Smith, 1994).
The response to cooling of Aspergillus repens can be seen on a
cryomicroscope(Fig. 1). Where ice is seen to nucleate within the hyphae as a
granulation of the cell caused by refraction of light by numerous small ice crystals, andon subsequent concentrationof the cytoplasm the precipitationof solutes and theformation of minute gas bubbles. On thawing the latter sometimes coalesce (Fig. ID)
and force their way out of the cell causing evacuation of the cell cytoplasm.
Observations made during many different cooling protocols can allow the selection of
the procedure that induces least observabledamage.
Thus improved coolingprocedures
can be determinedfor organisms to be used in freeze-drying
andcryopreservation.
Reorganizationof Collections

Rationalizationof collections
must begin nationally.
In the UK the Officeof Science and Technology commissioned a review of microbial resource collections(OST, 1994). The UK collections were considered to have been under-resourced.
The
review team suggested a reduction of the eleven collections
to 3 main centres with one
contact point. They also recommended the reduction of the nine funding bodies to one
and wished to keep the best of the existing
situation
where there is a wealth ofexpertise.
The latter would be attained by keeping 7 of the 8 collections
as satellite
collectionsof the 3 main centres, relocating
only one in its entirety.
This wasenvisaged
as resulting
in a more efficientuse of equipment and resources andcoordinationof marketing. The extent to which these proposals can be implemented

repens
AspergilIus

12

Figure 1.

photographedduring freezing and thawing

on the cryogenic

light

microscope.

A. Unfrozen at 4°C.
B. Extracellular
ice formation, the advance of the ice front (arrow).
C. Intracellular
ice nucleates at -10°C in the hyphae and channels of un frozen liquid outside the cell
be seen. On freezing, solutes come out of solution,gases reach saturation
point and minutebubblesaretrapped between ice crystals and compressed together (arrow).
D. On thawing gas bubbles coalesce both inside and outside the cell. Some redissolve
immediately
others migrate with the ice/liquidinterface.
E. Gas bubbles migrate to the vesicle (arrow) and out through the conidiogenous
cells. This causes
cytoplasm to follow.
F. This results in the complete evacuation of all cell cytoplasm and death.

can
gas
and
cell

13

will ultimately
depend on the resources that can be made available.
Microbial
resource collections
need to collaborate
with, and be treatedalongside,other genetic resource collections
to capitilize
on common storage
procedures. One prime example of this is the MAFF Genebank in Tsukuba, Japan. A
most positive national plan, where seeds (plant propagules)
and microorganisms are
kept at the same site.
Coordinationof collection

activities

worldwide

There are national and internationalorganizations
establishedto encourage
collaboration
between collections.
National Federations
exist in Japan, UK and USA.
These carry out useful work in their own right coordinating
training,
producing
publications
and representing
microbial resource collections
in societies,
unions andin governmental
surveys and discussions.
The European Culture Collection
Organization(ECCO) offers a forum for discussionof topics relevant to collections
and brings together collection
staff to initiate collaborative
projects.
It is publicisedby the Information Centre for European Culture Collections(ICECC) which is a sourceof informationon ECCO members and their full range of activities
and services. At
a world level the World Federation for Culture Collections(WFCC) aims to coordinate
microbial
resource collection
activities.
The WFCC includes committees on
Education, Culture Collection
Standards, Biodiversity,
Postal, Quarantine and Safety
Regulations and Patenting.
It too runs training courses, produces publications
and italso organizes an international
congress every four years. The next will be held in the
Netherlands in 1996. One of the WFCC responsibilities
is to oversee the activities
ofthe WDCM. This database is one of several initiatives
set up to aid microbioligists
andindustrialists
get access to microbial resources and is probably
the longest running.
Hitachihave a CD ROM entitled CD- STRAINS, the EU has sponsored the MINE
project and many databases are available through INTERNET and the Microbial Strain
Data Network (MSDN).
Informationon all these organizations
and databases can befound in The Preservation and Maintenance of Living Fungi (Smith & Onions, 1994)
or directly from the organizations(Table 1).
Despite the objectivesof the organizations
that serve microbial resource
collections
worldwide
none have executive power to influence
the individualcollections
they purport to represent.
They try to set standards
and influencecollections
but it is the owners of collections
that must collaborate
if collectionsareto unite to try to meet future challenges.
Standardsin Collections

The WFCC have produced the booklet Guidelines
for the Establishment
andOperation of Collectionsof Microorganisms(Hawksworth et al., 1990) which covers
the management of collections but also stresses the need for clear objectives
and longterm funding. The objectivesof a collection must include not only which organisms

14

Table. 1. Microbial

Resource Collection

Information

Sources

it will collect but how it will select the representatives
and a target number based upon
the facilities
and support available.
Acceptance of a strain into a collection requires careful evaluation.
It should
not be a duplicationof a strain in the same collection or that of another readily available
source. High priority should be given to strains that have unique propertiesof value15

to the collection,
represent a systematic or ecologicalgroup not currently held, are ofpotential interest and the facilities
must be available to handle the strain safely.
The MINE project has also set standards for their member collections(Hawksworth & Schipper, 1989).
Stevenson and Jong (1992) have reported the needfor quality control and standards within collections.
Such intitiatives
are necessary toincrease the quality in collections
by setting minimum standards.
Although this isnecessary for a good database there is the need to go much further. It is only by allconcerned working together that the enormous task of conservation of microorganisms
for sustained use for the benefit of humankind can be achieved. Owners of collections
are urged to collaborate
together through joint initiatives.
Governments must look totheir national policies for Microbial Resource Collections
and how these relate to their
National Biodiversity
Action Plans. A sound footing is required to ensure collections
continue to offer the service to science and industry that is required today and for the
future.
Summary recommendations:

Answers to questions

1.

collections

Are there sufficient
microorganisms?

All microorganisms - No.
With a focus on useful organisms

to preserve

posed
adequate

representationof- Yes.

Industrial
or economicallyimportant strains from unexplored areas withunique habitat
considering
diversity,
uniquenessand stabilityof the populations
should be targetted for collecting
and conservation.
2.

Are those that exist duplicatingYes.

effort?

3.

What could be done to rationalize
existing collections
at a national
Coordinate
activities,
marketing and supply.
Reduce unecessary duplication.
Share facilities.
Pool resources for reseach and development.
Centralize
and control funding.
Offer incentives.

4.

What can be done to coordinate the activities
of collections
worldwide?
Introduce
an accreditation
scheme ensuring standardization
andquality.
Coordinate
collections
though their owners.
Correlate national government policy to produce a world policy.
Fund collections
through international
programs of research andlevel?

16

development

givinginternational

coordinationand direction.

Conclusion
Currently
collections
are striving
independantly
to conserve microbial
diversity,
concentratingon their own aims and objectives.
This leads to duplication
and a waste of resources.
To achieve the enormous conservation
task ahead,collections
will have to work together.
Their own aims must be satisfied
but with
incentives,
such as adequate funding, they could take on responsibility
for thecoordinationof world microbial conservation.
References
Gams, W., Hennebert, G. L., Stalpers,
J. A., Jansens, D., Schipper,
M. A. A., Smith, J., Yarrow, D.
& Hawksworth, D. L. 1988. Structuring
strain data for the storage and retrievalof information
on fungi and yeasts in MINE, Microbial
Information
Network Europe. Journal of General
Microbiology
134: 1667-1689.
Hawksworth,
D. L. & Schipper,
M. A. A. 1989. Criteria
for considerationin the accreditationof
culturecollections
participatingin MINE, the Microbial
Information
Network Europe.
MIRCEN Journal 5: 277-281.
Hawksworth, D. L., Sastramihardja,
I. Kokke, R. & Stevenson, R. 1990. Guidelines
for the establishment
and operation of collectionsof cultures of microorganisms.
WFCC Secretariat,
Brazil.
Hawksworth, D. L. 1991. The fungal dimension of biodiversity;
magnitude, significanceand conservation.
MycologicalResearch 95: 641-655.
Hawksworth, D. L. & Colwell, R. R. 1992. Microbial
Diversity
21: biodiversity
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and Conservation
1: 221-226.
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controlled
cryogenic
light microscopy- an introduction
tocryomicroscopy. In The Effects of Low Temperatures in Biological
Systems , B. W. W. Groutand G. J. Morris, eds., Edward Arnold, London, UK, pp. 234-267.
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Culture Collections.
An independantreview commissionedby the Office of Science and Technology,
Her Majesty's
Stationery
Office (HMSO),
London.
Smith, D., Tintigner,N., Hennebert, G. L., de Bievre, C, Roquebert,
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Improvement of preservation
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for fungi of biotechnologicalimportance.
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Biotechnology
in the EC II, Detailed
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Magnien, eds., Elsevier, Paris, p. 89.
Smith, D. & Onions, A. H. S. 1994. The Preservationand Maintenance of Living Fungi, Second edition.
IMI Technical Handbooks No. 2, CAB INTERNATIONAL, Wallingford,
UK.
Stevenson, R. E. & Jong, S. C. 1992. Applicationof good laboratory
practice (GLP) to culture collections
ofmicrobial
and cell cultures. World Journal of Microbiology
and Biotechnology
8: 229-235.
Sugawara, H., Ma,J., Miyazaki, S., Shimura, J. & Takishima, Y. , eds. 1993. World Directoryof Collectionsof Cultures of Microorganisms, Fourth edition. WFCC World Data Center on Microorganisms,
Japan.
Thomas, V. & Smith, D. 1994. Cryogenic
light microscopy
and the developmentof long- term
cryopreservation
techniques
for fungi. Outlook on Agriculture
23: 163-167.

KEYNOTE ADDRESS II
CultureCollectionsin Japan:

TAKASHI NAKASE
Japan Collectionof Microorganisms
The Instituteof Physicaland Chemical
Wako, Saitama

History351-01,of culture

ResearchPresent

Statusand Future Plans(RIKEN)Japan

collections

Microorganisms
have long been used in Japan for the productionoffermented foods, beverages and seasonings. The production of these foods and relatedcommodities
was a big industry
even three hundred years ago and advanced
technologies
were developed in this field. At that time, engineers working in sake
(Japaneserice wine) breweries already knew the method for low temperaturesterilization(Pasteurization)
to lengthen the shelf life of sake though they had noknowledge of microbiology.
Aspergillus oryzae (Ahlburg)
Cohn, a mold used for the
production of "Koji", which was employed for saccharificationof rice starch in sakebrewing, has been maintained as pure culture in "Koji-ya", companies specialized
inthe productionof Koji. Koji, conidia and mycelia of A. oryzae grown in/on steamed
rice, was provided to sake breweries by the Koji-ya.
Modern microbiology
was introduced
to Japan about 120 years ago, byprofessors
from European countries and Japanese researchers who learned microbiology in Europe and the U. S. A. Studies on traditional
fermentation products weremajor targets of applied
microbiology
at that time. Sake brewing, especially,wasextensively studied by many microbiologists
because it was one of the most important
industries in Japan at that time, and about one third of national taxes came from tax on
sake. In addition,
sake brewing has microbiologicalinterest because of its uniquefermentation process.
In 1904, in order to maintain and increase the income from sake tax, thegovernment established
National Research Institute of Brewing (NRIB) in the Ministryof Finance, to develop the technology
for steady productionof sake. NRIB has
contributed greatly to the development of applied microbiology
and culture collectionsin Japan, and is still one of the leading institutionsin brewing. Microbial
culturesmaintainedat NRIB are now maintained at the IAM Culture Collection,
Center for
Cellular and Molecular Research, Institute of Molecular and Cellular Biosciences,
The
Universityof Tokyo, and Institute for Fermentation, Osaka (IFO).
The activities of the Government Research Institute of Formosa (GRIF) and18

the Central Research Laboratory, South Manchuria Railway Co. (CLMR) must not be
neglected when we refer to culture collectionsin Japan. The first catalogue of CLMR
entitled "CLMR Catalogue of Cultures of Fungi" was publishedin 1927 which is thefirst officially published catalogue of microorganisms in Japan. The subcultures of the
microorganisms maintained in these two culture collections
were transferred to IFO
before the end of the 2nd World War.
Nagao Institute(NI), in which the first private
collectionof microbial
culturesin Japan was established,
was founded in 1940. NI contributed
to thedevelopmentof basic microbiology
and microbial
culture collectionsin Japan. It
maintains fungal and microalgal cultures. A catalogues of cultures was published andadditional
cultures are listedin "Nagaoa", the official journal of the institute.
Unfortunately
it closed in 1971.
Towards the end of the Second World War, two great events occurred in thehistory of applied microbiology
and culture collectionsin Japan. The one was thestudy of the production of penicillin
in 1944-1945
and the other was the foundationof Koku Hakko Kenkyusho (Institutefor Fermentative Production of Aviation Fuel)in 1944. The former let leading scientists
in universities,
goverments and companies,
recognize the importance of applied microbiology,
and the latter was the establishmentof microbial
culture collection
for the purpose of productionof aviation fuel byacetone-butanol
fermentationof anaerobic bacteria. One year after its foundation,
Koku Hakko Kenkyusho changed its name to Institute for Fermentation, Osaka (IFO).
IFO has been steadily
developed and is now the largest general service collection
inJapan.
New trends in 1980s
In the 1980s a new trend began in culture collectionsof microorganisms inJapan. Several ministries and agencies of the Japanese government establishedculturecollections
as an essential infrastructure
for the promotion of life sciences including
microbiology,
biotechnology,
environmental studies, agriculture,
forestry and fisheries,
and for the the maintenance and effective
use of invaluable
microbial resources for
mankind.
In 1980, the Science and Technology
Agency establishedthe Japan
Collectionof Microorganisms(JCM) at The Instituteof Physicaland Chemical
Research (RIKEN),
for the promotion of life sciences in Japan. JCM is a generalservicecollectionof microorganisms
and maintains
bacteria,
actinomycetes,archaebacteria,
yeasts and yeast-likefungi, and filamentous fungi. In addition to theJCM, Life Science Research Information Section (LSRIS)
was established
at RIKEN
for bioinformatics
including information on microbial strains. In 1986, the World Data
Center on Microorganisms(WDCM) of World Federation
for Culture Collections(WFCC) moved to LSRIS from the Universityof Queensland,
Australia.
WDCM
publishes"World Directory of Collections of Cultures of Microorganisms"(Sugawara

19

et aL, 1993) and provides on- line service on world wide culture collections
and strain
data of microorganisms. Further, a gene bank was founded at the Tsukuba Life Science
Research Center of RIKEN. It collects and maintain animal and human cell lines, plant
cells and DNAs.
In 1983, Environmental
Agency established
NIES- collection
at National
Institute for Environmental Studies. NIES- collection is a general service collection
formicro -algae.
In 1985, Ministry of Agriculture, Forestry and Fisheries (MAFF)
establisheda Genetic Resources Center at the National Instituteof Agrobiological
Resources,
where the Center Bank of MAFF Collection is located. The MAFF collection collects
and maintains bio- resources which are important to agriculture,
forestry and fisheries.
The MAFF Collectionis the largest collectionof bio-resouces in Japan. A microbial
culture collection
is a part of MAFF-Collection.
Unfortunately,
governmental collections
for medicallyand industrially
important microorganisms have not yet been established.
Japan Society

for Culture

Collections(JSCC):

The core of cooperation

By international
standards, culture collectionsin Japan are not so large. Mostof them belong to parent institutions
or universities
and do not have sufficient
manpowerand budget. Therefore, cooperation among culture collections
is especially
important in Japan to effectively
collect and maintain microbial
cultures and fully
respond to the demand of users.
Japan Federation for Culture Collections(JFCC) was organized in 1951 forthe cooperationof culture collectionsin Japan. At that time, JFCC included only
affiliatedmembers. In 1974, JFCC was re- organized to permit the inclusionofindividuals
who were interested in culture collections,and sciences and technologies
which support
culture collections.
Ten companies have been supporting
JFCC assustaining
members.
In 1993, JFCC again re-organized
as the Japan Society
for Culture
Collections(JSCC) for further development of activitiesof the Federation.
Now, JSCC
has 341 members; 253 individual
members, 26 affiliatedmembers and 61 sustaining
ones. At the end of 1993 fiscal year, affiliatedmembers of JSCC held 105,692 strainsof microorganisms including viruses and cells lines of human and animals (Table 1).
In this fiscal year, they distributed
20,650 strains; 17,909 to domestic users and 2741
overseas (Table 2).
JSCC holds annual meetings, symposia, training
courses, and publishes
catalogues and an official journal. The latest edtion (5th ed.) of the catalogue (Kaneko,
1992)
contains
the holdingsof 25 culturecollections
includingbacteria,
actinomycetes,
yeasts, filamentous
fungi, mushrooms, microalgae, protozoa, viruses
and miscellaneous microorganisms. The total number of strains listed is about 27,500;
9,500 prokaryotes
and 18,000 eukaryotes.

20

Table

1. Holdingsof microbial

clutures

A ffilia te d

F ila m e n tou sm e m b ers

fu n g iY e asts

O UT

397

3 ,8 1 0

R IM D0

G IF UIM R Gof affiliatedmembers of Japan Society

A ctin o ‑Collections.

O th e rs

T o tal0

1 ,1 0 0

M icro ‑m y c ete s

B a c te ria

0

0

0

0

0

0

FERM

16

2

RIB

8 89

N IG

for Culture

V iru s es

a lg a e

1 ,1 0 0000

15 ,2 3 6

000

1 5 ,2 3 6

0

940

0

0

0

940

0

2

0

0

0

20

1 6 ,5 7 0

22

0

0

1 6 ,5 9 2

O

55 7

2*

787

15 6

4 ,9 9 4

129

0

0

0

IF M

2 ,4 4 6823

586

T IM M

N IE S1 ,1 7 5

55 9
3 ,8 5 5

1 ,8 4 2

3 ,7 0 3

0

0

0

0

0

IID

0

1

5

1 ,4 2 0

0

0

0

IA M

1 ,3 3 0415

30

1 ,0 8 2

0

5 82

0

0

A T U

720

295

1 17

403

N R IC4 22

1 ,4 1 5

9

2 ,7 2 97

5 ,5 4 5
1,4 2 6
3 ,4 3 9
1,5 3 5
4 ,5 8 2

N IA H56

0

7

2 ,1 9 3722

0

1 16 * 2

3 ,0 9 4

M A FF

7 ,2 6 8

472

16 6

5 ,0 2 8

537

2 37

6 6 *3

1 3 ,7 7 4

IF O

7 ,3 9 1

2 ,9 0 9

1,5 6 6

2 ,4 2 870

0

4 10 *

1 4 ,7 7 4

H U T

590

420

2 68

99

1,3 7 7

A H U

1 ,3 3 4

835

12 0

3 20

2 ,6 0 9

R IF Y

25

594

JC M

88 1

1 ,5 9 4

1,5 9 9

3 ,8 7 7

2 5 ,6 0 7

1 7 ,4 44

4 ,4 7 6

5 4 ,7 8 3

T o tal

4 40

1 ,0 6 1

1 ,3 5 80

54 *

8 ,0 0 5

1 ,3 7 6

64 8

10 5 ,6 9 2*1 protozoa;*2 protozoa 89, animal cells 27; *3 protozoa; *4 animal cells; *5 archaebacteria.
OUT= Faculty of Engineering,Osaka University; RIMD= Research Instituteof Microbial Diseases, Osaka
University;
GIFU= Gifu University
School of Medicine, IMRG= Gunma University
School of Medicine;
FERM= Fermentation Research Insitute;
RIB= National Research Institute of Brewing;
NIG= National
Instituteof Genetics, NBES= National Instituteof Environmental
Studies, IFM=Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba
Teikyo University;
IID= Instituteof Medical

University;
Sciences,

TIMM= Research Center for Medical Technology,
The Universityof Tokyo; IAM= Institute of Applied

Microbiology,
The Universityof Tokyo; ATU= Faculty of Agriculture,
Tokyo University;
NRIC= NODAI
Culture Collection,
Tokyo Universityof Agriculture;
NIAH= National Instituteof Animal Health, MAFF=
Genetic Resources Center, Ministry of Agriculture,
Forestry and Fisheries;
IFO= Instituteof Fermentation,
Osaka;

HUT= Facultyof Engineering,

University;
RIFY= The Insitituteof Microorganisms,
RIKEN.

Hiroshimaof Ecology

University;

and Viticulture,

AHU= Faculty
Yamanashiof Agriculture,

University;

Hokkaido

JCM= Japan Collection

21

Table

2. Microbial
cultures
distributed
from affiliatedmembers of Japan Society for Culture Collections
D is tr ib u t ion to

A ff i l ia te dm em be r s
OUT
R IM D
G IFU
IM RG

T o ta l
Dom e st ic u se r s

R IB
N IG
N IE S
IF M
T IMM
I ID
IA M
AT U
NR I C
N IAH
MAFF
I FO
HU T
AH U
R IF Y
J CM

188
72
53 5
2, 13 4
28
26
39 7
3 13
32 5
2 04
85
1 ,1 72
51
6 74
1 25
4 22
8 ,2 6936
2 22
22
2 ,6 09

T o ta l

17 ,9 09

O ve rs ea s u se rs
30
25

27 1

218
97
53 5
2 , 13 728
26
1, 53 3
36 2
42 9
20 4
86
1, 23 8
51
69 1
12 7
42 2
9, 29 939
2 26
22
2 ,88 02, 74 12 0 ,6 50

1, 13 6
49
10 4

66
17

1, 03 0"Microbiology
and Culture Collections" is the official journal of JSCC which
includes original papers and reviews on the technologies
and management for culturecollections,and papers on microbial systematics. It also contains technical protocols
and worldwide information on culture collections.
The domestic networking of culturecollections and future plans are discussed by the Culture Collection Committee of JSCC.
The role of generalservice collections:Past,

present

and future

Since the establishmentof the first recorded service culture collectionahundred years ago in Prague, the role of culture collections
has graduallychanged,accompanied by progress in the biologicalsciences and industries
where micro-

22organisms are used. General service collections
can be considered to evolve throughthree phases.
General culture collections in the first phase collect, preserve and distribute
microbial cultures. These are universal fundamental activitiesof culture collections
sothis is an important function even in modern collections. In order to fully perform thesefunctions, microbial taxonomy is an essential scientificbackground, because, most of
the users of general culture collections
choose strains for their research dependingon
the scientificnames of microbial strains. So, culture collections
have to distribute
microbial cultures with correct scientificnames.
In contrast to plants and animals, scientific names of microorganisms frequently
change accompanied by a change in the classification
system. These changes areinevitablebecause of the recent rapid progress in microbial taxonomy. So, curators inculture collections
have to follow up the recent progress of microbial taxonomy inorder to keep scientificnames up- to- date, and provide the list of synonyms for theconvenience of users. In addition,
division and unificationof species are frequently
proposed. In such cases, re-examination of taxonomic characteristics
are essential tocorrectly
assign strains of newly defined species. Curators should be experts in
microbial taxonomy and have to continuously check the propertiesof microbial strains
maintainedin culture collectionin order to quickly respond to changes in scientificnames.
In addition to the functions of culture collectionsin the first phase, culturecollectionsin the second phase have to play a role as a microbial systematics center.
It is a worldwide
trend that in universities
and colleges the activities
related tomicrobial
systematics
are on the decline. Culture collections
have to replace thefunction of universities
and colleges in this field. This is quite reasonablesince
culturecollections
routinely
carry out taxonomic studiesof microorganisms
maintained in their collections
as mentioned above.
Culture collectionsin the first and second phase also provide users various
kinds of services such as safe deposit, identification
service, microbiologicalcontrolof food products, isolation
studies, information
service, and varieties of contracted
researches in microbiology
and biotechnology,
in addition to the fundamental serviceof culture collections.
Most of the general culture collections
in developed countries
are nowin the second phase of evolution.
Culture collectionsin the near future have to enter the third phase. They haveto play the role of a general microbial
center including
the function as microbial
resources center. In addition to the functions of the second phase, culture collectionsin this third phase have to act as the center for the maintenance of microbial diversity
on the earth and the effective use of microorganisms which are indispensable
to themaintenance of global environment and human welfare.

23

Importanceof isolation

studiesof microorganisms"Microbial Diversity 21", the action statement, which was made in 1991 by
International
Union of Biological
Sciences and International
Union of MicrobiologicalSocieties(Colwell& Hawksworth, 1991), requested culture collections
to develop
networks of culture collections
and the increase of capacity for ex situ conservationof microorganisms. It is estimatedthat mankind knows less than 5- 10% of microorganisms living on the Earth. This means that microorganisms will become extinct
as the global environment changes due to the actions of man without a chance to be
knownto mankind, in contrast to plants and animals where most of species are alreadyknownto mankind. Culture collections
have to make every effort for the maintenanceand survey of diverse microorganisms on the earth and their effective exploitationforhumanwelfare as reproducible
resources for mankind. I think that culture collections
have to put more emphasis on the isolationand study of unknown microorganisms.
If we have the strong will to isolate new microorganisms and devise newtechniques
for their selective isolation,
we can easily isolate many unknown species
from the natural environment. A good example is the isolationof ballistosporous
yeasts which had long been believed to be a minor group of yeasts. "The Yeasts, A
Taxonomic Study", 3rd. ed., was publishedin 1984, includes 15 species of this kindof yeasts,
that is, 6 in Bullera (Rodriguesde Miranda,
1984)
and 9 inSporobolomyces/Sporidiobolus (Fell & Statzell
Tallman, 1984). Among them, noculture of Bullera dendrophila van der Walt et Scott is maintained and Sporobolomyces
holsaticus Windisch ex Yarrow et Fell is now regarded as a synonym of Sporobolomyces
salmonicolor (Fischer et Brebeck) Kluyver et van Niel. So, practically
13 species were
known at that time.
We carried out isolation
studies using an improved technique(Nakase &Takashima, 1993) of the ballistosporefall method of Derx (1930) for the selective
isolation of these yeasts. We found 36 unknown ballistosporous
yeast species, that is,
16 species of Sporobolomyces', 9 species of Bullera, 8 species of Bensingtonia, 2
species in Kockovaella and 1 species of Udeniomyces(Table 3). In addition
15 new
species have also recently been reported by researchers in other countries. As a result,the number of species in this group increased 4.8 times that listed in "The Yeasts, A
Taxonomic Study". Just 10 years ago, only 20% of presently recognized species were
known.

24

Table

3. New ballistosporousby yeast research
S pe c ies

yeasts foundgroup of JCM (1985-1995)
Y ea r

L oc a l ity

S p o r o b o lo m y c e s
1. S . s u b br un n e us
32 . S . oi rn yo zs i ct o plah i l u s

1 98 5
1 9 8 76Ja pa nJ a p a n , N e w Z e a la n d

4,
5.
6.
7.
8.
9.
10.
ll.
12.
13.
1 54 .

19 87
19 8 7
19 8 719 89
19 89
19 9 019 94
19 9 519 9 519 9 519 9 5J a pa n , T a s ma n ia
J a p a n , N e w Z e a la n d
J a pa n
J a pa n
J a pa n
J a pa n
J a pa n
N e w Z e a la n d
N e w Z e a la n d
N e w Z e a la n d
N e w Z e a la n d

1 6 . S . ta up o e n s is

19 9 5N e w Z e a la n d

B u i le r a
21 . B . po rs ye zu ad eo a l b a

1 9 8 56

J a p an

19 8 719 9 0*****J a p an , Ne w Ze a lan d
J a p anN e w Z e a la n d
N e w Z e a la n d
N e w Z e a la n d
N e w Z e a la n d
N e w Z e a la n d

19 8 6
1 98 7
1 98 7

Ja p anJa p a n
Ja p a n , N ew Z ea la nd

1 98 7
1 98 8
1 98 91 99 5

Ja p a n
Ca n a da
Ne w Ze a lan d
T h a i la n d

K o c k o va e l la
1 . K . tha i lan d i ca
2 . K . i mp e r a ta e

1 99 1
1 99 1T h a i la n d
T h a i la n d

U d e n /om y c e s
1. U . m eg a los p or us

1 98 6Ja pa n

3.
4.
5.
6.
7.
8.
9.

S . g r is e o f la v us
S . s a s ic o la
S . f a lca tus
S . r vb er
S . x a n th us
S . la c to p h i lu s
S . 1 ind e r a e
S . n o va z e a la n d ic u s
S . d imm e n a e
S . c o pr o s m ic o la
S . dc ro ap cr o ps hm ya el l u s

B . v a r ia b i l i s
B . m iy ag ian a
B . c op r o sm ae n s is
B . h a nn a e
B . h u i a e n s is
B . m ra k i i
B . un ica

B e n s i n g t o n ia
1 . B . in ter m e d ia
2 . B. m is ca n th i
43 . B . ns au gb ar no so e an s i s
5.
6.
7.
8,

B . y am a t oa na
B . y u c c i c o la
B. ing o l d i i
B . m u sa e

in p r e s s

25Collaborationof Asian Culture

Collections

Microorganisms have long been used for the production of fermented foods
and related products in Southeast Asian countries and neigbouring
regions. Uniquetypes of fermentation
product are produced and consumed in these countries andpeople are not reluctant to live with microorganisms.
These countries are able todevelop high technology bio-industries
following a similar course to Japan.
In the last 20 years, applied
microbiology
and biotechnology
rapidlydeveloped in these countries and now the level of research is close to that of developed
countries.
For further research progress, Asian countries have to develop culturecollectionsof microorganisms as an important component of their infrastructureforapplied microbiology,
biotechnology
and related fields. It is estimated that more thana half of the species of living organisms on earth are living in this region. This strongly
suggests that more than a half of the species of microorganisms on the earth are also
living in the region. Many of them must have diverse and useful functions for humanwelfare. So, the development of technologies
for the maintenance and the effective useof microorganisms is an urgent task in these countries where destructionof the naturalenvironment is progressing
rapidly, and rapid economic development is occurring.
In 1993, we proposed a collaborative
project on microbiology
among Eastand Southeast Asian countries. After a two years' feasibility
study, a project named the"Asian Network on Microbial
Researches" will start in fiscal year 1995 in theframework of "Global Research Network System" which is supported by the Scienceand Technology
Agency of the government. In the project,a network will be
constructedamong culture collectionsin Asian countries;
Indonesia,
Thailand,
Malaysia, Philippines,
Singapore,
China, Korea, and Japan. This network will act as
a promoter of researches in applied microbiology,
biotechnology
and related fields
where microorganisms are used. RIKEN promotes the project as the core institution
andJCM acts as the center of the culture collection network.
One of the main targets of the project is the isolationand study of microorganisms found in Asian countries. This will be carried out from the viewpoint of thediversity of microbial functions and the diversityof microbial species. In the former
studies, we expect the isolation of microorganisms having useful functions to produce
biologically
active substances, to degrade or concentrate harmful substances, andhaving excellent activities
for the effective use of agricultural
products. In the latterstudies, we expect the isolation of new microbial species which will contribute to therapid progress of microbial systematics. In the near future, this network will act as theregional
network for the study of microbial
diversityand collect basic data for
preparing an inventory of microbes in the Asian region.
The "Asian Network on Microbial Researches" is expected to be a model ofregional networking of culture collections.
Several regional networks are expected todevelop in other regions of the world in the near future. Then a worldwide network can
be implemented
for the maintenance of microbial
diversityon the earth and the26effective use of microorganisms. The continuous funding and training of scientificandtechnical
staff is critical to realise an effective network. I believe that WFCC will be
the core of the worldwide network of culture collectionsin the near future.
References
Colwell,R. R. and D. L. Hawksworth. 1991. Microbial Diversity
21: Action Statement.
International
Union
of Biological
Sciences and International
Union of MicrobiologicalSocieties.
Derx. H. G. 1930. Stude surles Sporobolomycetes.
Ann. Mycol. 28: 1-23.
Fell, J. W. and A. Statzell
Tallman. 1984. Genus Sporobolomyces. In The yeasts, a taxonomic study, 3rded., Kreger- van Rij, N. J. W. ed., Elsevier Sci. Publ., Amsterdam, The Netherlands,
pp. 91 1920.
Fell, J. W. and A. Statzell Tallman. 1984. Genus Sporidiobolus. In The yeasts, a taxonomic study, 3rd ed.,Kreger- van Rij, N. J. W. ed., Elsevier Sci. Publ., Amsterdam, The Netherlands,
pp. 532-540.
Kaneko, T. (Chief ed.). 1992. JFCC catalogueof cultures,
5th ed., the Japan Federation
for Culture
Collections,Tokyo, Japan.
Nakase, T. and M. Takashima. 1993. A simple procedure for the high frequency isolationof new taxa ofballistosporous
yeasts. RIKEN Review 3: 33-34.
Rodrigues de Miranda, L. 1984. Genus Bullera. In The yeasts, a taxonomic study, 3rd ed., Kreger-van Rij,N. J. W. ed., Elsevier Sci. Publ., Amsterdam, The Netherlands,
pp. 577-584.
Sugawara, H., J. Ma, S. Miyazaki,
J. Shimura, J. and Y. Takishima,eds. 1993. World directoryofcollectionsof cultures of microorganisms,
4th ed., World Data Center on Microorganisms,
Wako, Saitama, Japan.

TECHNICAL
SessionCollection

REPORTS
1

and Preservation

Chairpersons
Takashi Nakase
David SmithCollection,
Philippines

Evaluationand Use of Microorganisms

in theREYNALDO E. DELA CRUZ and ROSARIO G. MONSALUDNational Institutesof Biotechnology
and Applied Microbiology(BIOTECH)
UP Los Banos, College, Laguna, Philippines
Abstract
Collecting of microorganisms in the Philippines
is being conducted by many researchers fromthe academe, government research institutions
and the private sector. There are many repositories
ofmicrobial
cultures, but these are mostly small in size and not well funded.
The Microbial
CultureCollectionand Services Laboratory(MCCSL)of the National Institutesof Biotechnology
and Applied
Microbiology(BIOTECH) is one of the repositories in the country. It has a culture holding of 1,428 strainsdepositedby various researchers.
Evaluation to determine useful traits of microorganisms is being done by individual
researchers.
Traits evaluated depends upon their areas of interest and expertise.
In most instances, this is in responseto the country's needs and problems. Assessment is performed using standard laboratory
procedures.
There are many uses of microorganisms in the country. In general, they are used in agriculture
as biofertilizers(Rhizobium, Azospirillum, Azotobacter, mycorrhiza)
and biopesticides(Bacillus
thuringiensis),in the food industry(tofu, nata de coco), in the brewing and baking
industry(Saccharomyces species), feed products (tylosin,
mannanase), industrial
enzymes (pectinase,cellulase,lipase),in health (vaccines, antibiotics),
and in the control of environmental
pollution(bioremediation,
removal of heavy metals).
Most of the applicationsof microorganisms
are still conventional.
However,
there are now efforts to use modern biotechnologicalapproaches such as genetic engineering
to modify
organisms for the purpose of increasing
the rates of processes or to produce novel products.

Introduction
Microorganisms
play a major role in the life of human beings. They areimportant in the processing of food and feed products, fermentationof wine andbeverages, manufacture of enzymes, conversion of biodegradable
materials
into
composts, pollution control and in the production of vaccines. Microorganisms playa major role in the food industry,
manufacturing,human and animal health andagriculture.
Many organisms also cause a number of diseases.
This paper discusses protocols
for collection,
evaluation
and use of
microorganisms in the Philippines.
Collectingof Microorganisms

Microorganisms
are collectedroutinely by researchers,
microbiologists,
mycologists, and pathologist
from a number of research institutions,
universities,
collegesand from the private sector. The reason for collecting
microorganisms varies among

30

individual

researchers.
Collected microorganisms are deposited in a number of repositories(Table 1 ).
Most of these repositories are with the academe and/or government research institutions.
To date, no private culture collection
exists in the Philippines.
BIOTECH is one of the major repositoriesof microbial
cultures in thePhilippines
based in its Microbial
Culture Collectionand Services Laboratory(MCCSL).
The main function of the MCCSL is to collect and preserve microbial
strains which have been isolated or acquired by different
projects of BIOTECH andfrom other local and foreign sources. It also provides cultures to microbial laboratories
for research, instruction
and commercial uses.
A summaryof the microbial collections
deposited with the MCCSL is shown(Table 2). A total of 1,428 different
strains are now being maintainedin thislaboratory.
In 1990, BIOTECH publisheda Catalogue of Strains (Anon. 1990).
Table 1. Summary ofmicrobial

culture repositories

in the Philippines.

In s titu tio n

A fi liatio n

N a tio n a l In stitute s o f B io te ch n o lo g y a n d A p p lie d
M icro b io lo g y (B IO T E C H )U P L o s B aiio s

In stitu te o f B io lo g ica l S cie n c es (IB S )U P L o s B afio s

In d u stri a l T ec h n o lo g y D e v elo p m e n t In stitu te (IT D I)

D ep a rtm e n t o f S c ien ce an d T ec h n o lo g y (D O S T )In stitu te o f B io lo g ica l S cie n c es (IB S )U P D ilim a n

N a tio n a l S c ien c e R e se arch In stitu te (N S R I)

U P D ilim an

In stitu te o f P u b lic H ea lth (IP H )U P D ilim a n

R es ea rch In stitu te o f T ro p ic a l M ed icin e (R IT M )D e p ar tm e n t o f H e a lth (D O H )Table 2. Microbial

culturesdepositedin the MCCSL of BIOTECH as of October

7, 1994.
T y p e o f O rg a n is m s

N u m b e r o f S tr a in s

B a cteria

824

F ung i248

Y e a s ts

2 18

U n id e n tif i e d f u n g i

34

U n i d e n tif i e d b a c t e r ia

95

A lg a e

8

P r o to z o a

1

T o tal

1 ,4 2 831

Living microorganisms are stored in a number of ways at BIOTECH. They
can be overlaid with sterile mineral oil, stored at ultralow temperature (-70 °C) in 10%
glycerol, stored in sterile soil, in liquid nitrogen, or by liquid drying.
MCCSL is a member of the World Federation
for Culture Collections(WFCC).
It has communication linkages with culture collection
laboratories
in thePhilippines
and abroad.
One major constraint of Philippine
microorganism collections
is the lack of
coordination
among culture repositories
in the country. A networking system needs
to be instituted
to achieve some degree of collaborationand cooperation.
A proposal
has recently been submitted to the Department of Science and Technology (DOST) to
convert the MCCSL into the National Microbial
Strain Bank of the Philippines.
If
approved, this will make the MCCSL the National Repositoryof microorganisms
forthe Philippines.
Its mandate will be to establish
a network of activities
with the various
culture repositories
in the country. It will also be a linking laboratory
with other culturecollections
worldwide.
Evaluationof Microorganisms

Microorganisms
are evaluated by various researchers,
mostly from theacademe, to assess their potential
use in various aspects in agriculture,industry andmedicine.
In agriculture,most research deals with biologicalnitrogen fixation,
mycorrhiza and composting in an effort to substitute,
if not replace, our dependence
on chemical fertilization
to increase crop yields. Recently, with the growing awarenessand concern for the protectionof our environment, ways to control environmental
pollutionuseing microorganisms
are being studied.
Microorganisms
are also being
screened for enzymes, antibiotics
and for vaccine production.
Efforts to improve theinherent capabilitiesof these microorganisms to produce the desired end products arealso being undertaken through genetic engineering.
Some of the research conducted by various researchers in the country arelisted (Table 3).

32

Table 3. Fieldsof research undertaken

by various researchers

in the Philippines.
I n s t itu t io n

R e se a rc h F o c u s

R ese a rch e r

B i o lo g ic a l n itr o g e n f ix a t io n

E . S . P a te rn o , S . N . T ilo , I . J .
M a n g u ia t, D . M . M e n d o z a , F . G .
T o r r e s , M L . Q . S is o n , E . G a r c i a .
V . M . P a d ill a , T . S J . S a n t o s

N a tio n a l In s ti tu te s o f
B io te c h n o l o g y a n d A p p li e d
M i c r o b io lo g y ( B I O T E C H ) , U P L

M . U . G a r c ia

C o ll e g e o f F o r e s tr y , U P L B

A . A lm e n d r a s

V is a y a s S t a t e C o ll e g e o f A g r ic .
(V IS C A )R . E .d ela C ru z , M . B . B ro w n , E .
M . L u i s , J . T . Z a r a te , E . L o r illa ,N .A g g a n g a n

B IO T E C H ,U P L B

L . H ag

In s t it u te o f B io lo g ic a l S c ie n c e(I B S ) , U P L B

L .E .P adua

B IO T E C H , U P L B

R . G . D a v id e , R . Z o r ill a , D .
S a n t ia g o , R . G a r c ia

N a t io n a l C r o p P r o te c ti o n C e n te r
(N C P C ) , U P L B

L . H a g , D . L a p iz , W . B a r r a q u io ,V . C u e v a s , I . F . D a lm a c i o , A .
M a b e sa

IB S , U P L B

B . M . E s p ir i tu

B IO T E C H , U P L B

V .C uevas

IB S , U P L B

T . M . E s p in o , A . F . Z a m o r a

B IO T E C H , U P L B

E . J . d e l R o s a r i o , E . C a s ti ll o

I n s t itu t e o f C h e m is t ry ( I C ) , U P L

P r o d u c t io n o f a n tib io t ic s

A . K . R ay m u n d o

B IO T E C H , U P L B

P r o d u c t io n o f m e th io n i n e

W . G . P a d o lin a

IC , U P L B

L e c ti n p r o d u c ti o n

F . M e rc a

IC , U P L B

V a c c in e p r o d u c ti o n

H . A . M o li n a , F . S . M a s lo g

B IO T E C H , U P L B

V .Q .P han

B io lo g i e s C o r p .

E . J . d e l R o s a r io , J . C . M a m a r il ,B IO T E C H , U P L B

M y c o rr h iz a

B io l o g ic a l c o n t r o l o f p e s t s a n dd is e a s e s i n a g ri c u l tu r e

C o m p o s ti n g

E n z y m e p r o d u c t io n

E n v i ro n m e n ta l p o l lu t io n c o n tr o l

E . C . B u e a n te , J . F . S i m b a h a n
G e n e t ic e n g in e e r in g

A . K . R a y m u n d o , B . M . E s p ir it u

B IO T E C H , U P L B

Uses of Microorganisms
Microorganisms have many uses. The reported
workers in this field are listed (Table 4).

uses of microorganisms

and

33

Table 4. Summary of the reported

uses of microorganisms

O rg an ism s

and the researchersU s esusing these organisms.
W ork ers

A sp e rg illus o ry z a e(A h lb u re ) C o h nb io c on v ersio n of a g ri cu ltu ral
w astes fo r an im al fe e d s

C . B . P h a m , T . J . R am irez , S . A .
Sedanou4zo sp irillu m so p .

n itro g e n fix atio n in c o rn an d

M . U . G a rc ia , T . S . J . S a n to s

u p la n d ri ce
B a cillus s u b tilis(E h ren b erg ) C o h n

d ec o lo riz atio n o f b io g a s e ffl u e n ts

E . J . d e l R o sari o , E . C . B u g an tep ro te ase p ro du ctio n

T . M . E s p in ob io log ica l c o n tro l o f A sp e rg illus
in fec tion an d a flato x in

R . G arcia

c o ntam in atio n in c o m
B a cillus th u ring ie ns isb io in se ctic id e

L . E . P ad u a

B ea u ver ia b a ssian a(B alsam o ) V u ille m inb io lo g ic al co n tro l o f ric e b u g(L ep to c o rn is a sp .)D . R . S a n tiag o

B ra dy rh iz o b ium sp p .

n itrog en fix a tio n in le g u m e s

E . S . P atem o , A . A lm e n d ras , D .
M . M e n d o za , F . G . T o rres , M L .
Q . S is on

C a n d id a ly p o ly tic a

lip a se p ro d u c tio n

T . M . E sp in o

C h a eto m ium co ch lio d es
P allise r
C h a eto m ium cup re u m
A m esb io lo g ic al c on tro l o f A sp erg illu s
in fe c tio n a n d afla to x in

R . G a rc ia

D eb a ry o m y c es h a ns en ii(Z o p f) L o d d e r e t K reg e r v an R ij

b io lo g ic al co n tro l of A sp e rg itt us
in fe ctio n a n d afla to x in

B erl in er

co n tam in ation in c o rn
R . G arc ia

co n ta m in a tion in c o rnb io lo g ic al co n tro l o f p e a nu t ru st

L . L . H ag

p ath o g e n P u ccin ia m a c h id is
E n te ro b a c ter clo a ca e
(Jo rd an ) H o rm ae ch e a n d E d w ard

b io lo g ic al c on tro l o f p ea n u t ru st

G lo m us m oss a e(G e rd em an n & T ra p p e ) N ich .

m y co rrh iz al in o cu lan ts fo r

L . L . H ag

p ath o g en P u cc in ia m a c h id isag ro n o m ic c ro p s

M . B . B row n , R . E . d ela C ru z ,E . M . L u is , J . T . Z a rate , L . L . Ila

G lo m us fa s cic u la tu s
(T ax te r sen s u G erd e m a nn )G erd . e t T rap p eb io lo g ic al c on tro l o f fr u it d isea se s

M . B . B row n , E . M . L u is

L eu c on os to c m ese n te ro id es(T sen K o v sk ii) v an T ieg h e md ex tra n p ro d u ctio n

I. F . D a lm a cio , G . D . R ey e s

M eta rh iz iu m a n is op lia e(M e tsch n ik o ff) S o ro k inb io lo g ica l c on tro l o f n em ato de s

R . A . Z o rilla

M on as cu s a n ka v a r m b e llu s
S ato

p ro tea se pro du ction

T . M . E sp in o

P a e cilom y c es lila c im is(T h o rn ) S a m so nb io log ica l c o n tro l o f n em ato de s

R . G . D a v id e , R . A . Z o ri lla

e t G erd .

34
Table

4. Continuation....
O rg a n ism s

U s esW o rk e rs

P e n ic illiu m c h ry s og en u m
T h omp e n ic illin G ‑ p ro d u ctio n

A . K . R ay m u n d o

P e n ic illiu m ox a lic u mb iolo g ic al co n tro l o f n em a to d e s

R . A . Z o rilla

lig n in a se p ro d u ctio n

E . J . d el R o sario

P is o lith us tin cto riu s(P erso on ) C o k e r e t C ou ch

m y co rrh iz al in o cu lan t fo r p in es

R . E . d ela C ru z , J . T . Z a rate , E .
L orilla , N . A g g a n g an

R h iz o b iu m sp p .

n itro g en fix atio n in le g u m e s

S . N . T ilo , I. J . M an g u ia t, M . U .
G a rcia, D . M . M e n d o z a, A .
A lm en dra s, V . M . P ad illa

re m o v a l o f h e av y m eta ls

J. C . M a m a ril, J. F . S im b ah an

R h iz op u s d elem a r(B o id in) W eh m e r et H a n za w a

lip a se p ro d u c tion

T . M . E sp in o

R h iz op u s o ry z a e
W e n t e t P rin s en G e erlig s

afla to x in d etox ifica tion in c op ra

I. F . D alm ac io

C u rri e e t T h orn
P h a n ero c h a ete ch ry so sp o riu m
B u rd sallan d E u c a ly p tus

m ea lR h iz op us s to lon ife r(E h re n b e rg ex F rie s) L in d
R h iz op us sp .

S a c ch a r o m y ces c ere vis ia e
H an senb io c o n v ersio n o f ag ric u ltu ral
w astes fo r an im a l fee d s

C . B . P h a m , T . J . R am ire z , S . A .
S ed a n o

rem o v al o f h e av y m e tals

J . C . M am aril, J . F . S im b ah an

b io lo g ic al c o n tro l o f A sp e rg illus
in fe ctio n in a n d aflato x in

R . G a rc ia

co n tam in a tio n in co rn
S a c ch a ro m y ces ch e va liereb io log ica l c o n tro l o f A sp e rg illus

G u illie rm o n din fec tion an d a fl ato x in

R . G arc ia

co n tam in atio n in co rn
S trep to m y c es f ra d ia e
(W ak sm a n & C u rtis) W a k sm a n& H en ric i

cru d e ty lo sin p ro d u ctio n

A .K .R aym undo

S trep to m y c es sp p .

m a n n an ase p ro d u ctio n

A ‑ F . Z am o rab io log ic al c o n tro l o f A sp e rg illu s
in fec tio n an d afl ato x in

R . G a rc ia

c o n tam in a tio n in co m
T ric h o d e rm a sp p .

c o m p o stin gb io lo g ic al c o n tro l o f s oil‑b o rn e

B . M . E sp iritu
V .C uevas

p a th o g e n s o f cro p s
b io co n v e rs io n o f ag ric u ltu ra l
w a ste s for a n im a l fe ed s

C . B . P h am , T . J . R am irez , S . A .
S ed a n o35

Future Plans
Immediate plans of BIOTECH as far as microbial
culture collection,
evaluation and uses of microorganisms are concerned is the conversion of the MCCSL
into a National Repository for Microbial Collections.
Funding is the major constraint
in this conversion plan. The government gives low priority for this kind of activity.
Related activities
for the future are the following:1.
Conduct research on animal cell cultures-cultivationand preservation of cultures-detection/eliminationof mycoplasma-cataloging of animal cell lines
2.
Use of chemotaxonomic and molecular biologicalmethods for identificationof microorganisms
3.
Offer scientificconsultancy and training services in the following
areas:-isolation and cultivationof microorganisms-characterizationand identificationof cultures-preservation of cultures
References
Catalogueof Strains. 1990. Microbial
Culture Collectionand Services Laboratory.
National Institutesof Biotechnology
and Applied Microbiology,
UP at Los Banos, College, Laguna,Philippines.
Annual Report. 1991. National Crop Protection
Center. UP at Los Banos, College, Laguna, Philippines.
Annual Report. 1992. National Institutesof Biotechnology
and Applied Microbiology.
UP at Los Banos,College, Laguna, Philippines.
Inventory of UPLB Research Programs/Projects/Studies.
CY. 1993. Office of the Director of Research.
Universityof the Philippines,
Los Banos.
Director'sReport. 1993. National Institutesof Biotechnology
and Applied Microbiology.
UP at Los
Banos, College, Laguna, Philippines.
Hag, L. L. and L. P. Valencia. 1993. BiologicalControl of Peanut Rust. Terminal Report. Department
of Plant Pathology,
UP at Los Banos, College, Laguna, Philippines.

A Note on the Present State of Indonesian
MIEN A. RIFAI
Herbarium Bogoriense
Bogor 16122 Indonesia

Puslitbang

Microbial

Diversity

Biologi-LIPI

Abstract
To date the exploration
for and systematic study of the microbial world in many tropicalcountries, including Indonesia, has not been sufficient to enable scientists to undertake their meaningful
inventory. Nevertheless from the few selected groups which have been taxonomicallysurveyed it is evident
that the microbial biodiversityof Indonesia is extremely rich if compared to other parts of the world. It isalso known that the diverse Indonesian people have been using these rich resources. Local knowledge andlocal wisdom about biodiversity
use is manifest. The destruction of many habitats and ecosystem types
occurring in the country has resulted in the disappearanceof many microbes.
In the past few years the Herbarium Bogoriense and her associate institutes
have been actively
undertaking
exploration
for soil bacteria, fungi, and algae especiallythose of economic importancedemanded by the industrial sectors. In line with the government policy and supported by government grants,
a number of biological studies on certain selected groups have been undertaken to support the developmentof microbe use. The successes achieved and the failures experienced as well as the constraints
faced arediscussed. Plans for the future of microbial diversity
prospectings
are presented.

Introduction
For Indonesia it is indeed very difficult
to give an accurate estimate of the
microbial
diversity
occurring in the country, because until today, collecting
andsystematicstudies of microbes are hardly adequate to enable scientists
to prepare ameaningful inventory. Like many other tropical countries it is not practical to give a
microbiotic
account of the country due to the absence of extensive collections
uponwhich such an undertaking
can be soundly based. At best a checklist
based onpublished records and scattered studies, as was done by Semangun (1992) recently onplant parasitic
microorganisms,
can be undertaken.
From the few selected groups which have been taxonomicallysurveyed themicrobial
diversityof the country as well as surrounding
areas is extremely richcompared to other parts of the world. This is understandable
because the biodiversityof Indonesia can be considered a megacentre. Although Indonesia occupies less than
1.3% of the earth surface, evidence suggests that more than 15% of the worlds species
can be found in Indonesia. There are about 7,000 out of the 19,000 species of worlds
fish species, some 1,600 species of birds out of a total of 9,200, about 1,500 species offerns out of ll,300
believed to be found on earth, 28,000 species of flowering plants
out of approximately
250,000 total world angiosperm flora, which testifies
to thebiologicalrichness of the Indonesianarchipelago.
Since microbes are usually living
in close association
with these varied organisms, it follows that the diversityofIndonesian microbial world will also be tremendous.

38

The diverse ethnic groups of Indonesia have been using these rich microbial
resources. Indigenous
knowledge about microbial diversity
use is also voluminous(Rifai,1994). Unfortunately
this knowledge is diminishing
due to the destructionofmany ecosystems which has resulted in the extinctionof some microbes. In the light of
the global concern for the future safety of biodiversity,
this is unfortunate.
In this paper
the state of our understandingof Indonesian microbial
diversityand the extent of
microbial
use by local people, as well the efforts being attempted by a number ofIndonesian institutions
to document and conserve microbial diversity,
are presented.
Indonesian

Microbial

Diversity

According to Hawksworth (1991)
there may be some 1,500,000
species offungi in the whole world, and by interpolating
it may be safely assumed that inIndonesia
close to 200,000
species of fungi exist. This is a huge number,if we
remember it is equal to the total number of fungal species that were thought to occur
in the whole world based on earlier conservatively
estimated figures (Ainsworth&
Bisby's Dictionaryof Fungi). Since the already described and named species of fungiin the whole world is only about 72,000 (see Smith in this volume) a Herculean task
awaits Indonesian scientists
to inventory their fungal diversity.
Even supposing that
under the hypotheticalsituationthat we manage to solicit the services of the entire
world taxonomic mycologists
to perform the inventory, the task cannot possibly becompleted in the foreseeable
future.
On the other hand, if we take a more conservative
attitude,
there areindications
that a preliminary
enumeration of Indonesian microbial
diversity
can beachieved. The experience of studying Indonesian phalloids
would seem to indicate thatfor certain groups of fungi it will be possible to make a complete, albeit tentative,
inventory.
Since Prof. K. B. Boedijn publishedhis excellent
account of Indonesian
Phallales in the early 1930's no additional species has to be added to his list of accepted
species. Similarly
for the larger fungi belonging
to the genera Lentinus, Phellinus,
Microporus no new species have been describedin recent years. These examples,
however, may not be a good choice because the explorationsof these easily and hence
well collectedgroups may have been exhausted. Moreover their speciation
is clearlydelineated,their species concept is well understood and their diversity
is not that great.
With the less easily preserved larger fungi because their fragile fruitbodies
cannot be transported long distances the picture is slightly
different.
This is especiallytrue in the case of ectomycorrhizal forming fungi, because long residence in an area will
reveal the extreme richness in species of this economicallyand ecologicallyimportantgroup. Corner's (1972) study on tropical boletes produced much interesting
data; evenon the relatively
small island of Singapore there are more species of boletes than thewhole of the British Isles (Rifai,
1989). The Indonesian archipelago
has hardly been
explored for boletes and the number of species likely to be encountered are expected
to be great. The possible high number of mycorrhizal
species can be attributed
to the39

fact that there are thousands of species of trees which may act as host to mycorrhiza.
Smits (1994)
difficulty
in definitelydetermining
the identityof dipterocarp
treeectomycorrhizae suggests that the number of mycorrhizal species is very large indeed.
I believe that the high estimate given by Hawksworth (1991) is nearer to the possibletrue number of fungi given diversity of this kind of species we find in Indonesia.
Taxonomically complex species problems occur in Indonesia for example theGanodermaapplanatum (Pers.: Fr.) Pat. - - Ganoderma
philipsii Cooke complex,
Sclerodermanitidum Berk. - SclerodermaverrucosumPers. complex, as well as theconstantly smaller ascospore size of tropical Daldinia concentrica (Fr.) Ces. & de Not.
compared to its temperate counterpart.
Perhaps when mycologists
are faced withspeciation
problems they should recognize
the subspecies
entity,as is used byflowering plant taxonomists, where biogeographicalconsiderations
are being used tojustify this taxonomic category. DNA fingerprinting
will be a helpful tool to help sortout these taxonomic problems.
For micro fungi the situationis completely
different,
because the number ofnovelties
here is very large. Host specificfungal parasites
occur in abundance inIndonesia, almost every powdery mildew encountered in the field seems to represent
an undescribed species. Sooty moulds and black mildews are similarlycommonsights
on many tropical wild plants, both in the forests and in the rural areas, and can be seen
also infesting
numerous cultivatedplants in agricultural
centres. In the highly humid
areas saprophytic
Hyphomycetes occur in large numbers so that students assigned tolook into the more commonlarger genera often find undescribed
species. In Malaysia,
Nawawi (1985)
and his associates,
described many unusual aquatic Hyphomycetes
which again indicates the species richness of our region.
Those regularly attempting
to isolate tropical soil fungi will frequently
findthat their cultures
do not appear to match any described
species which often
discourages
them from further indepth
biologicalstudies.
Since from a genetic
perspective
there should be as many species of Trichoderma as there are species of
Hypocrea, the number of species of these ubiquitous
soil fungi in the tropics is alsohigh. Trichoderma isolates derived from various areas in Indonesia show a high degreeof variability
very different from those obtained in temperate regions.
The same can also be said of algal flora in the Indonesian waters and here
more intensive works are also needed. Unfortunately
very little is known about the
species of Indonesianbacteria, but it is probably
true for other parts of the world
because the biogeographicaldistributionof bacteria does not seem to interest anybody.
In this group of organisms it is more rewarding to study their molecular genetics,
biochemical
properties
and ultrastructure
because of their potential
for immediate
development in making biotechnologicalbreakthroughs.
Microbial

Use by Indonesians
Another

approachtoappreciating

the richnessof Indonesian

microbial

40

diversity
is to study the way the local people are using these groups of organisms. As
early as the sixth century there were written reports by Chinese travellers,
who
described
the abilityof Indonesians
to prepare alcoholic
beverages from the palm
inflorescence. Similarly from reports written on palm leaves, it can be concluded thatlong ago the Indonesians
had been using many kinds of microbes as sources of foods,
medicines, cordage, for occult purposes or as important elements in different life cycle
traditionalrituals.
The Indonesians are known to use unusual species of fungi (Rifai, 1989). The
Sundanese of West Java enjoy consuming raw vegetables including winged bean pods
infected
with Synchytrium psophocarpi (Rac.) Gaum., one of the major fungal
pathogens of this legume crop. Similarly
they enjoy eating the highly swollen seed ofmaize infected by the corn smuts Ustilago maydis (DC) Corda. Besides the familiar
Hirneola fuscosuccinea (Mont.)
Farl., Oudemansiella canarii (Jungh.)
Hohnel,Pleurotus anas van Overeem and Boletus subtomentosus (L.) Fr. as well as the much
cultivatedpadi straw mushroom Volvariella volvacea (Bull. : Fr.) Sing., the Indonesians
are known to consume the split gills Schizophyllum communeFr. , Lentinus sajor-caju
Fr., Polyporus udus Jungh., the operculate
discomycete
Galiella javanica (Rehm)
Nannf. & Korf and the ectomycorrhizal
puffballScleroderma sinnamariense Mont,
which otherwise are labelled as inedible and unsuitable for culinary purposes in many
standard European or North American mushroom field guidebooks.
Botryodiplodia theobromae Pat. is a notorious
fungal plant pathogen,however, people in Java find it useful in making cassava tubers more digestible
andbetter to eat. They turn the wound parasitic
habit of this fungus to advantage inconverting
the long carbon chains of the white starch biochemicallyinto the shorter
ones in the blackened fermented product locally called gatot. In this connection it isof interest
to note that the Indonesian
farmers use the severity of the attack by
Cercosporaleafspot on peanuts as the indicationthat nuts should be harvested!
The assistance rendered by fungi in food fermentation
represents one of themost welknown tropicalfungal phenomenon (Saono, Hull & Damcharee, 1986).
Mould- fermented soya bean cakes such as the black coloured tempe produced throughthe activity ofRhizopus oligosporus Saito, Rhizopus orysae Went & Prinsen Geerlings
and species ofMucor, as well as the pink coloured oncomprepared by inoculatingthe
Monilia state of Neuruspora sitophila Shear & Dodge on the soybean are familiar
sights in Java because there they become the poor men's meat and hence the mostimportant protein source for village people. Similarly
numerous kinds of yeastfermented glutinous
rice- based tapai and the cassava- based peuyeum are also
extensively used as means of varying the preparationof these much used carbohydrate
sources. As can be expected Saccharomyces cerevisiae Meyen, species of Candida,
Endomycopsis•E,
and a number of other filamentous fungi are employed here.
The preparationof alcoholic
beverages from palmyra palm, coconut andsugar palm known as tuak or saguer are traditional
fermentation
products of Indonesia.

41

These beverages have been much prepared by the local people for many hundreds ofyears. Unfortunately,
the yeasts involved in these fermented drinks have not been
subjected
to modern taxonomic research so that the kinds of species as well as thebiochemistryof the process are not yet wholly understood. Moreover in the preparationof these beverages the local people have been using barks of at least three tree species
as additives but their proper function has not yet been elucidated,but perhaps it has
something to do with preventing
undesiredmicrobes from taking part in thefermentation process.
Besides fungi, local people have been successful
also in securing theassistance of bacteria in many traditional
food fermentations.
The productionof theIndonesiansoya sauce kecap involves both fungi (mainly Aspergillus orysae(Ahlburgh)
Cohn in the solid state fermentation
phase) and bacteria (the brine state
fermentation phase). Terasi is a bacteria-fermented
shrimp paste which represents animportant ingredientin Indonesiancooking. The buffalo milk-based
dadih is a solid
side dish produced through the activity of bacteria. Tempoyakand kuyuk are other typesof bacteria fermented foods, but in this case the raw material used are durian fruits andbamboo shoots respectively.
Kuyuk is prepared by burying the bamboo shoots in rivermuds, soliciting
natural inoculum from the soil. Very little is known about the active
bacteria involved, and no real study has been undertaken to understand the biologicalor chemical processes that are taking place in their preparation.
Burying bamboo culms in river muds is also much practiced by the local
people for the purpose of preserving the culm by making it hard and insect resistant.
Biochemical analyses of preserved culms indicated that the starch content decreases in
mud.Bacteria apparently break down the starch present in the culms and insects do notattack culms not having starch.
In the past people in Java were using the giant puffball Langermannia bicolor(Lev.) Demoulin & Dring to combat certain type of stomach disorder. The same people
used also the scarlet polypore
Pycnoporus sanguineus (L.) Merrill
in traditional
medicines to relieve patients suffering from venereal diseases. The water contained in
the fruit body oiXylaria tabacina (Kickx) Berk, was used as remedy for certain eye
diseases, whereas powder prepared from the fruitbodies
of Xylaria obovatumBerk, wasapplied to skin of those suffering from burns. Lichenes, especially
species of Usnea,
have also been extensively used in traditional
medicines to cure burnwound as well as
diarrhea. In the old days people in the Moluccas used to hang the fruit body ofGanodermaamboinensePat. to deter evil spirits from entering the house and causing
spiritualmaladies. Today youths in Bali love to incorporate
certain dung inhabiting
speciesof Psathyrellain their omelletteand other cookings
because of thehallucinogenic
effects.
These numerous traditional
uses, some of which involve the cultivationofmicrobes, may have lead to a wide range of genetic variation during the course of theirlong time use by the local people. Although local people have not always realised theexistence of these variations
because the variation is less obvious than those of plant

42

cultivars or animal breeds. On the other hand, their knowledge about the variationsisexpressed in the kind of products offered. For example, in Indonesia there is tempe
bogor, tempe malang, tempepurwokerto, tempe bandung and so on, each having
different
quality,taste, appearance, texture, and aroma, even though they have been
preparedfrom the same type of soya beans. As well as the differencesin certain
methods for their preparations,
every kind of tempe has its own specifically
prepared
inoculum. Similarly
one can detect differences
in tapai from different areas based on
their alcoholicor sugar contents. The same can also be said for the bacteria fermented
shrimp paste because certain areas produce terasi with different
colour, texture, aroma,
taste and so on.
Even in the larger fungi traditionallycultivated,such as the padi straw
mushroom Volvariella volvacea, genetic variation
can also be demonstrated.
This
variation
has increased in the last few years because of the introductionof speciallyraised cultivars from Taiwan.
Expandingthe Horizonof Microbial

Use

The experience and knowledge passed on to us by our ancestors in harnessing
the microbial
world for the benefit of mankind, opens a new vista because we cancontrol the activity of microbes for many other uses. Therefore in the past few years the
Herbarium Bogoriense and her associate institutes
have been actively undertaking
theexplorationand collectionof soil bacteria,
fungi, and algae especiallythose ofeconomic importance. In line with the government policy and supported by government
grants, scattered biologicalstudies on certain selected groups have been attempted tosupport the development of their use.
These activities
have been prompted also by the demand from the industrial
sector. Some years ago a group of scientists
from the Bandung Institute of Technology
managed to isolate and purify one of the fungi present in ragi or yeast cake traditionallyused as a starter in inoculating
tempe. The fungus, Rhizopus oligosporus , appears to beable to produce better quality tempe compared to those traditionallyprepared by using
mixed inoculum contained in the yeast cake. Based on this experience, now and again
certain local producers of fermented foods come to the Herbarium Bogoriense foradvice as well as to obtain information
on ways of improving their products.
To
anticipate
these demands our scientists
have been isolatingstrains of microbes used inmany local industries
and study their biologicalproperties
with the objectiveof
selecting superior strains.
Recommended strains have been available for some of the products based on
the results of the study on how to produce better quality oncombogor,peuyeumcitatah,
tapai kuningan and so on. However, because the spectrum used in screening thesevarious strains apparently was not wide, the results obtained often have not been verysignificant.
The traditional
inoculum of certain types of tapai, for example, cannot be
replaced without jeopardizing
the taste already expected by the consumers. The very

43sweet tasting tapai kuningan produced by researchers is not always very popular
because some people found the flavour too strong and the alcohol content is high;moreover it was tinged with the acrid taste of rose apple leaves which some people
found unpleasant. The requirement from the local producers and their customers,
therefore,
are not always in line with the suggestions
and recommendations
fromresearchers.
A number of Indonesian researchers now are soliciting
the lignocellulolytic
capabilityof certain fungi to decompose lignin produced in large quantity as waste
products in many industries such as saw mills and palm oil factories. The activity ofthese fungi will enable us to obtain white coloured cellulose fibers, which otherwise
would have to be bleached using polluting chemicals, of suitable length to be used in
the paper industry. Some scientists associated with the Herbarium Bogoriense are also
busy screening for the most effective strains of Trichoderma having high cellulolytic
activity capable in degrading rice straws in the rice fields in order to shorten the period
from one rice planting to the next one. So far we have managed to select two or threestrains of Trichoderma harzianum Rifai capable of reducing the 36 day interval ordelay normally needed into a 20 day period.
Foresters have been successful in cultivating
native pine species on a large
scale, due to the availabilityof suitable
ectomycorrhiza.
Since Indonesian
forests
contain stands of hundreds of species of dipterocarp
trees, ectomycorrhizal
fungi areneeded to cultivate seedlings of dipterocarp species. It is necessary to learn much about
the dipterocarp/mycorrhizal
symbiosis(Smits,1994). Much effort is directedtomastering the use of endomycorrhizal
fungi for many agricultural
crops, because
Indonesian soils are low in phosphorous content and the country has no natural depositof this element.
Green manurehas been popular with the farmers so that strains of Rhizobium
have been produced to improve the nitrogen fixing abilityof certain legume crops.
Some of these strains are already produced commercially.
Similarly
the use of blue
green algae in association with certain aquatic ferns are being introduced to farmers toassist them in minimizing the cost of nitrogen fertilizers
they needed in the rice fields.
Collectionsof strains of Bacterium thuringensis are being made from soils from various
places all over Indonesia
to be used in biologicalpest control using sophisticatedbiotechnologicalapproaches.
A number of foreign pharmaceuticalindustries
approached the Herbarium
Bogoriense to undertake microbial diversity
prospectings.
The aims of these ventures
are mostly directed towards the isolationsof microbes with anti-cancer potential
andnovel antibiotics.
Working permits are being processed to enable us to undertake thissurvey but it will take some time before it can be executed. The government now isthinkingof setting up an agency to undertake the "marketing" of microbial and othertypes ofbiodiversity,
to be managed by giving certain rights to explore a specific area
to a private company in close cooperation with a government research establishment,
and to regulate the sharing of the results for mutual benefits.

44

Looking Ahead
One of the major handicaps
of working with the Indonesian
microbial
diversity
is the absence of a national culture collections.
The living isolates obtained
during our explorations
have been kept in many associating
institutes
which maintaincultures according to their respective field of specializationand interest, for example
the PhytopathologicalResearch Institute,
the Veterinary
Research Institute,theMicrobiological
Division of the BiologicalResearch and Development Center, andmicrobiologicallaboratories
of a number universities.
There has been a concensus thatthese separate institutes
which now are only loosely cooperating
shall form a kind offederationin the future while awaiting the establishmentof a national
culturecollection.
The section of microbial collectionsin the Herbarium Bogoriense keep thepreserved specimens and continue to undertake taxonomic studies of the Indonesianand the Malesian flora. Beside being charged with the task of undertaking
research formaking an inventory of the Indonesian flora as a whole, the Herbarium Bogoriense has
also been requested to provide data and information that can be used by the government
to formulate a utilizationand conservation
policy. Conservation
is indeed needed
because as far as the microbial world is concerned, microbial extinctionis also taking
place. The specialized
fungi which are highly host specificappear to be the first to
disappearcompletely
when the hosts are no longer available.
Complete
habitat
destruction resulted in species titRussula disappearing from the formerly forested lands
in Java. Old records indicated that the local people made use of Russula for culinary
purposes. We are left with only records of their local names in the old literatures,andmodern Javanese people do not have any knowledge or remember these vernacularnames.
The government fully realizes that at present the country does not have themastery, the technology
or even the knowledge to undertake appropriate
microbial
diversity
conservation. As in many developing countries, conservation is not popularwith the politicians,the planners and the decision makers as well as with the public atlarge especiallyin the areas close to the conservationsites. The policy adopted
therefore
is to preserve the ways microorganisms
are used because if we do want tocontinuously make use of them on sustainablebasis certainly we must be sure that theresources concerned should be always available.
Consequently
conservation measure
will be achieved indirectly
by subtle steps which does not antigonized
the party who
may have strong feeling about this politicallymuch abused word.
There is a general feeling nationallythat the conservationof microbial
diversity
is a must (Kantor Menteri Negara Kependudukan dan Lingkungan Hidup
1992).
Everybodyseems to agree that in the future the much advertised
biotechnologicalbreakthroughthrough the spectacular genetic engineering
approachwill not be wholly successful without the availabilityof biodiversity
to work with.
What little
we achieved today is being directed
towards that future promising

45

possibility.
References
Corner, E. J. H. 1972. Boletus in Malaysia. Singapore Botanical Gardens, Singapore.
Hawksworth, D. L. 1991. The fungal dimension of biodiversity:
magnitude, significance,and conservation.
Mycol. Research 95 (6): 641-655.
Kantor Menteri Negara Kependudukan
dan Lingkungan
Hidup. 1992. IndonesianCountry Studyon Biological
Diversity.
Kantor Meneg KLH, Jakarta.
Nawawi, A. 1985. Aquatic
Hyphomycetes
and other water- borne fungi from Malaysia.
MalayanNature Journal 31: 75-134.
Rifai, M. A. 1989. Astounding fungal phenomena as manifestationsof interactions
between tropicalplantsand microorganisms.
In Interactions
between Plants and Microorganisms,
Lim, G. and
Katsuya, K., eds., National Universityof Singapore, Singapore,pp. 1-8.
Rifai,M. A. 1994. A discourseon the biodiversity
utilizations
by local Indonesians.
In Workshop
on Indonesian Biodiversity,
3- 5 November 1994, National
Research Council, Serpong, Jakarta.
Saono, S., Hull, R. R. & Dhamcharee, B. 1986. A Concise Handbook of Indigenous
Fermented
Foods in the ASCA Countries. LIPI, Jakarta.
Semangun, H. 1992. Host Index of Plant Diseases in Indonesia.
Gajah Mada Univ. Press, Yogyakarta.
Smits, W. T. M. 1994. Dipterocarpaceae:
Mycorrhizae
and Regeneration.
Tropenbos
Foundation,
Wageningen.

Collection,
Resources:

Preservationand Use of Microbial Genetic
Activitiesof the MAFF Gene BankHISATOSHI KAKUNational Instituteof Agrobiological
Tsukuba, Ibaraki 305, Japan

Resources

Introduction
Microorganisms are of great importance as genetic resources, because theyplay an important role as useful biotic agents in the productionof wine, fermented
foods, and antibiotics.
Microorganisms also include pathogens of human, animals andplants. Even if they are pathogenic, they are useful, since they are essential for vaccine
preparationand development of control methods. In addition,certain kinds of plant
pathogenic bacteria can be used as gene transfer vectors. Microorganisms have great
potential
to improve many aspects of our life. Therefore,
it is very importanttomaintain microbial
strains for the their use in industry as well as research.
In this paper, the current status of the microorganism section of the MAFF
Gene Bank is introduced with special reference to collection,
evaluation, preservation
and use of microbial genetic resources.
What is the MAFF Gene Bank
The MAFF Gene Bank Project was initiatedin 1985 for the purpose ofcollecting
and preserving genetic resources of microorganisms, plants , animals, forest
trees and aquatic organisms. The Genetic Resources Center of MAFF was establishedin the National Instituteof Agrobiological
Resources (NIAR) in December, 1986.
The MAFF Gene Bank Project completed the first phase in March, 1993, andthe second phase of 8 years started in April, 1993.
NIAR plays the role of the central bank for microorganisms,
plants andanimals, and these three areas are coordinatedby the Genetic Resources Coordinatorbased at NIAR. Accordingly,
the Genetic Resources Center of MAFF is primarilyincharge of the preservationand supply of microbial
strains, plant seeds, animal
reproductive
cells and embryos.
Organization

National
subsidiaryof Microorganism

Sectionof the MAFF Gene Bank

The organization of the MAFF Gene Bank consists of the central bank at theInstituteof Agrobiological
Resources (NIAR) as described above and 14
banks (sub-banks)(Table1). These sub-banks include 92 laboratories
and

48
Table

1. Organizationof MAFF Gene Bank for

Microbial

Genetic

Resources

Section2 departments, where exploration, collection, introduction,
classification,
identificaion,evaluation,
multiplicationand preservationof microorganisms
are conducted. It
reflects
the diversityof microorganism
to be collected
and preserved that so manylaboratories
joined the MAFF Gene Bank Project. The first and second editionsofcatalogue of microorganisms
in the MAFF Gene Bank was publishedin 1989 and
1993, respectively.
In Japan, genetic resources of microorganism are mostly collected,
deposited

49

and preserved in culture collectionsof national and public institutes,
universities,
corporations
and the private sector. These culture collections
are organized by theJapan Society of Culture Collections(JSCC)(Kaku,
1993). The MAFF Gene Bank was
included in JSCC. The society publishes
catalogues and bulletins,
as well as holding
meetings and symposia annually. JSCC has joined the World Federation of Culture
Collections(WFCC), which holds an international
meeting every four years. The
World Data Center (WDC) of WFCC was set up at JCM (Japan CollectionofMicroorganisms)
of RIKEN in 1986, and it plays
a role in the exchange of data
concerning microbial strains among the culture collectionsin the world.
Each culture collection
has a specificfield,
i.e, Research Center for
Pathogenic
Fungi and Microbial
Toxicoses, Chiba University(IFM) is for medical
microbes, Tokyo University of Agriculture(NRIC) for fermentation
microorganisms,
and MAFF for agricultural
microorganisms.
Collection,
Exploration
MAFF Gene Bankand Introductionof Microbial

Genetic

Resources in the

Various kinds of microorganisms are collected by the laboratories
associatedwith the MAFF Gene Bank Project. Therefore, bacteria, actinomycetes,
yeasts, fungi,
viruses, protozoa, nematodes, algae, animal cell lines and DNA/RNA have beencollected,
introduced
and preserved by the MAFF Gene Bank Project. All the
microorganisms collected and preserved are associated with agriculture,such as plant,
animal and insect pathogens, soil microorganisms,
food microorganisms,
mushrooms,
and marine microorganisms.
In addition,
overseas and domestic explorationand collectionof microorganisms has been conducted every year since 1988. By overseas collection,
nitrogenfixationbacteria
in Thailand,
protozoa in Indonesia,
microorganisms
related tofermented foods in Nepal, hyperparasitic
fungi in Thailand, edible mushrooms in New
Zealand, salt resistant bacteria and yeasts in Thailand, rice blast fungus in China andthermotolerant lactic acid bacteria have been collected between 1988 and 1993 (Table
2.).

Generally,
governments have quarantine systems to protect agricultural
plants and animals from infection by pathogens from overseas (Khan, 1977). In Japan,
most pathogenic
microbes can be introduced for research with special permission of
the minister of MAFF.
Domestic exploration
has been conducted since 1987. The target area fordomestic exploration was concentrated on Kyushu and Hokkaido.
A total of 21 teams
have been dispatchedfor domestic exploration,and many kinds of microorganisms
have been collected (Table 3.).

50

Table

2.

Table

Overseas
exploration
andBank project(1987-1992)

3. Domestic

explorationcollection

and collection

by

MAFF

Gene

by MAFF Gene Bank project(1987-1992)

51

Preservationof Microbial

Genetic

Resourcesin the MAFF Gene Bank

Up to 1992, 7,507 strains have been preserved in the base collectionin the
MAFF Gene Bank. Of the base collection,
3,223 are bacteria, 2,767 fungi, 393 viruses,
361 yeasts, 111 actinomycetes,
33 mycoplasma, 62 protozoa and 27 nematodes. The
number of the active collection
for distribution
is 4,350 ( bacteria
2,144, fungi 1,826,
viruses 192, yeasts 102, actinomycetes
44, phages 18, mycoplasmas 17 and protozoa
6 (Table 4.). Detailed information is available in the second edition of microorganisms
in the MAFF Gene Bank (NIAR, 1993).
Long- term preservation of microbial germplasm is essential for research anduse. At the MAFF Gene Bank, bacteria and actinomycetes are preserved by lyophilization (freeze- drying), L- form drying, and freezing at low temperature (-80 °C)(Table5.). For lyophilization,
microorganisms are mixed with a freeze protection solution suchas 10% skim milk plus 1% of L- glutamic acid. For fungi, freezing under ultra- lowtemperature of liquid nitrogen is used as the method for long-term preservation(Table5.). However, there remain many problems concerning preservationof some kinds ofmicroorganisms. For the routine research work, slant cultures are kept in the cool room.
In such cases, however, transferring many times can cause mutations and for pathogenic
microorganisms a loss of pathogenicity.
Evaluation of Microbiai
Genetic Resourcesfrom evaluationof pathogenic
bacteria

in the MAFF Gene Bank - examples

The collected strains have been evaluated for their characteristics
by the mostsuitablemethods, such as taxonomic markers, pathogenicity,chemical tolerance,
antagonism, infectivity,
toxin production,
gene analysis, enzyme activity.
Recently,
Table

4. Microbial

M icroorgan ism s

genetic

resourcesA c t iv e c o l l e c t i o npreserved

in MAFF Gene Bank(1992)

B ase co llection

Work ing co llec tion

Tot al

B a c t e r ia
Actinomy cetes

3,176
10

4 , 42 080

7,48 996 3l l, 9 0 91, 0 4 3

Yeast
Fung i

10 2
3,938

44 9
5 , 5 146 70
1, 8 5 4 2

1, 1 1 924, 056

Viruses
PhagesM y c o p la sm a s

28 1
10
50

50 0
12
75

78 8
0
2 33

1, 4 8 812308

R i c k e t ia
R e c o m b .B a c .

0

470

1 ,4 2 0

1,890
45
29

DNA
A n im a l c e l l s

0

27

40
2

Protozo a
Nemato des

13
0

66
32

30
99

96
13 1

7,58 1ll,65330,48042 ,133

Tota l

52

Table
M

5.

Preservation

icroorganism

Bacterium,

Plant

for

microorganisms

Preservation
Actinomycetes

Virus

virus

Lyophi1ization
Low-temperature
L-form drying

Gene

Bank

(Freeze-drying)(-80*0)(Liquid

Lyophilization
Low-temperature(-80*0

Low-temperature
Infected
plant(-80^)
tissueLow or ultra-low(-80^,
Liquid

N

Soil

with

MAFF

methodProtozoon

ematodein

SlantcultureUltra-lowtemperature

FungusAnimalmethods

nitrogen)(-80cC)

temperatue
nitrogen)plant

newmethods such as 16S rRNA sequence analysis and RFLP analysis have also beenused. RFLP analysis of phytopathogenic
bacteria has been conducted in the MAFF
Gene Bank.
Classificationand identificationof bacteria have been based on similarities
in phenotypiccharacteristics.
However, this method has not been precise enough todistinguishsuperficiallysimilar organisms or for determining phylogenetic
relationships among the bacterial groups. The identificationof bacteria is laborious and a timeconsuming process due to the number of physiologicaland biochemical
tests necessary
for acculate identification.
In addition,
plant pathogenic
bacteria are grouped into
pathovars and races on the basis of their pathogenicity
to plant species and varieties.
A rapid differentiation/identification
method by RFLP analysis was developed
using rRNA (16S+23S)
of Escherichia coli and fragments of genomic DNA of thephytopathogenic
bacterium as probes. With the probe of rRNA of E. coli, type strainsof ll species and 14 pathovars of phytopathogenic
pseudomonads were analyzed for/findlll fragments of their genomic DNA. Type strains of 1 1 species of pseudomonads
showed different RFLP patterns, and those of the pathovars could also be differentiated.
In addition,
Pseudomonassyringae pv. lachrymans and P. syringae pv. tabaci weredifferentiatedby their RFLP patterns, though they were indistinguishable
bacteriologically and serologically.
RFLP analysis was also conducted for 19 pathovarsof
Xanthomonascampestris using the same probe and EcoRl as restriction
enzyme. All
the strains tested showed RFLP profiles characteristicof each pathovar.
Bacterial
blight of rice is a disease of world- wide importance. The causal
agent of the disease, Xanthomonascampestris pv. oryzae, is grouped into many races

53

on the basis of their pathogenicity
to rice cultivars. RFLP analysis was conducted for
the Japanese strains representativeof races I, II, III, IV, V and VII using the probe
pJELlOland restriction
enzymes &0RI, Clal, HinAlW and BamHI.The probe isderived from genomic DNA fragment of the Philippine
race 2 and contains highly
repetitive
sequence. Six representative
strains were differentiatedby the RFLP patterns
irrespectiveof the restriction
enzymes used (Kaku, 1994). The strains belonging to thesame race showed RFLP patterns similar to the representativeof the race that they
belongto.
As described above, RFLP analysis is applicable
to differentiation/identification species, pathovars and races. A given bacterium will be identified
rapidly andsimply by refering to the RFLP pattern data-base of known phytopathogenic
bacteria.
In addition, the relationshipamong strains is also clarified
by cluster-analysisof RFLP
patterns. Phytopathogenic
pseudomonads are grouped into category containingallpathovars of P. syringae and that of other pseudomonads. In the future, RFLP, 16SrRNA sequence and RAPD all will be able to provide a clearer picture of therelationships
among bacteria.
Distribution

and Use of Microbial

Genetic

Resourcesin the MAFF Gene Bank

Microbial strains registeredin the Central Bank are listed in the catalogue(NIAR, 1993). They can be distributed
to national institutes,
universities,
prefectural
institutes
and private companies in the country as well as overseas.
Among the microbial resources requested have been Agrobacterium spp.
(biotechnology),
pseudomonads (biocontroletc.), xanthomonads (pathogenicityetc.),
Rhizoctonia spp. (taxonomy),fusaria (taxonomy and chemical development),
viruses(vaccinationand disease protection),and yeasts (fermentation).
Approximately
2, 100
microbial strains have been distributed
between 1987 to 1992. Of these, 952 and 1,055
were bacteria and fungi, respectively.
Future Prospects
Resourcesof the MAFF Gene Bank Projectfor Microbial

Genetic

After the success of the first phase of the MAFF Gene Bank Project, thesecond phase of 8 years has started. Recently, a DNA Bank was establishedin theGenetic Resources Center of NIAR. The MAFF Gene Bank is rapidly increasing
its
coverage of genetic resources and the accumulation of data related to genetic resources.
For microbial genetic resources, development of new, rapid and accurate
methods for classificationand identification
are expected in the future. Currently, they
are often time- consuming and laborious. Therefore, the MAFF Gene Bank Project has
the objective of promoting conservation of genetic resources from a global perspective
as well as supporting
related research (Nakagahra,
1993). International
cooperation
among culture collections
worldwide
will help promote research and reduce

54

duplicationof effort and enhance conservationof microorganisms.

References
Kaku, H. 1993. Microbial
Genetic Resources in Japan. In Proceedings of the Japan-Russia
Workshop on
Genetic Resources and Biotechnology,
Tsukuba, pp. 39-43.
Kaku, H. 1994. Bacterial
Leaf Blight of Rice in Southeast Asia. 2. RFLP analysis oiXanthomonas
campestris pv. oryzae. In Proceedings
of JIRCAS Workshop Papers No. 1, Present Situation
and Future Development of Research Activities
Concerning Crop Plant Diseases in theDeveloping
Countries, Tsukuba, pp. 40-46.
Khan, R. P. 1977. Plant Quarantine:
Principles,
methodology
and suggested approaches. In Plant healthand quarantine
in internationaltransfer of plant genetic resources, W. B. Hewitt and L.
Chiarappa,
eds., CRC Press, pp. 289-308.
N1AR 1993. Catalogue of Microorganisms,
2nd ed., MAFF.
Nakagahra, M. 1993. Activities
on Plant Genetic Resources in Japan. In Proceedingsof the Japan-Russia
Workshop on Genetic Resources and Biotechnology,
Tsukuba, pp. 17-24.

55

Questionsand Answers Session 1

Q.
Are there any difficulties
and Indonesia by foreign scientists

to collecting
in cooperation

microbial
strains in the Philippines
with local scientists?
(Suzui)A.
Indonesia:
Cooperative
researchwith foreign
government
research
establishments
have been permitted so far. However, with respect to foreign private
sector establishments
we are still waiting for new government regulations
with regards
to the rights and sharing of results.
(Rifai)
A.
Philippines:
In the Philippines
we are still formulating
policy on this issue.
With respect to biosafety,
the guidelines
to be adopted for the release and testing of
microorganisms, particularly
geneticallymodified microorganisms, needs to be spelled
out. (dela Cruz)C.
National property rights is an extremely important issue. This was discussed
during the Convention on Biological
Diversity
once the latter was ratified,we willbegin to see legislation
with regard to remunerationof the country of origin for
exploitationof that countries biodiversity
by a third party/country.
This will cause
difficulties
for collections
if they sell cultures that originatedfrom other countries.
New legislation
has been introducedin the U.K. as a result of European
Union directives formulated in 1992. This puts in place inhibitory
rules for collecting
activities.
It requires registrationof a laboratory
which will work with geneticallymodified organisms three months before they are received. This means that collections
will not be able to receive such organisms unless solicited
3 months in advance.
Collections
are only able to distributegeneticallymodified organisms to registeredlaboratories.
(Smith)Q.
Dr. Rifai, what do you know about collaboration
between the Netherlands
through the Centraal Bureau voor Schimmelcultureswith collections
in Bogor? I
understand that a building has been built for a collection but the project has since cometo a halt. (Smith)A.
This work came to a halt for politicalreasons.
this project can be linked to funds for Bogor BiologicalCenter promised by the Japanese Government. (Rifai)

In the future it is hoped that
Research and Development

Q.
Dr. dela Cruz, what is the most important problem to resolve
development of culture collectionsin the Philippines?
(Nakase)A.

Funding (Government),

(dela

Cruz)

to promote the56Q.
discussed

Have you any plans to achieve the new initiatives
and researchin your paper. Howwill you try and achieve your goals? (Smith)

you haveA,
Funding may be difficult
since large amounts will be required.
We arethinking
in terms of project based funding of national initiatives
from government
agencies. There are no plans at present to coordinate with other countries,(dela Cruz)
Q.
Dr. dela Cruz, is your institute(BIOTECH)
working on the identificationofmicroorganisms associated with traditional
fermented foods and beverages? Do you
publish a catalogue of this? (Tamang)A.
catalogue

Yes we do work on the identification
was produced in 1989. (dela Cruz)

Q.
After the Convention on Biologicalinitiatives
in the Philippines
and Indonesia
Government level? (Nakagahra)of such microorganisms.

Our latest

Diversity,
have there been any new
on microbial genetic resources at theA.
Philippines:
Two initiatives
have been taken. (1) The creation of thePhilippine
Council for Sustainable
Development under the National Economic
Development Authority(the highest
economic body of the government) with themandate to spearhead plans in using biodiversity
for development. (2) The creation of
the National Institute of Biotechnology
and Biodiversity.
This is a senate initiative
toconvert BIOTECH into the National Institute for Biotechnology
and Biodiversity
withthe mandate of expanding biotechnologicalwork to include biodiversity,
its use andpreservation,(dela Cruz)
A.
Indonesia:
It is hoped that the government will assign money to initiate
development in culture collections.
We are hoping to have a Biodiversity
meeting in
Bogor in 1995 where such issues will be raised and discussed.
(Rifai)Q,
Are there any plans to organizein the Philippines?
(Nakase)a domestic

federation

for culturecollections

A.
Yes, there is a plan to establisha Philippine
Federation
for culturecollections.
The major constraint is the funding requirements
in terms of setting up
networking agreements, (dela Cruz)
Q.
Do you have any data on the genetic erosion of microorganisms
in thePhilippines
and Indonesia? Is it in fact occurring? If so in what areas is it most serious?
(Vaughan)A.

Indonesia:

Yes we have some evidence of genetic

erosion. One example is

57

the loss of Russula spp. in West Java. (Rifai)
A.

Philippines:

We have no data on genetic

erosion,Q.
Are there any collections
of VA- mycorrhiza
programs to use them for crop production in the Philippines

(dela

Cruz)

(endomycorrhiza)and Indonesia?

and any(Yoshida)

A.
Indonesia: Staff of my institute have been recruited and are now collecting
VA-mycorrhiza. We are seeking collaboration
with the U.K. (Rifai)
A.
Philippines:
We have 27 species of VA- mycorrhiza in our collection
and add
about 8 new accessions each year. The biggest challenge is to build up a database on
crops, conditions
affecting
VA- mycorrhiza use and other relevant data. Lack offunding,again, would be a major inhibiting
factor. The private sector is currently
testing organisms, (dela Cruz)
Q,
To what extent are the activitiesof the 92 laboratories
Genebank system controlled?
Do they have centralized
information

under the MAFF
storage?

A.
The active and base collections
can be stored at the Central
registration,
except animal pathogens and some other limited organisms.

Bank after(Kaku)Q.
Do you verify the identificationof microbes before depositionin thegenebank?
Do you impart training
to foreign researchers
on modern methods ofmicrobial identificationand preservation?
(Tamang)A.
Yes, we identify at least to the genus level. We welcome foreign trainees.
However funding is limited. STA fellowships
and some other funds are available,
if thequalificationsof the candidate are good. (Kaku)

TECHNICALREPORTS

SessionNitrogen-Fixing

2

Microorganisms

Chairpersons
Tomio Yoshida
J. K. LadhaCollection and Maintenance of Germplasm of Nitrogen-Fixing
Organisms from Lowland Rice Ecosystem
J. K. LADHA, T. VENTURA, and R. SO
Soil Microbiology/Soiland Water Sciences Division
The International Rice Research Institute
Manila, Philippines
Abstract
Realizing the importance of nitrogen- fixing organisms in maintaining
soil fertility
and sustaining
yield in rice lands, the International
Rice Research Institute(IRRI) maintains the world' s largest collectionof N2- fixing organisms such ds Azolla, blue- green algae (BGA), aquatic legume-rhizobia,
and free-living
bacteria useful for rice culture. Collection,
evaluation,
maintenance and disseminationof the biofertilizer
germplasm have been regarded as important activities
to support research on biologicalnitrogen fixation(BNF) in rice at IRRI and elsewhere. A manual describing
the collection,
main characteristics,
and methodsof conservation/mailing
was published in 1992 (see Watanabe et aL, 1992). This paper provides an updateon the germplasm collectionof various N2-fixing organisms at IRRI.

Introduction
Modern rice varieties have contributed
significantly
to food productionindeveloping countries. However, the high yielding
varieties require heavy applicationof nitrogen
fertilizer
which is an expensive input in rice production.
In many
developing countries, fertilizer
is subsidized
in order to encourage its use for growing
modern rice varieties.
Fertilizers
although subsidies
put a burden on national budget.
In spite of it, a large number of farmers in Asia apply little or no nitrogen fertilizer
because of financial constraints
or non-availabilityof the fertilizer.
Even under suchsituations,soil fertility
in wetland paddy soils is maintainedeven though a substantial
amount of nitrogenis removed by the rice crop, or is lost throughleaching,
denitrification,
and ammonia volatilization.
Long term fertility
trials in Japan and thePhilippines
have shown that as much as 50 to 75 kg N per ha is added every year towetland ricefields (Koyama and App, 1979; Greenland and Watanabe, 1982). Thoughsome of this nitrogen is contributedby exogenous sources like rainfall,
irrigationwater, and atmospheric ammonia, BNF is recognized as the major source.
Compared with dryland soils, wetland rice soils have a high N2-fixing
potential.
This is because the wetland rice ecosystem comprises of water/soil,
photic/non-photic
zones, and oxidized/reduced
environments and this diversity
permits all major groups of N2- fixing organisms to grow in wetland rice fields.
Nitrogen-fixing
microorganisms in rice fields comprise:(a)heterotrophic
bacteria associatedwith the rice plant, and free- living
heterotrophic
and phototrophic
bacteria in the soil;(b)free-living
phototrophic
BGA; and(c)symbiotic
BGA, viz., Anabaena associated with Azolla.

62

Table 1. Range of estimates
potential

S y st e mof N2 fixed by various agents in wetland ricefields

and assumptions(modified

and theoreticalmaximumfrom Roger and Ladha 1990).

R e p o rt e d r a n g e

M a x im u m p o te n tia l( k g N /h a p e r c r o p )(k g N / h a p e r c r o p )A s su m p tio n s

R ic e r h iz o s p h e r e

10 ‑3 045**3CA 0lfl mbo wagc Nteo fria
isl fi.5axrte/hdN/ag2 ‑fCi .x e r s .

S o il/fl o o d w a t e r

30 ‑8 080

詛D詛P
*H5 rimot mmreg ainshridyae tenupceroreoistodruafopcBpptio
Ghlie
icAn dNo(toCfisO/Nso.5fi xil.
=1e Cd7 /g/h
) ar eassidBu Ge .A .

A z o lla G M

4 0 ‑15 0

220*T NO wdn feoaAAa zz‑o8olla
0lla% sct.raon pd sinag rec rog rop wis n 1p4 e0 r krgic eN c/hr oa p. .

a: Ndfa =N derived from the atmosphere.

The quantities of N2 fixed by these diverse diazotrophs
in rice fields have been
estimated with reasonable accuracy. The ranges of determined values and the theoreticalpotential
maximum are shown (Table 1).
Realizing the importance of N2-fixing organisms in maintaining
soil fertility
and sustaining yield in rice lands, IRRI establisheda collection of N2-fixing organisms
L eg u m e G M
4 0 ‑2 5 0
2 50* S . r o s tr a ta o r S . c a n n a b in a is u s e d a s G M .
(biofertilizer)
for rice. A manual describing
the collection,
main characteristics,
and(4 5 ‑ 6 5 d )(5 5 d )26900% k .g N /h a is a c c u m u la t e d w it h N d fa o fmethods of conservation/mailing
was publishedin *1992(see Watanabe et al, 1992).
This paper provides an update on the germplasm collection
at IRRI, now the worlds
largestfor N2- fixing organisms. A summary of IRRI's collectionof N2-fixing
organisms is shown (Table 2).
AzollaThe Azolla collection
at IRRI contains 529 accessions originatingfrom 58
countries. About 57% of the accessions are from Asia and Oceania, 24% from North
and Latin America, 7% from Africa and 5% from Europe. Thirty-five
percent of the
Asian accessions are hybrids,
progenies of selfed plants, mutants, and redistributed
nonindigenous
strains. Sexual hybrids and algal hybrids occupy 23% of the Azolla
collection (Table 3). The collection is composed of 7 species, of which, Azolla pinnata
is the most common.
Normally,Azolla is preserved in the vegetative stage in liquid cultures undercontrolled
conditions.
However, this requires a large amount of space and labor. To

63

Table 2. Summary ofBiofertilizer
IRRI, as of October

Germplasm at
1994.

N u m b er o f A cc ession s
T o talG en eraS p e cie s

B acteria

58

5

10

B G A

203

10

S ev eral

A zo lla529

L eg u m es

91

3

43

R h izo b ia

394

3

(h o st3 sp
7 e cie s)

Table 3. Summary of the /lz0//ff germplasm,

S p e c ie s

as of October

1994.

R e g u la r

A n a b a e n a ‑ fr e e

H y b ri d so b t a in e d b y

T o talN o . ofst ra i n s

st r a in sa lga na sf
trl er

h y sb eri xduiza al tio n M u ta n t s

ns tor.a oinfs

csoo uu rc
n tre ie s

A . p in n a ta v dLV . im b r ic a ta

90

1

0

0

0

9 1

20

A . fi li c u lo id e s

79

13

4

6

18

12 0

24

A . m ex ica na

26

0

0

3 0

0

56

A . c a r o lin ia n a

63

2

0

7

0

72

A . m ic r o p h y l la

53

4

19

53

1

130

A . p in n a ta v a r . p in n a ta

39

1

0

0

0

4 0

U n c la s s ifi e d ( ? )

8

1

0

2

2

13

365

22

23

98

2 1

52 9

17

A . n ilo tic a
A . ru bra

T O T A L

a: Includes

Anabaena-free Azolla obtained

from sexual hybrids. Sexual hybrids include

putative

20

103

ones.

save resources and to lessen the chances of cross contamination,
the shoot-tip cultureofAzollamethod
preservation was adopted (Fig. l). By this method, intervening
time
interval between sub- culturing is increased substantially- two to three weeks in liquid
culture vs. 20- 24 weeks in agar shoot- tip culture. Currently,
about 90% of the total
IRRI collection
is propagatedthrough shoot-tip cultures. Success of aseptic transfer
Azolla varied from species to species.
filiculoides seemed to be well adapted to the

and

64

Cutting of Azolla tips
* Cut off 5-mmfragments that include

meristems

and several leaves.

* Enclose cut fronds in a small net bag.
* Wash with running tap water for 30 minutes.

Sterilization* Sterilize

with 2% NaOCl and 1% Triton X 100 solution* Wash with sterilefor 1-3 minwater ten times.

Inoculation* Inoculate

15-20 piecesof the cut tip portion

into a 50-ml flaskwith 20 ml. IRRlAzo/Ia medium and 0.5% agar.

Cultivation* Incubate

at 26/18°C under 5-8 klux.

* After 2-3 months of growth,
InoculateFig. 1. Shoot-tip

cut the tip portionof the Azolla fround.

again into another medium.

methodof Azolla preservation.

method while s, microphylla
A. nilotica were difficult
to maintain as shoot- tip
cultures.
At least two other institutions(FujianAcademy of Agricultural
Sciences,
China and Universite
Catholiquede Louvain, Belgium) maintain original/duplicate
accessions of Azolla and are available on request.
Blue-green

Algae

The blue- green algae collection
contains 203 strains from 21 countries. About
45% of the strains are from Africa and 40% from Asia (Table 4). The strains areclassified
into 12 genera. Most strains are classified
as belonging to Nostoc^Anabaena,
and Calothrix genera. Although
IRRI's BGA collection
is not large, it represents
rice field BGA. The primary purpose is to make available a range of reference strains
for field and laboratory
experiments. Most strains are preserved as unialgal material
and are not bacteria-free.
The collection
is maintainedon agar slants and subcultured
every 3 months.
The strains may also be maintained as soil-based and dry-powdered inocula, which

65

Table

4. Number and origin of the blue-green

G enus

S e n e g aAl fricaO th e rs

algalcollection,

as of October

P h ilip p inAesias O th e rs

1994.

E u ro p ereO thg ioern s

T o ta l

A na baena

13

5

7

15

5

5

50

C a lo th r ix

23

2

ll

7

1

1

45

F is ch e re lla

4

2

4

3

1

0

14

28

9

12

17

4

1

71

69

21

37

49

12

195

76

21

37

49

13

2 03

G lo e o tr ic h ia
N o d u la r ia
N o s to c
S cy to n e m a
T o lyp o th rix
N 2‑fi x in g
N o n ‑N 2‑fix in g
T o talwere found to be satisfactoryfor long-term preservation.
A simple method ofconserving BGA on paper strips has been tried. This method is simple and convenient
but good only for short-term preservation.
Aquatic

Legume-Rhizobia

IRRI's germplasm of aquatic legumes includes green manure or fodder crops
adapted to lowland rice- based cropping systems. No other organizationcollects
primarily
aquatic legumes. At least three genera of aquatic legumes are known Aeschynomene,Neptunia,and Sesbania. Currently, there are 9 1 accessions composedof 3 genera and 43 species collected from 24 countries (Table 5). The collection
also
includes 55 accessions of stem-nodulating
legumes.
Three hundred and ninety- four strains of rhizobia have been collected from43 legume host accessions (Table 6). Rhizobia
strains with the ability
to produce
bacteriochlorophyll
a were isolated from stem- nodulating Aeschynomenespecies.
Several representative
strains have been characterizedbased on numerical taxonomy
of a large number of phenotypic
traits, fatty acid profile and 16SrRNA sequencing(Ladha and So, 1994; So et al., 1994). Results suggest the need for the creation of new
species in Bradyrhizobium to accommodate the pigmented rhizobia (Fig. 2).

66
Table

5. Summary of aquatic legumes collection,

G enu sas of October

SN po e.coief s

A cNc eos.soiofn s

21

47

17

S e s b a n ia

21

42

15

T o ta l43

9 1( 5 5 )"24

A eschy n om e ne

1 994.

S o Nu roc.e oCf o u n tri e s

N e p tu n ia

a: Stem-nodulating

legumes

Table 6. Summary of the rhizobia

collection,

L egu m e h o st

as of October

1994.

S ym b ion t

A e schy n om en e afrasp era

o Tf otalstrains

S oulationiso
rce of

B ra dyrh izob iwm sp.

27

S N /R N /W L R S

A . afr asp era

B ra dyrh izob ium e lkcm if

7

S N /W L R S

A . a m erican a

B ra dyrh izob ium sp .

3

R N /W L R S

A . a sp era

B ra dyrh izob ium sp .

9

R N /W L R S

A . d en ticu la ta

B ra dy rh izob ium. sp

1

S N /W L R S

A . e ven ia

B ra dyrh izob ium sp .b

5

S N /W L R S

A .f lum in en sis

B ra dy rh izob inm. sp .b

7

RN AVLR S

A .in d ica

B rad yrh izob ium sp .b

34

S N A VL R S

A .n ilo tica

B ra dy rh izob ium sp .b

16

S N /W L R S

A .p ra tem es

B ra dy rh izob ium sp .b

18

S N A vL R S

A . ru dis

B ra dy rh izob ium sp .b

4

S N /W L R S

A . sca bra

B ra dy rh izob ium sp .b

13

S N /W L R S

A . sch im p erii

B ra dy rh izob ium sp .

3

S N /W L R S

A . sen sitiva

B ra dy rh izob ium sp b

13

S N /W L R S

A z orhizo biu m ca ulin oda n s

16 1

S N /R N A VL R S

S .ca n na b ina

R h izo biu m sp .

78

R N /W L R S

S .ex a lta ta

R hizo biu m sp

1

R N /W L R S

S .sesb an

K hizo biu m sp .

44

R N /W L R S

S .sp ec io sa

R h izo biu m sp .

14

R N /W L R S

S . sp iritus

R h izo biu m sp .

6

R N /W L R S

S . tetrap teraR h izo biu m sp .

5

R N A VL R S

N ep tun ia o lera ecea

K hizo biu m sp .

9

R N A VLR S

C ro to laria j un cea e

B rady rh izo biu m sp .

55

R N /W L R S

G yt cine m a x

B ra dy rh izo bium j ap on icu m

12

R N /W L R S

M a crotiliu m sp .

B rady rhizo bium sp.

3

R N A VLR S

S esba nia rostra ta

a: SN-stem nodules;
b: photosynthetic-type
c: non-photosynthetic-type

RN-root nodules;
belonging

WLRS-wetland rice soils.

to a new species based on numerical and fatty acid data and phylogeny.

clusteredwith B. elkanii group based on phylogeny.

67

T =type strains
Fig. 2. Phylogenetic
constructed

Free-Living

tree derived from 16S rRNA gene fragment data. The tree wasby using the neighboringjoining
method, (from So et al., 1994)

N2-Fixing

Bacteria

The collection has 58 strains of N2-fixing bacteria that were isolated at IRRI
from the roots, stem, and decaying straw of wetland rice (Table 7). A new species,
Pseudomonas
diazotrophicus (accession IRBG 183), was described to accommodatea predominant N2-fixer from the rice root. In addition, IRRI maintains 120 reference
strains (33 genera) of N2- fixing and non- N2-fixing bacteria obtained
from othersources. All the strains are preserved under lyophilized
condition.

Evaluationof N2-Fixing

Organisms

IRRI has played a leading role in research on BNF in the rice agroecosystem.
The BNF program at IRRI has the following
objectives:(1) to identify
N2-fixingorganisms in rice soils, quantify their N2-fixing ability, and identify environmental and68

Table 7. Summary of the free-living
G e n u s/sp e cie s

N2-fixing

bacteria

as of October

1 994.

o fT strao talin s

S o u rc e o f iso latio n

179

22

R ic e so ils, w etla n d

1

R ic e ro o ts, w e tlan d

E n te ro b a cter c lo a ca e

194

10

R ic e rh iz o sp h e re

K le b s ie lla p la n tico la

1852

R ic e ro o ts, w e tla n d

P s e u d o m o n a s d ia z o tr op h ic u s

1832

R ic e ro o ts, w etla n d

P s e u d o m o n a s so .

2 26

1

R ic e ro o ts, w etla n d

R h o d op s e u d o m o n a s s p .

207

2

D e c o m p o sin g ric e straw

R h o d op s e u d o m o n a s sp .

208

ll

R ic e rh iz o sp h e reR h o d op s e u d o m o n a s sp .

20 5

7

R ic e s o ils , w e tla n d

A z o sp irillu m lip of er u m

Rstrain
ep ressen(IRtaB tivG e)collection,A z o sp ir illu m lip of er u mmanagement factors that influence their propagation;(2) to develop, standardize,andapply new methodologies;(3) to develop management practices to enhance BNF; (4)to establisha BNF related research network in rice growing countries;(5) to train
scientists
and strengthen national BNF research programs; and (6) to collect, maintain,
and disseminate microbial germplasm.
Several methodologies
have been developed, standardized
and refined to study
soil microbiology
in rice soils and plants. They include the methods for assessing
microbial biomass, microbial counts, ARA and 15N methodologies
to study nitrogen
fixation, preservationof microorganisms among others.
The biofertilizer
germplasm at IRRI is being extensively
used in the BNF
research at IRRI (see for reviews Kundu and Ladha, 1995; Ladha and Reddy, 1995;
Ladha etaL, 1992; Ladha et al., 1990; Whitton and Roger, 1989; Roger etaL, 1987;
Roger and Watanabe ,1986; Watanabe, 1982; Watanabe et aL, 1980). IRRI's biofertilizer
germplasm has been also widely utilized by researchers and extension workers for their
BNF programs world over (Table 8). For example, more than 3,000 accessions ofAzolla were supplied to researchers and extension personnel in 22 countries. It is hopedthat the free and easy availabilityof this germplasm will encourage its use in developing
sustainable
rice production
technology.

Table 8. Summary of Biofertilizer

Germplasm Requests(1991

to 1994).

References
Greenland,

D. J. and I. Watanabe. 1982. The continuing
nitrogen enigma. In Wither Soil Research, Trans.
12th Int. Congress Soil Science 5: 123-137.
Koyama, T. and A. App. 1979. Nitrogenand rice. International
Rice Research Institute,Los Banos,
Philippines,
pp. 95-104.
Kundu, D. K. and J. K. Ladha. 1995. Enhancing
soil nitrogen use and biologicalnitrogenfixationinwetland rice. Experimental
Agriculture(in press).
Ladha, J. K., R. P. Pareek, R. So and M. Becker. 1990. Stem nodule symbiosis
and its unusual properties.
In Nitrogen Fixation: Achievements and Objectives,
P. M. Gresshoff, L. E. Roth, C. Stacey , W.
L. Newton, eds., Chapman and Hall, New York, pp. 633-640.
Ladha, J. K. and P. M. Reddy. 1995. Extension of nitrogen fixation to rice- necessityand possibilities.
Geojournal(in press).
Ladha,J. K. and R. B. So. 1994. Numericaltaxonomy of photosynthetic
rhizobia
nodulating
Aeschynomene species. Int. J. Syst. Bacteriol.
44: 62-73.
Ladha, J. K., A. Tirol- Padre, K. Reddy and W. Ventura. 1992. Prospects
and problems of biologicalnitrogen fixationin rice production:
A critical assessment.^
An invited paper presented in the
9th Int. Congress on Nitrogen Fixation, Cancun, Mexico, Dec. 6-12, 1992.
Roger, P. A., S. Santiago-Ardales,
P. M. Reddy, and I. Watanabe. 1987. The abundance of heterocystous
blue- green algae in rice soils and inocula used for applicationin rice fields. Biol. Fertil. Soils 5:98-105.
Roger, P. A. and I. Watanabe. 1986. Technologies
for utilizing
biologicalnitrogen fixation in wetland rice:
potentialities,
current usage, and limiting
factors. Fertilizer
Research 9: 39-77.
So, R. B., J. K. Ladha and J. P. Young. 1994. Photosynthetic
symbionts
of Aeschynomene spp. form acluster with Bradyrhizobia
on the basis of fatty acid and rRNA analyses. Int. J. Syst. Bacteriol.
44: 392-403.
Watanabe, I. 1982. Azolla-Anabaena symbiosis- its physiology
and use in tropicalagriculture.
In
Microbiology
of Tropical
Soil & Productivity,
Y. Dommergues and H. Diem, eds., Martinus
Nijhoff
Publisher,
Hague, pp. 169-185.
Watanabe, I. , P. A.Roger, J. K. Ladha and C. van Hove. 1992. Biofertilizer
germplasm collections
at
IRRI. IRRI, Los Banos, Philippines,
p. 66.
Watanabe, L, C. Lin, C. Ramirez, M. T. Lapis, T. Santiago-Ventura
and C. C. Liu. 1989. Physiology
andagronomy ofAzolla- Anabaena symbiosis. In Nitrogen Fixation with non-legumes, Skinner et70

Watanabe,

Whitton,at , eds., Kluwer Acad. Publishers,
Hague, pp. 57-62.
I., R. So, J. K. Ladha, Y. Katayama- Fujimura, H. Kuraishi.
1987. A new nitrogen- fixing
speciesof Pseudomonad:
Pseudomonas diazotrophicus
sp. nov. isolated
from the root of
wetland rice. Can. J. Microbiol.
33: 670-678.
B. A. and P. A. Roger. 1989. Use of blue-green algae and Azolla in rice culture. In Society ofGeneral Microbiology
Vol. 25, Microbial
inoculationof crop plants, R. Campbell and R. M.
Macdonald,eds., IRL Press, Oxford Univ. Press, Oxford, pp. 89-100.

Collection
and Use of Tropical
Legume Yield in Thailand

Rhizobia

for Increasing

BUNHARN TANGCHAM,PRECHA WADISIRISUK and CHIRASAK AROONSRI
Soil Microbiology
Research Group, Soil Science Division,
Department of Agriculture,
Bangkok, Thailand
Abstract
The collection and the use of rhizobium for increasing the yield of leguminous crops in Thailand
is mainly conducted by the Department of Agriculture.
The results of much research already done has
shown that when rhizobia are applied both in the greenhouse and field, leguminous crops have an increased
yield.
The studies of rhizobia
which have been conducted by the Departmentof Agriculturearesummarized in this paper.

Introduction
Thailand is located in tropical monsoon Southeast Asia. The major crops ofThailand are rice, corn, cassava, sugarcane and legumes. The main legume crops are
the soybean, peanut and mungbean. These legume crops are economicallyimportantin Thailand. Soybean and peanut are produced for domestic consumption and export.
Research activities
into biologicalnitrogen fixation have investigatedthe changes inyield of soybean and other leguminous crops. Legumes are well known for their ability
to fix atmosphericnitrogen when they live symbioticallywith the bacterial
generaRhizobium, Bradyrhizobium and Azorhizobium. The qualityof nitrogen
fixed
geneticallydepend upon legume species, Rhizobium strains and environmental
conditions.
Soybean, mungbean and peanut are the most important legume crops inThailand while cowpea, rice bean and other legumes are less important. The havested
area and production of these crops is shown (Table 1).
Soybean harvested area has increased over the past ten years. In the crop year
1991/92
its production
was about 436,000 metric ton but the demands was about
800,000 metric tons. The average yield of soybean was 950 kg/ha in 1980/81
and 1369
kg/ha in 1991/92.
Peanut harvested area and production increased a little from 1980 to 1991. The
average yield was 1425 kg/ha in 1991/92.
The harvested area of mungbean increased up to 1985/86
and then declined. The
production in 1991/92 was 304,000 metric tons. The average yield was 731 kg/ha.
Rhizobia

Researchin Thailand

Research on Rhizobia
in Thailand
has been done largelyby the Soil
Microbiology
Research Group, Division of Soil Science, Department of Agriculture,

72

Table 1. Harvested area and production of soybean, groundnut and mungbean for crop years1 980/8 1
to 1991/92 (Office of Agricultural
Economics, 1 992).
Y ear

H a r v e s te d a re a (h a x 1 0 0 0 )S o y b e an G ro u n d n u t M u n g b e a n

19 8 0 /8 1
19 8 1/8 2
19 8 2 /8 3
1 9 8 3 /8 4
1 9 8 4 /8 5
1 9 8 5 /8 6
1 9 8 6/8 7
1 9 8 7 /8 8
1 9 8 8 /8 9
1 9 8 9 /9 0
1 9 9 0 /9 1
1 9 9 1 /9 2

10 5
12 5
10 1
156
193
24 1
282
30 3
392
502
40 8
3 18

10 0
117
117
12 0
12 5
12 1
125
117
118
120
117
110

39 5
458
4 44
448
4 83
52 9
49 3
43 8
46 2
49 6
42 8
4 18

P r o d u c t io n ( to n s x 1 0 0 0 )S o y b ea n G ro u n d n u t M u n g b e a n

10 0
13 2
113
179
2 64
3 09
3 56
3 38
517
6 72
530
4 36

12 9
14 7
14 5
14 7
172
17 1
169
162
164
16 1
16 1
157

26 1
284
28 1
2 88
3 52
32 3
30 1
26 7
33 3
3 56
30 3
3 04

Ministryof Agriculture
and Co- operative.
The Departmentof Agricultureis
responsible
for the production of rhizobium inoculum. The Department of Agriculture
Extension is responsiblefor distribution
inoculant. Using training and on farm trials
the Department of Agricultural
Extension also promotes the use of inoculum. The
collection
and use of Rhizobium for increasing legume yield in Thailand was studied
as for:1.soybean
2. mungbean
3.peanut
4. legume trees
1. Soybean
The research on Rhizobium/soybsan symbiosis was studied more than 20 years
ago. Rhizobial
strains were collected
all over Thailand and selected for the mosteffective strains for recommendation and use in inoculant production. An example ofrhizobial
strain selection for soybean is shown (Table 2). Good response to the use of
Rhizobium inoculum under field conditions
was found in Northeast Thailand (Table3). Inoculation
increased soybean seed yield equivalent
to the applicationof nitrogen
fertilizer
at the rate of 75-150 kg N/ha. Liming is important to increase seed yield insome areas (Table 4). The amount of nitrogen fixed was 32 to 161 kg N/ha depending
on Rhizobium strains, locationand management. Rhizobial
inoculation
increasedsoybean cultivar SJ5 seed yield in many on farm trials between 1989 - 1992 as shown(Fig-1)-

2. Mungbean
Rhizobialin the greenhousestrains suitable for mungbean in Thailand were collected and tested
and in field conditions.
The results are shown in Table 5 (Siripin,

73
Table 2. Seed yield(kg ha"1) of soybean inoculatedwith various strains

1 rhizobia(Wadisirisak,1988).

SJ 2

SJ4

S trainK h o n k ae n

R o i‑e t

TH A I
TH A 2
TH A 3
TH A 4
TH A 5
TH A 6
TH A 7
U SD A 76
U SD A 94
U SD A llOU S D A 6 1 1 0M
U SD A l17
U S D A 12 2
U S D A 8 ‑0
U S D A 8 ‑T
U S D A 1 5‑7
U n in o c% cv

38 1 ab
435 a
384 ab334 ab45 1 a
400 ab‑394 ab30 1 be347 ab402 ab347 ab357 ab355 ab37 1 ab37 1 ab
266 c

334 a
347 a
2 2 0 a bc
2 5 8 a bc
347 a
2 11 be‑352 ab205 be236 abc
3 12 a b
284 abc
365 abc
16 8 c306 ab306 ab18 0 c20

M ah a sarak ram

N ak h on ratch a sim a

R o i‑c t

198 b
19 2 b138
2 25

205 ab205 ab23 1 ab

12 4
160
14 3

247 a

14 5

235 ab23 5 ab23 5 ab235 ab2 13 a b
ll

1 64
2 34
173
1 64
116

differentat the 0.05%

2 4 7 ab
2 5 6 ab
2 2 6 ab2 2 2 ab2 2 0 ab
2 2 8 ab‑254 ab207 ab2 15 a b
245 ab209 ab275 a
252 ab
2 3 5 'a b
235 ab18 3 bValues within a column followed by the same letter are not statisticallylevel using Duncan's multiple range test.
Table 3. Seed yields of soybean (kg ha"1) grown at different
Rhizobium inoculation and N fertilizer application

locations as affected(Vasuvat, 1 976).

N o r th e a st
T h a ila n d

C e n tr a l
T h a ila n d

N o rt h
T h a ila n d‑N+N

1 07 5
14 4 41840
2090

12 0 4
1380

‑N
+ N

2 006
1963

2096
2 1 151354
14 8 8U n in o c u la te d

I n o c u la t e dTable 4. Seed yields of soybean (kg ha*1) grown at different locations asaffected by liming and Rhizobium inoculation(Vasuvat, 1976).
N o r th e a s t
T h a ila n d

N o rthT h a ila n d‑ R h iz o b iu m
+ R h iz o b iu m

9 63
1 6 94963
1 15 0‑ R h iz o b iu m
+ R h iz o b iu m

12 8 8
1763

963
13 56

N o lim in g

L im in gby74

Fig. 1. Comparison

Tableof yield

5. Screening of'Bradyrhizobium
VC 2768/A/1560
D (Siripin,

Averageyield

of soybean

cultivar

SJ5 from year 1989-1992.

sp. (Cowpea
1992).

group)

strains

Values within a column followed by the same letter are not statisticallyusing Duncan's multiple range test.

for high N2 fixationdifferent

in mungbean

at the 0.05 level75

Bradyrhizobium

Fig. 2. Screen Bradyrhizobium sp. strains

Bradyrhizobium

Fig. 3. Nitrogenase

sp. strains

for high N2-fixationin Mungbean.

sp. strains

activity.

1992).
Cowpea rhizobial
strains were not specificwith mungbean line because
mungbean is a promiscous legume. Most of them can form nodules and fix nitrogenon mungbean roots. Significant
amounts of nodule formed by some Bradyrhizobium
strains and their nitrogenase activities
are shown (Figs. 2 and 3).

76

3.Peanut
Fifty five soil samples were collected
and checked for their indigenous
rhizobial populations. The results showed that from 17 soil samples Rhizobium could
not be detected(Table 6) but more than 500 isolatesof peanut rhizobia could beobtained from the other 38 soil samples. Good responses to Rhizobium inoculation on
peanut were found under sterile conditions in the green house. The amount of nitrogen
fixed by peanut Rhizobium ranged from 100 to 150 kg N/ha depending on cultivars.
The Rhizobium population varied with location and cropping history. The population
is high in the fields with standing legume. The effect of cowpea Rhizobium inoculationon peanut yield on farm trial is shown (Table 7). The use of cowpea Rhizobium
inoculum increased the yield of peanut.

Table 6. Most population number (MPN) of indigenous
systems (Wadisirisuk,
1 992).

rhizobia

in soil of different

N o . o f s o i l s a m p le
C o u n t a b leU n d e t e c te d

C r o p p in g s y s te m

cropping

M P N o f r h iz o b ia in c o u n ta b l e s o i l
R an g e
A v e ra g e
c e ll s p e r g s o il

L eg u m e s
O th e r c r o p s
L e g u m e s ‑ o th e r s
U n c u lt iv a t e d‑ w it h le g u m e s‑ w it h o u t le g u m e s
T o ta l

Table

15
7
4

1
13
1

2 8 .8 ‑ 4 6 1 0
2 8 .2 ‑ 1 5 0 0
2 5 .0 ‑ 6 4 0 0

1 1 3 0 .0
3 6 8 .92 9 4 6 .2

9
3

0
2

1 0 2 .0 ‑ 9 6 0 0
2 8 .2 ‑ 2 8 .2

2 9 9 8 .9

38

17

7. Effect of Cowpea Rhizobium inoculation
on farm trial (kg ha 1) (Aroonsri,
1993).

T re atm e n t

T a in a n 9

K K C 6 0 ‑1

K K C 6 0 ‑3
469

14 12

8 72

R

178 7

12 4 4

R + P

1694

R + PK
R + L

‑
16 19

R + L + P
PK

172 5

N PK

on Peanut yield

82 6
102 5

12 5 2

13 16

1172

98 7

1152

106 0

77

4. Legume trees
The collection of Rhizobium for some legume trees has conducted by the DOA.
Rhizobial
inoculation
could increased top dry weight and acetylene
reductionofPterocarpusi/tdicus Willd. , Acacia mangiumWi\i&. ,A. auriculiformis Cunn.? Sesbania
glandiflora L and Sesbania samanMerr. in glasshouse experiments.
Rhizobium

inoculants

production

Rhizobium inoculants
are mostly produce by the Department of Agriculture
but some quantities
are produced by private companies. The inoculant production
factory of DOA has a capacity of 200 metric ton per year. The production of inoculant
by DOA nearly met its full capacity
in 1993 (Table 8). In the same period privatecompanies produced 60 metric ton of inoculant.
The DOA control the qualityofproduct. The rhizobium inoculum were distributed
to the farmers through the DOAE.
They are sold along with the seed to the farmers at the price of 0.40 US$ per 1 packageof 200 gram peat inoculant in 1994. The farmers use 5 bags of inoculum per hectare.

Table 8. Rhizobium inoculant production
the Department of Agriculture.
Y ear

P (to
ro dnu s)
ctio nand distribution

by(to n Ds)istrib u tio n (b a g s )19 7 7

5 .0 03 .3 6

1 ,8 1 5

19 7 819 7 9

1 0 .5 9
7 .4 2l l .5 5
5 .7 75 7 ,7 5 3
2 8 ,8 4 419 8 019 8 1

4 .9 2
7 .4 85 .6 4
7 .3 6

2 8 ,1 9 0
3 6 ,8 1 3

19 8 219 8 3

6 .5 814 .3 6

6 .6 4
1 3 .7 9

3 3 ,2 2 4
6 5 ,7 4 0

19 8 45

43 86 .7
.1 76

43 63 .5
.7 91

21 36 28 ,5,9 78 75

19 8 619 8 7

7 .8 .0 0
8 1 .6 3

7 4 .7 8
7 9 .7 93 7 3 ,9 1 1
3 9 8 ,9 7 3

19 8 8

1 4 0 .7 0

1 3 6 .2 3

6 8 1 ,1 7 8

19 89
19 90

1 3 4 .2 7
1 2 6 .3 5

1 2 5 .3 0
1 1 7 .6 7

6 2 6 ,5 2 3
5 8 8 ,3 5 0

19 9 119 92

7 3 .7 8
9 8 .4 4

7 2 .3 0
9 2 .8 1

3 6 1 ,5 0 6
4 6 4 ,0 7 5

19 93
19 94

1 7 9 .7 8
2 0 0 .0 0

1 7 1 .5 3
2 0 0 .0 0

8 5 7 ,6 8 4
1 ,0 0 0 ,0 0 0

One bag contains

200 g inoculant

at cost 10 baht (US$= 0.40)78

Conclusions
The collection and selection of tropical rhizobia for increasing yield of tropicallegume crops has been successful for soybeans. The results for mungbean and peanut
are less successful
in field conditions.
Researchers are continuing
to try and findeffective strains and methods of inoculation
for mungbean and peanut. The researchon rhizobia for legume trees is insufficient.
However, to decrease chemical fertilizer
use and to promote sustainableagriculture
this research must be speeded up.
References
Aroonsri, C, N. Boonkerd and P. Wadisirisuk. 1993. Field Trial of Bradyrhizobiumjaponicum
inoculation
on soybean. Technical annual meeting for Soil Science Division,
Department of Agriculture,
Ministry of Agriculture(in Thai).
Officeof Agriculture
Economics. 1992. Agricultural
Statisticsof Thailand,
Crop year 1991/92.
Ministryof Agriculture
and Copperative,
Bangkok, Thailand.
Siripin,
Settha.
1992. Selecting
and Breeding for Enhancement of Nitrogen Fixation
PotentialinMungbean. Ph. D. thesis, Kasetsart University,
Bangkok, Thailand.
Vasuvat, Yenchai. 1976. The importance of legume inoculation
in increasing
soybean seed yield. Thai J.
Agric. Sci. 9: 171-177(in Thai).
Wadisirisuk, P. 1988. Biofertilizer
for Cropping in Poor Soil-Rhizobium
for Soybean Production.
In Theproceeding of "The cultivation
on Poor Soil in the NortheasternPart of Thailand Seminar" 2327th May, 1988, Khon Kaen ADRC, Khon Kaen, Thailand, pp. 226-238.
Wadisirisuk,P., A. Nantagij.,
S. Kotepong and N. Boonkerd. 1992. Population
and N2-fixing
Effectivenessof Indigenous
Peanut Rhizobia Sampling from Different
Croping Systems. In
Proceeding of llth Peanut Research Seminar May 17-20, 1992, Ranong, Thailand (in Thai).

nod

to

Genetic
Rhizobia
ThailandVariabilityof the Common Nod Gene in Soybean
Strains with Particular
Reference to Strains fromand Japan

TADASHI YOKOYAMAx and SHOTARO ANDO2
1) National Instituteof Agrobiological
Resources2-1-2, Kannondai, Tsukuba, Ibaraki, 305, Japan
2) Japan International
Research Center for Agricultural
1-2, Oowashi, Tsukuba, Ibaraki, 305, Japan

Science

Abstract
To determine the taxonomic relationshipbetween Thai soybean rhizobia
and soybean rhizobia
from other regions, a total of sixty-two Bradyrhizobium strains were isolated from Thai soils by using a
trap- host of Glycinemax cv Soja 5 (Thai local variety).
The genetic diversityof the strains was examinedwith reference to forty-six Japanese and fifteen USDA strains. The degree of sequence divergence in andaround commonnod gene regions of the one hundred twenty- three strains were estimated by RFLP
analysis using the B. japonicum USDA 110 common nodDYABCgene probe. The phylogenetic
groupingof the strains resulted in four major clusters (Cluster 1 to 4). The rate of nucleotide
substitutionsin andaround the common
region among the four major clusters was estimated to be over 11%. Cluster 1
comprised the Japanese and USDA strains, which originatedin temperate regions, whereas the Clusters 3
and 4 were of tropical Thai strains. The results suggest that the common nod genes of Bradyrhizobium
strains have ecogeographic
specificity.
Cluster 1 comprised the DNA homologous groups Gpl and Gpla,
and hence could be classified as B. japonicum. Cluster 2 strains were in GpII, and hence were classified
as
B. elkanii. Cluster 3 and 4 strains, however, did not correspond to any known DNA homology groups.
These results indicate that Thai soybean rhizobia are distantly
relatedB. japonicum and B. elkanii andprobably
warrant recognizing as new species.

Introduction
Members of the genus Bradyrhizobium are slow- growing, gram- negative
soil bacteria which invade and form nitrogen-fixing
nodules on the roots of specificlegumes. Bradyrhizobium japonicum and Bradyrhizobium elkanii are the root nodule
microsymbionts
of soybean. Genetic characterizationof soybean Bradyrhizobium
strains on the basis of DNA base composition analysis showed that twenty-five strainstested could be classified
into three DNA homology groups (I, la and II)(Elkan,
1969;
Elkan etaL, 1971; Hollis etal. , 1981).
Stanley etai (1985) using a presumptivenodgene probe, classified
ten strains into two symbiotic
genotypes (STI and STII) based
upon the degree of base substitution
in and around the nod genes. Devine etal. (1988)
found that rhizobitoxine
could be induced only by strains in GpII, and La Favre et al.
(1986),
Minamisawa (1990) also showed rhizobitoxine-producing
strains belonged to
specific
seroldgicalgroups (serogroups
31, 76, 94 and 130). Subsequently,
genetic

80

characterizationof several US serogroups have been substantiallystudied (Huber et
aL, 1984; Keyser et al., 1987; Kuykendall
et al., 1988; Sadowsky et al, 1987;
Sadowsky et aL, 1990).
In Asia, soybeans are grown in temperate regions and the tropics. Sawada
et al. (1989) applied the USDA antisera to determine serologicalproperties
of Japanesestrains. They found that more than 85 % of the Japanese strains reacted to the USDA
antiseraand possessed well- known cross- reaction patterns of US native soybean
rhizobia.
Kang et al. (1991)
reported that serogroup 123 was predominant in Korea.
Keyser et al. (1982) isolated fast- growing strains of soybean-nodulating
rhizobia fromthe northernpart of China and Sadowsky et al. (1987)
examined serologicalrelatednessof fast- growing soybean rhizobia to other rhizobia. Thompson et al. (1991)
examined serologicalproperties of fifteen hundred root-nodule bacteria isolated fromfive different hosts in northern Thailand, and showed that the response to antisera of
USDA strains was generally weaker than to those from than to those from Thailand.
There are few studies of genetic characterizationof Asian native soybean
rhizobia. Therefore, in order to understand the taxonomic affinities
among Asian native
soybean rhizobia,
genetic variabilityof common nod gene of Thai soybean rhizobia
was examined with reference to Japanese and USDA strains.
Materials
Isolationand Methodsof Bradyrhizobium strains from Thai soils
Soil samples were collected from fourteen soybean fields in Thailand
during1988. No bacterial
inoculations
had been made in the fourteen soybean fields,
therefore,
the collectionof strains was considered to be representativesof the native
population. A ten- fold dilution of the soil suspension was used as an inoculant. Seeds
of Glycine maxcv. Soja 5 (Thai local variety) were surface-sterilized
by immersing
in 70% ethanol for 30 sec, and then in 3% sodium hypochlorite
for 3 min. Four seeds
were sown on sterilizedvermiculitein 200 ml glass jar. Ten ml of the inoculant wasapplied to each jar together with 40 ml of sterile plant nutrient solution. After 1 monthof cultivation
under the conditionof 14 hr daylength
at 28°C and 10 hr darkness at
20°C, all the nodules on the roots were collected and washed in running tap water toremove vermiculite. The nodules were surface- sterilized
by immersing in 70% ethanol
for 30 sec, and then in a 3% sodium hypochlorite
solution for 3 min. Subsequently,
thenodules were washed at least five times with sterile water. Each nodule was crushed
to obtain a turbid suspension and an aliquot of the suspension was streaked onto thesurface of an yeast extract- mannitol agar (YMA) plate (Somasegaran
and Hoben,
1985). The plates were set upside down and were incubated at 30°C for 2 weeks. Wellseparated
single colonies were restreaked into fresh plates to obtain pure cultures.
These strains were reinoculated
to test for their nodulating
ability to Glycine maxcv.
Soja 5 and Glycinemax on.Enrei. And forty- three strains listed in Table 5 were further
tested for their nodulating
ability to Glycine soja, Arachis hypogaea , Vigna radiata ,81

and Macroptilium atropurpurem.
Bacterial

strains, cultivation
media, and temperature
Atotal of one hundred twenty- three strains of Bradyrhizobium tested (Table1). Fifteen
strains of B. japonicum and B. elkanii with different
serotypes
wereprovided by the US department of Agriculture BeltsvilleRhizobium Culture collection(USDA)(Keyser
and Griffin, 1987). Forty-six strains of Japanese B. japonicum and B.
elkanii were also provided by Dr. Yasuo Sawada of the National Instituteof AgroEnvironmental
Science (NIAES)(Sawada
et aL, 1989). The forty- six strains wereisolated from non-inoculated soybean fields, therefore, these strains were considered
to be native strains in Japan. All the strains obtained were cultivatedin yeast extractmannitol broth (YMB) at 30°C for one week and kept on YMA slants. Escherichia coli
DUl {recAl, endAV,gyrA96, thi- 1, A?dM7, supE44, relA1, F ) containing the plasmid
pRjUTIO, carrying the nodulation
genes of.fi japonicum USDA 110 (Nieuwkoopetal.} 1987; Russell et al, 1985), was provided by Dr. Gary Stacey, The UniversityofTennessee.
Genomic DNA isolationand its Southern blotting
Genomic DNA was isolated from stationary
phase cells grown in YMB at
30°C according to the method of Schmidt et al. (1986).
DNA (l#g) was digested with
50 units of the four different
restriction
enzymes; BamUI, /find III, Pst I, and EcoR I(Nippon Gene Inc. Toyama, Japan) for 3 hr at 37°C. The digested mixtures were run
in a 0.7% horizontal agarose gel (14 by 15cm) containing
40 ug/ml ethidium bromide
at 25 volts. Digested mixtures were subsequently transferred to Hybond N+(Amersham
International
pic, UK) by using a vacuum blotting unit (Pharmacia LKB Biotechnology
Inc., Sweden) for hybridization.
Preparationof peroxidase-bonded
common nodDABCgenes probe
PlasmidpRjUTIO,
carrying the nodulation
genes of B. japonicum, wasisolated from^ co/iDU1 by alkaline lysis (Sambrook et a/., 1989). The pRjUTIO wasdigested with /find III to obtain a 3.9Kb Hind III fragment containing the nodDYABC
gene (Nieuwkoop
et aL, 1987). This fragment was further purified
by the freezesqueeze method of Tautz and Renz (1983),
and was bonded to peroxidaseusing an
ECL gene detection system (Amersham International
pic, UK).
Hybridizationof peroxidase- bonded commonnodDABCgenes probe with isolated
genomic DNA
Horseradish
peroxidase- labeled commonnodDABCgenes probes (20ng/l
ml of hybridization
solution)were hybridized
to the Southern blots of enzymedigested genomic DNA on a nylon membrane by the ECL gene detection system (RPN
2101 version 2, Amersham International
pic, UK).

82
Table 1. Bradyrhizobium
S tr a instrains

testedP la ce o f Iso latio n

C o u n trys e ro gro u p "R F L P typ e *c lu ster "B raz i l
U S D A

12 2

M iss iss ip p i,
M ary lan d ,Iow a ,

U n ited S ta tes
U n ited S tates

12 5 ‑ 12 6
7 6 ‑ 12 7 ‑ 12 3

Iow a,U SD A

12 3

M iss iss ip p i,
Iow a,

U n ited S tates
U n ite d S tates

F lorid a,
W isco n sin ,U n ite d S tates
U n ite d S tates
Ja p anN e w Je rsey
A lab a m a ,U n ite d S tates

A riz o n a
C a lifo rn i a,
N o rt h C aro lin a ,U n ite d S tates

N IA E S

3139

K u m am oto ,Ja p a n

N IA E S

3146

T otto ri ,Ja p a n

N IA E S
N IA E S

3152
3 16 6

F u k u sh im a ,N a g as ak i,

J ap a n
Ja p a n

N IA E S
N IA E S

3 18 83 19 7

H iro sh im a ,K a g os h im a,

J ap a n

N IA E S
N IA E S

3200
3204

K a g o sh im a,
H o k k a ido ,J ap a n
J ap a n

N IA E S
N IA E S

3205
3124

H o k k a id o ,T o ya m a,

J ap a n
J ap a n

N IA E S

3 17 9Fukuoka,

J ap a n

N IA E S
N IA E S

3 18 7
3 15 1

H iro sh im a,
F u k u sh im a ,J ap a n
J ap an

N IA E S

3 15 5

K u m am o to ,J ap a n

N IA E S
N IA E S

3 17 3
3 19 0E h im e,
M ie,

Ja p a n
J ap an

N IA E S

3 13 4K y o to,J ap a n

N IA E S
N IAE S

3 14 03 14 5T o tto ri,
T o tto ri,

J ap a n
J ap a n

N IA E S
N IA E S

3 14 7
3 15 4

T o tto ri,
K u m am o to .

J ap a n
J ap a n

N IA E S

3 15 9H iro sh im a ,J ap a n

N IA E S
N IA E S

3 16 03 16 1

K u m am o to ,K u m am oto ,J ap a n
J ap a n

N IA E S

3 16 2A ich i,N IA E S
N IA E S

3 16 43 16 5

N ag as ak i,
N ag as ak i,

J ap a n
J ap a n

N IA E S

3 16 9K a g aw a,

J ap a n

N IA E S
N IA E S

3 17 03 19 2E h im e,
M ie,

J ap a n

N IA E S

3 14 3T o tto ri,

J ap a n

N IA E S
N IA E S

3 15 03 19 1F u k u sh im a,
M ie,

J ap a n
J ap a n

N IA E S

3 18 5

H ok k a ido ,J ap a n

N IA E S
N IA E S

3 13 1
3 17 5A o m or i,
K u m am oto ,J ap a n
J ap a n

N IA E S
N IA E S

3 13 2
3 12 6A o m or i,
W ak a y a m a ,J ap a n
J ap a n

N IA E S

3 13 6

T o k u sh im a,

J ap a n

N IA E S
N IA E S

3 14 2
3 15 8T o tto ri,
H iro sh im a ,J ap a n
J ap a n

N IA E S

3 17 2E h im e,

J ap a n

N IA E S
N IA E S

3 19 3
3 19 6

N iig ata,
K a g o sh im a,

J ap a n
J ap a n

N IA E S

320 1

O k in a w a,

J ap a n

N IA E S

3203

K a g o sh im a,

J ap a n

S a n se i,

T h a ilan d

T A R CT A R C

10 2
10 5

B a n gk h e n ,B a n gk h e n ,T h a ila nd
T h a ila nd

T A R C

13 0

S a ra b u ri,
S a ra b u ri,
S a ra bu ri,

1 2 2 ‑1 2 9

T h a ila ndcontinued on following

page

a Serogroup of USDA and NIAES strains were taken from reference of Keyser and Griffin( 1987)b Number of each strain in this column corresponds with RFLP types in Table 2.
c Number of each strain in this column corresponds with Clusters in Fig. 1.

and Sawada et al.( 1 989).

Comparison of sequence divergence in and around commonnodDABCgenes
among Thai, Japanese and USDA strains
The degree of sequence divergence in the specific regions of the genomes ofBradyrhizobium strains can be estimated by hybridizationof probe DNAs to Southernblots of restriction
digests of their genomic DNAs (Hadley et al., 1983; Hartmann et84

aL, 1992; Kuykendall
etaL, 1992; Minamisawa, 1990; and Stanley et al., 1985). The
fraction of conserved restriction fragments for all pairwise combinationsof strains was
obtained from hybridizationof four different
restriction
digests ( BamH I, /find III, Pstl, £coR I) with the 3.9 Kb USDA 110 common nodDYABCgenes probe. The expected
proportion of shared DNA fragments (P) was able to be estimated by
F=2mxy/(mx+mY),

where mxand mYare the numbers of restriction
fragments from strain X and Y,
respectively,
whereas mXY
is the number of fragments shared by the two strains. For
each pair of strains, Fvalues were calculated from the number of conserved fragments
and the total number of fragments generated by all the four restriction
enzymes. The
expected proportion of nucleotide
substitution
in and around a given probed sequence
was derived from the fraction of conserved restriction
fragments according to themethod of Upholt (1977).
Genetic relationships
among isolates were determined byusing the unweighted
pair group method with average clustering
analysis from therectangular data matrix of the expected proportion of nucleotide substitutions
betweenall pairs of strains.
Results
Hybridization
patternsof the Japaneseand the USDA strains with common
nodDABCgenes probe
The forty- six Japanese and the fifteen USDA strains were tested forhybridization
patterns of the four restriction
enzyme digests with the B. japonicum
USDA 110 commonnodgenes probe. The Japanese strains tested were classified
into
thirteen RFLP types (Table 1 and 2). Nine strains belonging to the serogroups 4, 6, 62,
123- 129, and 125- 127 were confirmed to possess the same fragment of the RFLP type1. Twenty- one strains belonging to serogroup 110 were separated into seven markedly
different
RFLP types (RFLP types 3, 6, 7, 8, 9, 12, and 13). Two- thirds of strains
belonging serogroup 110 were confirmed to possess the same fragments of the USDA
110. Nine strains belonging
to the three serogroups 31, 46, and 76 were categorized
into B. elkanii and shared the same fragments of the RFLP type 14.
The fifteen USDA strains tested were separated into five different
RFLP
types (Table 1 and 2). The following six strains, USDA 115, USDA 122, USDA 125,
USDA 127, USDA 129, and USDA 138 possessed the RFLP type 1 fragments (Table
2). USDA 110 and USDA 123, were categorized
into the RFLP type 6 and ll,
respectively.
Seven strains USDA 31, USDA 39, USDA 40, USDA 46, USDA 71a,
USDA 76 and USDA 94 categorizing
into B. elkanii were separated into two different
RFLP types (RFLP types 14 and 15).

Table2. Restrictios
fragments
sizes in kilobase
USDA 1 10 strain of B. ianonicum

Genomic DNAs of the one hundred
and the southern blot of each digest

hybridizing

to the 3.9Kb

common nod DYABC genes oftwenty-three strains were digested with four different restrictioon
was hybridized to the 3.9kb commonnodDYABC genes isolated

enzymes,from USDA 1 10.

86Hybridization

patternsof Thai strains with common nodDABCgenes probe
The sixty- two Thai strains were tested for hybridization
patterns of the four
restriction
digests with the B. japonicum commonnod genes probe. The Thai strains
were classified
into fourteen different RFLP types (Table 1 and 2). Out of sixty-two
Thai strains, twenty- four strains possessed the RFLP type 14 fragments. Only one
strain of TARC 80 had the RFLP type 1 fragments. The remaining thirty-sevenstrains
were categorized into twelve different RFLP types which were unique to Thai strains.
Thus, about sixty percent of the Thai strains tested (37/62)
did not share common
hybridization
fragments with the Japanese and the USDA strains used as reference
strains.
Sequence

divergencein and around common nod genes
Sequence divergencein and around commonnod genes were examined byusing four different
restriction
enzymes, i.e., BamUI, /find III, Pst I and EcoR I. The
similarity
matrix derived from the fraction of conserved restriction
fragments for all
pairwise combination of strains and the expected proportion of nucleotide
substitution
in and around commonnod genes are shown (Table 3).
Thai strains of eleven RFLP types, i.e., RFLP type 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, and 26, did not share the common hybridization
fragments with theremaining RFLP type strains (Table 2 ). Therefore, the values of % similarity among
them were estimated at approximate zero % (upper right in Table 3).
The one hundred twenty- three strains clusteredin to four major groups(clusters
1 to 4)(Fig.
1). In general, the degree of genetic divergency between two
DNA sequences correlatedwith the proportionof DNA fragments shared by them.
With regard to thej&z/tzH I and Hind III digests (Table 2), a 9.3Kb DNA fragment wasshared by both cluster 3 and 4, and 3.9Kb DNA fragment was shared by both cluster
1 and 2. Of the remaining restriction
enzyme digests, no DNA fragments was sharedby the four major clusters. From the results of the average unweighted pair groupmethod of clustering
analysis, the rate of nucleotide
substitution
in and around thecommon//^ region among the four major clusters was estimated to be over 11%(lower left in Table 3).
Of the forty- six Japanese strains, the thirty- seven were categorized
intocluster 1, the remaining nine strains were grouped into the cluster 2 (Table 4 and Fig.
1). Of the sixty- two Thai strains, the thirty- seven strains were categorized
into thecluster 3 and 4 (Table 4 and Fig. 1). The remaining twenty-four strains were grouped
into the cluster 2. Only TARC 80 was classified
into the cluster 1.
Nodulation

performance of strains categorized
into the four clusters
Thai strains, twenty- two NIAES and four USDA strains (Table 5) were tested
for nodulating
abilityof SJ5 (Thai local variety),
Enrei (Japanese
local variety)and
AGS129 (Asian vegetable Research Center Variety) of soybean. As a result of this test,
weconfirmed that all strains tested had nodulation abilities
to the three soybeans.

Table 3. Similarityand percent sequence divergence
into the 26 RFLP types

matrix for different

pairwisecomparisons of the Bradyrhizobium

The values on the upper right are percentages of similarity, and the values on the lower left are percentages
based on the fraction of conserved restriction fragments for all pairwise combinations of strains.
*Number of each strain in this column corresponds with RFLP types in Table 2.

strains

categorizedof sequence divergenceFig. 1 Sequence
forty-six

divergence

Japanese strains)the data obtained

among one hundred twenty-three
categorizedstrains(sixty-two

Thai, fifteen USDA, andinto twenty-six RFLP types. The dendrogram was derived fromwith nod DYABC gene by using the unweighted

aNumber of each RFLP type corresponds

pair group method.

with RFLP types in Table 2.

etal.,

89

Table 4. Frequencies of the Japanese and Thai strains tested
in each of four clusters
C lu ster ty p e
N o .o f
O riginisolatete sted s

1

Jap a n46

37

T h ailan d

62

1

24

36

To determine if strains categorized
into the four clusters were restrictedfornodulation
by Glycine so/a, Arachis hypogaea, Vigna radiata, and Macroptilium
atropurpurem, we inoculated the four leguminous plants with cultures of the fortythree strains (seventeen
Thai, twenty- two NIAES, and four USDA strains) listed inTable 5. Results of this study indicatedthat B. japonicum USDA 110 strain, twentytwo NIAES strains and the Thai strains of cluster 3 and 4 were not restrictedin theirnodulation
ability.
B. e/Jtanii USDA 31, USDA 94, NIAES 3201, and NIAES 3203
strains did not produce nodules in Arachis hypogaea , while USDA 76, NIAES 3193,
and NIAES 3196 did not produce nodules in Vigna radiata. Thai strains grouped into
cluster
2 did not induce
nodulesin either
Arachis hypogaea or Vigna
radiata.

Discussion
In this work, we used restriction fragment length polymorphisms
to evaluate
the genetic relationshipof soybean nodulating
Bradyrhizobium originatedin Japanand Thailand with reference to USDA strains. The commonnodYDABC
genes which
we used as a DNA probe in this study are structurally
and functionallyconserved
among all rhizobia and are absolutely required for both root hair curling and infection(Kondrosi
1984; Lerouge et al., 1990; Roche et al., 1991; Sanjuan et al., 1992).
And we demonstrated that the commonnod genes of B. japonicum tested have a wider
diversity
among Japanese and Thai strains.
We have known that Bradyrhizobium japonicum consists of at least three
related groups (DNA homology group I, la, and II). Using various DNA probes,
including presumptive nodgenes and nifDHgenes, Stanley et al. (1985) showed there
are RFLP differences between DNA homology group I and II. Using probes of nifDKand nifE genes from USDA 110, Minamisawa (1990)
also suggested that the twodifferent DNA homology groups were two highly divergent evolutionarylines. Younget al. (1991)
showed that clear phylogenetic
differences
exist between the DNA

Table 5. Nodulation

performance of strains categorized

into the four clusters
L e g u m in o u s p la n t te ste d

U S D A 1 1 0 , N IA E S 3 13 1, 3 13 4 ,
3 1135 91,, 3 16
14 65 , 3 114706 , 3 17
14 97 , 3 1 85
50 ,G lyc vc .inseJ m5 a x

G ly
c vc.inE en mre aix

cGv ly. Ac inG eS m1 a2 x9

G slyojc ain ehAy prao cg ha ise ar Va digian ata

aM traocpruoprptiliu
u r emm

+

+

+

+

+

+

+

+

+

+

+

+,

+,

+

+

3 19 0
T8 8A ,R8 9C ,21812, 4,21,4
1 33,,16244,,7149 ,28 4 , 8 6 ,'T A R C 79

4

+

+

+

+

3U 2S0D1A, 332 10 ,3 U S D A 9 4 , N IA E S

1

+

+

+

+

U S D A 7 6 , N IA E S 3 19 3 , 3 19 6

2

‑ f‑

‑f

+

‑f

‑f‑

+

+

+

+

+

‑

T13A6R C 1 6 , 17 , 2 7 , 7 3 , 7 5 , 7 7 , 13 2 ,+, Positive,

more thantwentynodules;-, negative,

no nodule;+/-,

none or less

+

+

/ ""

+

+ / "

+

than

two nodules.

91

homology group I and II based on sequencing of a 16S rRNA gene segment.
Kuykendall et al. (1992) proposed that the DNA homology group II organisms should
be categorized
as a new species. Thus, classificationof B. japonicum based on
differencesof RFLP by using /z//genes and presumptive nod genes as DNA probes
were directly connected to differencesof phylogenetic
relationship.
Cluster 1 strains directly connected with the DNA homology groups I and la.
For example, difference in the substitution
between USDA 122 and USDA 110 was
0.8%, and this value agrees well with that reported by Stanley et al. (1985 ). USDA 31,
USDA 46, USDA 76, and USDA 94 ofB. elkanii were all categorized
into the cluster
2. Thai strains of cluster 2 reacted strongly with an antiserum prepared against USDA
31 by ELISA and the agglutination
test, and those strains showed the same restriction
enzyme digestion patterns as the USDA 31 (data not shown). These results indicatedthat Thai strains of cluster 2 connected with the DNA homology group II. Thus, RFLP
analysis of soybean bradyrhizobia
using the common nod genes probe distinguishedbetween B.japonicum and B. elkanii.
The clusters
3 and 4, however, did not correspond to any known DNA
homology groups. Concerning host range of the cluster 3 and 4 strains, those strains
were identical with USDA 110, as reported by both Keyser et al. (1982) and Chamber
et al. (1988).
These results show almost all Thai strains of the cluster 3 and 4 arecategorized into B. japonicum based on the theory of bacteria-plantcross-inoculationgroups, while based on the degree of sequence divergence in and around commonnod
gene region, those Thai soybean rhizobia are distantly
related to B. japonicum and B.
elkanii. Therefore, these strains probably warrant recognizing
as new species of thegenus Bradyrhizobium.
Cluster 1 contains only temperate strains except TARC80. Only Thai strains
were found in clusters 3 and 4. Recently we collected soybean nodules from both
Malaysia and Indonesia, and are testing their soybean rhizobia for genetic diversityof the commonnodgenes. Wefound cluster 3 strains in Indonesia,
and cluster 2 strains
in Malaysia(data not shown). However, we did not find any cluster 1 strains. This
leads us to think that Bradyrhizobium japonicum is the predominantgroup intemperate soybean rhizobia.
While, cluster 3 is a predominantgroup in tropicalsoybean rhizobia. The two groups diverged in response to climate. However B. elkanii
was found in cluster 2 which contains strains from both the temperate and tropicalregions. Strains of this cluster show no regional specificity
in distribution.
Ourfindings help to clarify the taxonomic relationshipof Thai soybean rhizobia.
Acknowledgments
Wethank Dr. Nantakorn Boonkerd and his staff (BNFRC, Thailand)
for theirco- operation.
We thank Dr. Yasuo Sawada and Dr. Kiyotaka Miyashita(National
Instituteof Agro- Environmental Sciences, Japan) for their technicaladvice and helpful
suggestions. We thank Dr. Donald L. Keister and his staff (USDA, Nitrogen Fixation

92

and Soybean
Gary Stacey

Genetics
Lab.) for providing
and Dr. W. Mark Barbour

for providing

E. co/i DU1containing(The

USDA serotype
strains.
We also thankDr.
Universityof Tennessee,
UnitedStates)the plasmid

pRjUTlO.

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M. A., Iruthathas,
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95

Questionsand Answers Session 2

Q.
How do you determine different
strains of rhizobia
etc. and has DNA
sequence analysis been used? If yes, what were the results? ( Bunyard)A.
Wedetermine different strains by serological
techniques.
We do not analyse
DNA sequences. However, Dr. Yokohama is working in this area. (Tangcham)Q,
When your institutedistributes
is the distribution
system in Thailand?

rhizobia( Nakagahra)

what do you charge farmers?

What

A.
Farmers are not charged. The Department of Agriculture sells Rhizobia to the
Department of Agricultural
Extension which give Rhizobia to farmers free of charge.
(Tangcham)
Q,
What is the origin of USDA 110? What is the ecology of strain USDA 110,because it survives in many areas of the world and can compete with native strains?
(Kaku)A.
USDA 110 is from BFN, Hawaii University.
In Thailand USDA 110 cannot
survive in the field because of the soil temperature after soybeans are harvested is toohigh. (Tangcham)Q.
Did you examine the nodule occupancy of inoculated
situ and ex-situ environmental
conditions?
(Yoshida)

legume crops under in-

A.
field

Yes, it depends on the cropping history. The inoculant will be effective
which has no legume crop in the previous season. (Tangcham)Q.
plantsHave you tried to inoculate
arbuscularwith inoculationof Rhizobium1} (Iwase)

mycorrhizal

fungi

in theto leguminousA,
Yes, inoculation with VA-mycorrhiza and Rhizobium to leguminous plants
should be better than inoculationof only VA- mycorrhiza or Rhizobium alone.
(Tangcham)Q.
Do inoculatedha? (Suzui)

of inoculaperA,
Yes strains do survive in the field. However we do not check the length
time the strain survives. The cost per ha is about US2.5. (Tangcham)ofQ.

Phosphorous

strains

is defficientsurvivein the field?

in most tropical

What is the pricesoils.

Biologicalnitrogenfixation

96

requires phosphorous. Howdo you solve this phosphorous
Rhizobium*} (dela Cruz)

requirement

A.
Yes, phosphorous is essential to get good growth and N2 fixation
Wehave shown that in Sesbania- rice rotation if we apply phosphorous
weget good growth and N2 fixation. When this Sesbania is incorporated
the availabilityof phosphorous to rice increases.
(Ladha)Q.
nitrogen

We have heard much about 15 ton rice. Realisticallysources help produce a 15 ton rice crop? (Vaughan)

when you useof legumes.
to Sesbania
into the soil

how can biologicalA.
If we have rice which would produce 15 tons/ha, it would require about 300400 kg N/ha which cannot be meet from existing biofertilizer
system. That is why
IRRI has initiated a project on assessing opportunities
for symbiotic N2 fixation in rice.
Although it may be difficult
to achieve symbiotic nitrogen fixation in rice, if achieved,
the returns may be very great. (Ladha)

TECHNICAL
SessionPlant Pathogenic

REPORTS
3

Bacteria

Chairpersons
Hisatoshi
Kaku
Baharuddin SallehDiversityof The Genus Agrobacterium and Its Relatives

fflROYUKISAWADA
National Instituteof Agro-Environmental
Sciences
3-1-1, Kannondai, Tsukuba, Ibaraki 305, Japan(NIAES)

Introduction
The incidence of crown gall and hairy root diseases affecting
a wide rangeof crops is a serious problem (Burr, 1988; El- Fiki and Giles, 1981; Horst, 1983;
Munnecke et al., 1963). It is assumed that the frequent occurrence of these diseases
may result from transmission
by contaminatedpropagationmaterials,
planting
into
infestedsoil, wounding of infected vines associated with freezing temperatures andother factors (Burr, 1988). This makes detailed
epidemiologicalstudies using reliable
methods for differentiationof the pathogen necessary to analyze the disease cycle.
Chromosomal markers (e. g., physiological,
serologicalor chemotaxonomic
characteristics)
are often used to develop methods for discriminating
phytopathogenic
bacteria(Schaad,1988). However, althoughboth the crown gall bacterium,
Agrobacterium tumefaciens (Smith
and Townsend)
Conn, and the hairy root
bacterium, A. rhizogenes (Riker, Ban field, Wright, Keitt and Sagen) Conn are highly
heterogeneous with respect to chromosomal markers and are separated into several taxa
(biovars
1, 2 and 3) based on them (Kersters and De Ley, 1984), differences
among
these biovars remain to be fully determined
and there are some anomalies left in thetaxonomy of the genus (Kersters and De Ley, 1984; Bradbury, 1986) which hamper thedevelopment of reliable methods for differentiating
them.
Bacterial taxonomy has become increasingly
objective
with the introductionof molecular techniques and analysis of nucleic acids. It was demonstrated that DNADNAhybridization
is essential
for the designationof bacterial species (Wayne et al.,1987).
It is also generally
recognizedthat nucleic acid sequencing
currently
providesa sound basis for determining
phylogenetic
relationships
among bacteria(Woese, 1987; Murray et al., 1990; Graham et a/., 1991).
In this study (Sawada et al., 1993),
phylogenetic
analysis was performed
using 16S rRNA sequencing and DNA-DNAhybridization
techniques
to clarify thediversityof the genusAgrobacterium and its relatives
and to resolve problems related
to the taxonomy of this genus, as a first step to developinga method for differentiating
them.

Sequence analysisof 16S ribosomal

RNA

There are three categories of RNAs in bacteria;
the short- lived messenger
RNA(mRNA), and the stable forms, i. e., transfer RNA (tRNA) and ribosomal RNA

100(rRNA). Analysis of RNAs for taxonomic purposes has centered on the three rRNAs;
5S, 16S and 23S molecules.
Due to the conserved functions of the rRNAs, they have changed very little
during evolution.
Thus, rRNAs from even the most taxonomicallydistant bacteria,that share little DNA homology, will have sequences in common, and, therefore,
relatedness can be assessed. Murray et al. (1990)
reported that 16S rRNA sequencingof 1,000 or more bases is the optimal
method for determining
phylogenetic
relationships
above the species level.
Until the mid- 1980s, it was not feasible to determine complete 16S rRNA
sequences.Therefore,
16S rRNA had been characterized
by oligonucleotide
cataloguingor the DNA-rRNA hybridization
method. Thereafter,
the need
to obtain more informationfrom 16S rRNA led to the developmentof reverse
transcriptase
sequencing (Lane et al., 1985), which is a rapid and relatively
easy
method. However, this method is already being replaced by direct sequencingofthe polymerase chain reaction (PCR) - amplified
16S rRNA gene. Various techniques
are being developed for direct sequencing of PCR products, enabling the comparisonof large numbers of rRNA sequences.
DNA-DNAhybridization
Determining
the extent to which single-stranded
DNA fragments
fromone bacterial strain hybridize
with single-stranded
DNA from another strain has
been used to examine DNAhomology. Generally, bacteria within the same genomic
species are considered to have DNA homology values above 60% (Johnson, 1984) or70% (Wayne et a/., 1987).
To conduct
hybridization
experiments,
DNA is labeledwithradioactive
substancesby nick translation
or random primed labeling,which israther complicated. However, recent advances have made it possible
to label DNA withnonradioactive
materials
such as photobiotin,
and fluorometric
DNA-DNAhybridizationin microdilution
wells has recently been developed as an alternativetoearlier methods in which radioisotopes
were used, thus enabling information on DNA
homology to be more easily obtained (Ezaki et a/., 1989).
Taxonomic studiesof the genus Agrobacterium by use of nucleic

acid techniques(1) Problems relating to the taxonomy of the genus Agrobacterium and its relatives
In Bergey's Manual of Systematic
Bacteriology
vol.1 (Kersters and De Ley,
1984),
the genus Agrobacterium was included in the family Rhizobiaceae
togetherwith Rhizobium, Bradyrhizobium and Phyllobacterium(Table1). All of themembers of the genus Agrobacterium except A. radiobacter are phytopathogenic
bacteria,
and the members of the genus Rhizobium are well- known nitrogen- fixing
nodule-forming
bacteria
associatedwith legumes. However, most of the genes
associated with phytopathogenicity
and nodule formation are located on plasmids (e.

101g., Ti, Ri or Sym plasmid), and therefore the present system used for definitionof thegenem Agrobactermmand Rhizobium is not based on chromosomal DNA upon which
the taxonomy of bacteria
should be grounded. Moreover, a close relationshipbetween the two genera has been recognized based on various chromosomal markers(Jarvis et al., 1986). These facts required the reassessment of the definitionof the two.
The delineation of species in the genus Agrobactermm(Table 1) is also basedon plasmid- mediatedcharacteristics,
namely, phytopathogenicity
and the type ofsymptoms inducedon plants. A. tumefaciens and A. rubi induce cortical
hypertrophyof the upper parts of roots and A. rhizogenes induces abnormal root
growth, whereas A. radiobacter is non- pathogenic.
As mentioned above, someinvestigators
have questioned
the validityof the use of these characteristics
astaxonomic criteria of species. On the other hand, three biovars (biovars
1, 2 and 3)
have been differentiatedon the basis of various chromosomal markers, and it has beensuggested that these biovars should be elevated to the species level (Kersters and De
Ley, 1984; Ophel and Kerr, 1990).

Table

1. Members of the family Rhizobiaceae
listed in Bergey's Manual of Systematic
Bacteriology
vol. 1 (1984)a
Genus I Rhizobium
R. leguminosarumbiovar trifolii
biovar phaseoli
biovar v/ceae
R. meliloti
R. loti
Genus n Bradyrhizobium
B.japonicum
Genus HI Agrobacterium
A. tumefaciens
biovar 1, 2, 3
A. radiobacterbiovar 1, 2
A. rhizogenes
biovar 1, 2
A. rubi
Genus IV Phyllobacterium
R myrsinacearum
R rubiacearuma : Azorhizobium caulinodans, Rhizobium
fredii, R. galegae, R. tropici, R.. huakuii,
R. etli, Bradyrhizobium elkanii were added
after this version was published.

102

(2)

Phylogenetic
Analysis Using Nucleic Acid Techniques
The complete 16S rRNA sequences of seven representative
agrobacteria,
eight representative
rhizobia and type strains of Bradyrhizobium japonicum Jordan
and Azorhizobium caulinodans Dreyfus, Garcia and Gillis were determined in termsof direct sequencing of PCR products. The amplified
16S rRNA gene was purified by
the glass bead method. Then, the purified DNA was sequenced with a Sequenase kit for
35S-dATP (United Biochemical,
Inc.).
A matrix of the levels of sequence similarity
for the representative
strains
is shown (Table 2). Similarities
among Agrobacterium taxa and Rhizobium taxa
exceeded 94%, whereas similarities
between members of the genera Agrobacterium
and Rhizobium and members of the B. japonicumlAz. caulinodans group ranged from86.6 to 92.1%.
The genetic distances between the sequences were estimated using the Knucvalues (Kimura, 1980). Then, a phylogenetic
tree was constructed by the neighborjoining
method (Saitou and Nei, 1987) by using K coli as the root organism. The
topologyof the reconstructed
phylogenetic
tree was evaluated by the bootstrap
sampling method (Felsenstein,1985).
The phylogenetic
tree derived from the sequence similarity
values is shown(Fig. 1), which showed that the 15 agrobacteria
and rhizobia formed a compact cluster
clearly
separatedfrom the other members of the alpha subclass of the classProteobacteria including
Bradyrhizobium and Azorhizobium species. However,agrobacteria
and rhizobia were phylogeneticallyentwined with one another in thecluster, and the two genera could not be separated.
These results suggest that thepresent nomenclature of the two genera does not reflect the true phylogenetic
relationship.
However, the data are not sufficient
to conclusively
reassess the definitionof these genera. Therefore, it is concluded
that at present Agrobacterium andRhizobium species cannot be combined into one genus.
Another problem is found in the taxonomic treatment of biovars. Each ofbiovars 1, 2 and 3 was different
in its complete 16S rRNA sequence from the others
and were clearly separated in the phylogenetic
tree (Fig. 1). The three biovars also
exhibitedhomogeneity
in their phenotypic(Kersters and De Ley, 1984; Sawada etaL, 1990, 1992a) and chemotaxonomic characteristics(De Ley, 1974; Sawada et aL,
1992b).
Fluorometric DNA-DNAhybridization
developed by Ezaki et al. (1989) was
performed to determine the DNA homology among the three biovars. Hybridizations
were carried out at 45°C for 3 h in a hybridization
mixture containing
formamide
and dextran sulphate. The results of DNA- DNA hybridizations
using DNAs from
representative
strains of the biovars as references are shown (Table 3). The levels ofhomologous within each biovar were high (63 to 106%), whereas the levels ofhomology were much lower (1 to 11%) between biovars, indicatingthat each one
forms a distinct
homology group. These results mentioned above (phenotypic,
chemotaxonomic,
genotypic
and phylogenetic
characteristics)
and information
availablefrom the literatures(reviewed by Kersters and De Ley, 1984) suggest that

Table 2. Similarity

matrix for 1 6S rRNA sequences of Agrobacterium and Rhizobium species and their relatives.
H o m o lo g y w ith :B io v ar 1 B io v a r 2 B io v a r G

A .r u b i

K ‑A g ‑3

C h ‑A g ‑4

T ax onA g ro b a c te r iu m

R h izo b iu mA z o r h iz o b iw n

N C PPB
16 5 0R le g u m i‑ R m e li‑ R f r e d ti R
n o sa r u m
lo ti

lo ti R g a leg a e R tro p ic i O K 5 5

B io v a r 1
B io v a r 2
B io v a r 3
A .ru bi
K ‑A g‑3
C h‑A g‑4
N C P P B 16 50
R le g u m in o s a ru m
R . jm r e dliloii ti

9 6 .39 6 .59 8 .59 6 .09 6 .39 8 .69 6 .29 65 .2.99 5 .89 5 .59 9 .71 0 0 .0
9 5 .69 8 .69 7 .3.5

9 6 .89 5 .79 5 .89 6 .6
9 4 .89 5 .9.4

9 5 .39 5 .59 9 .79 5 .1
9 5 .6.3

9 9 .79 5 .49 8 .39 7 .0.2

9 5 .69 8 .69 7 .3.5

9 5 .1
9 5 .7.4

9 77 .0.2 R

9 9 .0R . lo ti

9 64 .8.3

99 56 .2

9 64 .7.4

99 56 .0.8

9 65 .0

9 65 .2

9 65 .8.2

9 65 .4.3

9 56 .4

9 56 .1.0

9 5.1

R . tro p ic i
O K 55A z . c a u lin o d a n s

9 5 .79 6 .89 1 .39 9 .69 6 .29 1 .79 5 .49 6 .3
9 0 .79 5 .09 6 .89 1 .9

1 0 0 .0
9 5 .99 1 .69 9 .69 6 .19 1 .79 5 .19 6 .89 1 .99 8 .19 6 .892 .1

9 6 .99 5 .6
9 0 .89 4 .69 5 .5
9 0 .99 4 .69 5 .49 1.1

9 5 .79 9 .49 1 .39 5 .79 1 .29 1 .2A z .x a u li‑ B .j ap o n i‑
n oda ns
cum

104

Fig.

1. Phylogenctic

tree derived

from the whole 16S rRNA sequences for members of thegenerea Agrobacterium and Rhizobium and their relatives.
£. coli was used as the root
organism. Positions
70 to 100, 181 to 219, 447 to 487, 1004 to*1036, 1133 to 1141, andspecies1445 to 1456 (£. coli numbering)were not used for the comparison

structures

among the strainsof these regions

variedthe estimatedconfidence

limit

determinedby bootstrap

analysis.

should be established(expressed

because the secondary

compared. The value on each branch is

as a percentage)

for the positionof the branch as

for each biovar.

(3) Determinationof Taxonomic Positions of Unclassified
Kiwi fruit and Cherry Isolates
Phytopathologists
sometimesfindagrobacteria
withphenotypiccharacteristics
that are intermediate
between the designatedboundaries
for thedifferent taxa. It is necessary to determine the taxonomic position of these intermediate
isolates
to clarify
anomaliesin the taxonomy of the genus and to refine it.
The taxonomic positions of kiwifruit isolates (K- Ag- 3 and 4) and cherry isolates (ChAg-4, 5, 7 and 8), which the author isolated and treated as unclassified(Sawada et aL,

105

Table 3. DNA relatedness

at 45°Ca among the strainsof the genus Agrobacterium.

D N A relate dn ess (% ) w ith strain :N C P P B 24 3 71 TF O 13 2 577 K err 8 4 N C P P B 3 55 4T IF O 13 2 6 1T
Strain(B iov ar 1)

B iov ar 1
N C P P B 2 4 3 77
10 0
M A F F 3 0 17 24
83
IA M 120 4 8T
87
B io v ar 2
N C P P B 23 0 3
6
IF O 132 5 7T
4
A T C C 1 58 34
4
K err 84
B io v ar 3
N C P P B 2 5 62
6
G ‑A g ‑2 7
N C P P B 35 54 1
9
A g.ruIFIF
ro Obbiac13
13terium
2266 011 sp . (kiw ifru it105iso late)(B io var 2 ) (B io va r 2 )

9
ll

9

94
10 0
88
88

84
77
71
10 0

7

9

7
35

ll
32

K ‑A g ‑3 1
K ‑A g ‑4
12
A g ro b ac terium sp . (cherry iso late)
C h‑A g ‑4 1
C h‑A g ‑5
C h‑A g ‑7ll
C h‑A g ‑8

19
24

17
23

14
16
21
ll

14
17
25
12

a [Comparisons were made in triplicate.
was arbitrarily
defined as 100 %.
b: T, Type strain for the species.

The level of binding(B iov ar 3)(A . ru b i)

3

10

76
63
100
I 43 I

10 60

between homologous

strains

1992a, 1992c), were investigatedon the basis of DNA homology by using fluorometric
DNA-DNAhybridization
method.
When the type strains of Agrobacterium taxa were used as references, lowvalues (1 to 25%) were obtainedwith unclassified
isolatesof both (Table
3).
Similarly,
the unclassified
isolates as references exhibited
low values (1 to 41%) withthe type strains of Agrobacterium taxa (Table 4). Furthermore,
kiwi fruit isolates
and cherry isolates
formed distinct DNA homology groups, respectively.
On thebasis of their genotypic
distinctness
which coincidedwith other phenotypicandchemotaxonomic characteristics(Sawada et al., 1992a, 1992b),
it is concluded
thatkiwi fruit isolates
and cherry isolates should be assigned to two new species of thegenus Agrobacterium.

106

Table 4. PNA relatedness at 45°C among Agrobacterium taxa andagrobacteria
isolated from galls on kiwi fruit and cherry.
D N A r e la te d n e s s (% ) w ith str a in :S tra inK ‑A g ‑3 1 K ‑A g ‑4

B io v a r 1
N C P P B 2 4 37 1
5
10
IA M 1 2 0 4 8 T
B io v a r 2
IF O 13 2 5 7 1
18
10
K e rr 8 4
18
17
B io v a r 3
N C P P B 35 54 7ll
12
A . ru b i
IF O 13 2 6 1 T
A g r o b a c te riu m sp . (k iw ifru it is o la te )A g r ocKb ah‑Ac‑Ate
g g‑4
‑3
riu
‑41mT sp . (c h e rry
1 80l lis
20 o late ) 1 081 906

C h ‑A g ‑ 5
C h ‑A g ‑7
C h ‑A g ‑ 8

18

16
23
19

C h ‑A g ‑ 5 C h ‑A g ‑7

C h ‑A g ‑8

4

14
13

15

16

8

12

I l O21O51

11263

T 2o1 76o

100
116
97

88
100
107

95
1 14
10 0

a: Comparisons were made in triplicate.
The level of binding
homologous strains was arbitrarily defined as 100 %.
b: T, Type strain for the species,
c: _, Comparison not done.

between

References
Bradbury,J. F. 1986.Agrobacterium Conn 1942. In Guide to plant pathogenic
bacteria,
CAB International
Mycological
Institute,Kew, pp. 6-15.
Burr, T. J. 1988. Crown gall. In Compendium of grape diseases,
R.C. Pearson and A. C. Goheen,eds., Amer. Phytopath.
Soc. Press, St. Paul, Minnesota, pp. 41-42.
De Ley, J. 1974. Phylogeny
ofprocaryotes.
Taxon 23: 291-300.
El-Fiki,K. and K. L. Giles. 1981. Agrobacterium
tumefaciens in agriculture
and research. In International
review of cytology, supplement
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Ezaki,T., Y. Hashimoto and E. Yabuuchi. 1989. Fluorometricdeoxyribonucleic
acid-deoxyribonucleic
acid
hybridizationin microdilution
wells as an alternative
to membrane filter hybridizationin whichradioisotopes
are used to determine
genetic relatedness
among bacterial
strains. Int. J. Syst.
Bacteriol 39: 224-229.
Felsenstein,J. 1985. Confidence limits on phylogenies:
An approach using the bootstrap. Evolution
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Graham,P. H., M. J. Sadowsky, H. H. Keyser, Y. M. Barnet, R. S. Bradley, J. E. Cooper, D. J. De Ley,
B. D. W.Jarvis, E. B. Roslycky, B. W. Strijdom and J. P. W.Young. 1991. Proposed minimal
standards for the descriptionof new genera and species of root- and stem-nodulating
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Int. J. Syst. Bacteriol.
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R. K. 1983. Diseases
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AmericanPhytopathologicalSociety, St. Paul, Minnesota, pp. 23-26.
Jarvis,B. D. W., M. Gillis and J. De Ley. 1986. Intra- and intergenericsimilarities
between theribosomal ribonucleic
acid cistrons of Rhizobium and Bradyrhizobium
species and some related
bacteria. Int. J. Syst. Bacteriol. 36: 129-138.
Johnson, J. L. 1 984. Nucleic acids in bacterial
classification.
In Bergey's manual of systematic
bacteriology
vol.1, N. R. Krieg and J. G. Holt, eds., Williams& Wilkins, Baltimore,
pp. 8-ll.
Kersters, K. and J. De Ley. 1984. Genus IE Agrobacterium
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vol.1, N. R. Krieg and J. G. Holt, eds., Williams& Wilkins, Baltimore,
pp. 244-254.
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rates of base substitutions
throughcomparative studies ofnucleotide
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Lane, D. J., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin and N. R. Pace. 1985. Rapid determinationof 16S
ribosomal RNA sequences for phylogenetic
analyses. Proc. Natl. Acad. Sci. USA 82: 6955-6959.
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and M. P. Starr. 1963. Hairy root (Agrobacterium
rhizogenes)offield roses. Phytopathology
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isolatedin Japan.
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Sawada, H. and H. Ieki. 1992a. Phenotypiccharacteristicsof the genus Agrobacterium.
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Diversityof Pseudomonassolanacearumin China

LI-YUAN HESKLBPI, Instituteof Plant Protection,
Yuan Ming Yuan Rd, Beijing 100094,

CAAS, West
P. R. China

Abstract
Pseudomonassolanacearumis the causal agent of the widespreadand destructive
bacterial
wilt
disease which infects many cultivated plants in China. Several new hosts of economic importance havebeen observed to be infected by this pathogen. A number of strains of/! solanacearum collected from avariety of host plants of different provinces or regions were compared for their pathogenicity
and physiobiochemical, serological and other characteristics.
Most strains were classified
as race 1 or race 3 based on
Buddenhagen, Sequeira and Kelman's classification
scheme, and as biovar 2, 3 or 4 according to Hayward's
subgrouping category. Some strains from mulberry, however, were identified
as race 5 or biovar 5 because
they were different from the established subclassification
units in their weak or non-pathogenicity
to somesolanaceous plants and their ability in oxidation of three disaccharides
and mannitol. Significant
differencesin specificity
to hosts were present among race 1 strains and, therefore, some pathotypes
were proposed.
Biovar 2 is a predominantstrain on potatoes in China. No representativesof race 2 are present in thecollected strains. Attempts were made to serotype strains of the pathogen using polyclonalor monoclonal
antibodies,but, no substantial
correlations
were found between the serologicalproperties
and othercharacteristics.

Introduction
Pseudomonas
solanacearumE. F. Smith is the causal organism of bacterial
wilt, a widespreadand destructive
disease of crops. This pathogen is distributed
worldwide but primarily
in the tropics, subtropics
and some warmtemperate regionsof the world. It is recorded to be able to infect several hundreds species in 44 plantfamilies,
involving
many cultivatedplants of economic importance, such as potato,
tobacco, peanut , tomato, eggplant, pepper, ginger, banana, teak , cassava (Agrios,
1989; Hayward, 1986) Since Erwin F. Smith first described
the pathogen on potatoes
near 100 years has passed. This pathogen and bacterial
wilt it causes have been thesubject of thousands of publications.
However damage and crop yield losses due to thisorganism are still increasing(Persley,1986).
Pseudomonassolanacearum is a variable organism in pathogenicity
to hostplants,culturalcharacters,
physio- biochemical
reactions,serology
and other
properties.
There are several systems of classification
for this organism at thesubspecificlevel on the basis of different
criteria.
Of them, two subclassification
systems,i.e.
race classification
and biovar (or biotype)classification,
are currently
widely accepted. The race classification
was proposed by Buddenhagen, Sequeira andKelman (1962) based on host plants origin and host range of strains and 3 races weresuggested.
The biovar classification
scheme was proposed by Hayward (1964)
accordingto the abilityof strains to oxidise 3 disaccharides(lactose,maltose andcellobiose)and 3 hexose alcohols (mannitol,
sorbitol and dulcitol)and 4 biovars were110

suggested.
The Spreadand New Hosts of P. solanacearum in China

Bacterial wilt due to P. solanacearum was first reported on tobacco in China
in 1940 although it was observed by local farmers much earlier (Lou, 1940). Until late
1950s this organism was recorded only to cause damage to a few crops, including
tobacco, tomato, eggplant, pepper, peanut and sweetpotato,
and it was limited to thesouthern part of China, mainly along the Yangtze River valley. Since late 1970s theinfected area has greatly expanded and some new host plants have been observed. For
instance, the bacterial wilt (or blast disease ) of sweetpotato was reported only in a few
countiesof Guangdong and Guangxi provinces before 1960s, but now it can beobserved in Fujian and Zhejiang
provinces (He and Hua, 1983; Zhen and Fan, 1962).
Bacterial
wilt of potato was first reported in the late 1960s and was limited to small
areas of Hunan and Sichuan provinces by late 1970s, but now it has spread northbeyond the Great Wall (He, 1986).
In China, this pathogen
attacks not only
solanaceous and other herbaceous
plants,but
also some woody plants, including
horsetailbeefwood (Casuarina equisetifolia L.), commonolive (Olea europaea L )and mulberry {Momsalba L.) and causes substantial
losses (He and Hua, 1983; Wu
et aL, 1986; Liang and Chen, 1982; Sichuan Instituteof Forestry, 1977). In recent
years this pathogen is reported to affect some cultivatedmedicinal
plants, such as
Pogostemoncablin Benth and Kaempferiagalanta L , a forage crop Symphytumsp.,
a fibre crop Hibiscus caunabinus L. and some important woody plants, such as
Eucalyptus spp. and Gossampimus
malabarica (Dc.) Morr (Liao et al, 1982; Wen andZheng, 1984; Zhang and Zhang, 1992; Wu and Liang, 1988) (Table 1). These hosts
have not been reported from other countries.
Despite the wide host range of P. solanacearum, marked differences
havebeen observed in its specificity
to various hosts and in epidemic characters in different
Table

1. New hosts of P. solanacearum in China.

H o st n am e

P ro v in ce o r re g io n

R e fe re n ce

S w e et p o t a to (Ip o m o e a b a ta ta s)
M u lbe rry (M o ru s a lb a )C o m m o n o liv e {O k a e u rop
e a ea )H o rsetail b ee fw o o d(C as u a rin a e q u ise tif o lia )B o eh m er ia n iv ea (U r tica n iv e a )S y m p hy tu m s p.
P o g o s tem o n c o b lin
K a em p f eria g a la n ta
E u c a ly p tu s sp p .
G o ssa m p in u s m a la b a r ica (D c .)M o rr
H ib iscu s c a n n a b in u s h .

G u an g d o n g , G u a n g x i, etc .
G u an g d o n g
H u b ei ,G u an g x i
G u an g d o n g , etc .

Z h en e t a l , 1 9 6 2
H e etal,1983
H e et a l , 1 9 8 3
H e et a i.9 1 9 8 3

Z h ejia n g , e tc .
S ic h u an
H ain a n
H a ina n
G u an g d o n g H ain a n
H a ina n
F u jia n

H ua etal,1984
L iao et a i , 1 9 8 2
W e n et a l , 19 84
W e n et a l , 19 84
W u e t a L , 19 8 8
Z h an g e t a l , 1 9 8 2

Illregions. For instance, P. solanacearum was reported to severely attack tobacco in
Fuqingcounty of Guizhou, but it was not observed in fields in Luiliang
county of
Yunnan where tobacco was grown surrounded by severely infected potatoes. Sesame
was reported to be infected by the pathogen in Jiangxi province, but was not observedin Huong- an county of Hubei province, where farmers usually used sesame as a
substitution
crop in bacterial wilt killed peanut fields (He and Hua, 1983). Moreover,according to Zhen and Fan (1962), the strains from sweetpotato were not virulent on
peanut and the strains from peanut were not virulent on sweetpotato. It is evident there
may exist different strains with host specialization
within this species of pathogen, andsome of them may be indigenous or introduced but adapted to local environments.
Races and Pathotypes
To examine the variation of P. solanacearum in pathogenicity
to host plants,
cross inoculationsof 38 representative
isolates on 13 different plants were conducted.
Results
indicatedthat all isolates
except those from mulberry could highlyormoderately
infect black nightshade,
eggplant,
tomato, potato and heart- shaped
tobacco. On commontobacco, pepper, peanut and sesame, however, a number ofisolates appeared to be aviruleqt.
To sweetpotato, ginger and Urtica nivea only thoseisolatesthat were collectedfrom the respective
host plants were virulent.
Twomulberry isolates (M2, M8) showed high virulenceon mulberry, low virulence on
eggplant, black nightshade
and other solanaceous plants. All non-mulberrry isolates
failed to cause wilting symptom on mulberry (Table 2). A trail of inoculationof 43
potato isolates on the respective plant and tomato demonstrated that all of them werevirulent on these plants.
Based upon reactions of inoculated
plants most isolates from different hosts
and different
locationsin China could be classified
as race 1 and 3 according
Buddenhagen et al. (1962).
Race 1 included 37 isolates from a wide range of hosts.
Most race 1 isolates showed high virulence on solanaceous plants examined. Isolatesof race 3 were mostly from potato and showed high to low virulence on potato andtomato and were somewhat virulent on eggplant, black nightshade
and heart-shaped
tobacco. The two mulberry isolates
differedfrom all establishedraces and areconsidered to belong to a new race, designatedas race 5. (He etal. 1983; He, 1986)(Table2).
Marked differencesin pathogenicityof P. solanacearum were observed
among isolates of race 1. Some isolates showed specificity
to their original hosts. For
instance, ginger isolates
and Urtica nivea isolates appeared virulent
only to theirrespective hosts. It is reasonable
to designate these strains as pathovars, or at least, todivide them into pathotypes
based on difference in their specificity.
Xu et al. (1980)
suggestedthat strains on peanut from various regions be divided
into different
pathotypes
based on the differencesof virulence on different varieties.

112

Table 2. Pathogenidty

of 38 isolatesof P.solanacearum

to 13 host plants

and classificationof them. 'P ath o g en icity on
B lac kIso late

n ig h tsh a d esh
H ae part‑ed
E g g p la n t T o m a to

C o m m on

P o tato

to b a cc o

P ep p er

to b a cc o

S e sa m e

U rt ica

Sw eetP ea n u t

n iv e ap o ta to

L

O

O

O

O

l

G in g e r M u lb e rry R ac e

H2
P o7
tb l
P7
C4

H

H

H

H

H

H

L

M

H

H

H

H

H

M
ND

M

L

ND
H

ND
Tm 6
E2
P ol
C2
CI
P e2
E I
Tm l
Ol
P 16
P el
PI
P6
P 19
P 20
P9
SI
Tm 2
S nl
B2
B4
Z2
Z3
B nl
Tm 7
Po3
Po2
P olO
M 4
M 8

1 Results
2 H=high

H

H

H

H

H
H
H
H

H
H
H
H

H
H
M
H

H
H
H
H

M
M
H
H
H
N D

ND

L

M

L

O

O

O

O

l

M
H
M
M

O
M
O
O

L
O
O
O

L
M
H
H

O
L
O
O

O
O
O
O

O
O
O
O

O
O
O
O

l
l
l
l

ND
H
H

H
H

M
H
M

H
H
ND

M
M
M
ND

L
O

O
L

O
O

H
H

O
O

O
O

O
O

O
O

l
l

H

H

H

H

H

M

L

H

O

O

O

O

O

l
N D
ND

O

O

O

O

O

O

O

O

3

L ‑M

ND

M

L ‑M
L
O ‑L

M

M

O ‑L

based on average disease indices(4.1-5.0),
M=moderate (2.6-4.0),

Biovars

and Other

ND
ND
ND

of more than 5 plans 15-21 days after inoculation.
L=low (1.0-2.5),
Onone (1.0), ND=not determined.

Variationin Physio-Biochemical

Properties

There were marked differencesamong isolatesof P. solanacearum inoxidationabilityto three disaccharides
and three hexose alcohols(Table 3). In
accordance with the Hayward scheme (1964),
these isolates except those on mulberry
were identified
as biovars 2, 3 and 4; 74 isolates from potato belonged to biovars 2, 3
and 4, respectively.
The potato isolate PolO differed from others in its inabilitytooxidise three disaccharides
and dulcitol
and in its lack of ability to oxidise mannitoland sorbitol.
Since this isolate
was similar to those of biovar 2 in all othercharacteristics,
it was considered to be a subtype of biovar 2. Seven of eight mulberry
isolates
were classified
as biovar 5 and one of them was identified
as biovar 3. The

113

Table 3.

Oxidation

H o stof carbohydrates

Q u an tity o f iso la teby isolates

L ac to seofP. solanacearuntM a lto se C e llo b io se D u lcito l M a n n ito l S o rbito l B io v ar

T o m ato2
2
P ep p er
E g g p lan t
P otato

+
‑

+
.

+
.

+
+

+
+

+
+

3
4

‑

‑

‑

+

+

蝣 +

4

4

.

.

.

+

+

+

4

2

.

.

.

+

+

+

4

+

+

+

4

‑

+

‑

5

69

P otato1
T o b ac co
B lac k n ig h tsh a d e
P ea n u t
15
G in g er
S esa m e
S w eet po ta to
U rtic a n iv ea
H o rseta il b e efw oo d
C o m m o n o liv e
1
M u lbe rry
7+ positive,

+

+

+

- negative

isolatesof biovars 5 showed delayed oxidationof 3 carbohydrides
and mannitol inwhich a marked acidic reaction
was observed two weeks after inoculation,orsometimes even later. It was unexpected that biovar 1 strain was not observed among
the numberous isolates tested.
Sixty nine out of 74 potato isolates
belongedto biovar 2 and werecharacterised
by growing at a relatively
low optimum temperate (27 °C). It should be
noted that this strain is spreading not only in the areas of higher altitude
but also inlowlands in southern China, and as far north as 41 °C latitude where the main potatogrowing regions of northern China are located.
Many isolates in our tests belonged to biovar 3 which was found on 14 of 16
natural host plants we investigated.
This biovar was reported also to exist on
Pogostemoncablin Benth., Kaempferia galanta L. and Eucalyptus spp. (Wen andZheng, 1984; Wu and Liang, 1988). It appears this is the predominantbiovar in China.
Biovar 4 was reported only on ginger and tomato in other countries, but it is observedon peanut, potato, eggplant,
horsetailbeefwood, commonolive, sweet potato, andKaempferiagalanga L. as well as ginger and tomato in China. Obviously,
this is an
economically important strain in China.

114

Table 4. Variationof P. solanacearum in some physio-biochemical
B io v ar 2

R ea ctio n(4 )B io v ar 3

properties.
B iov ar 4

M o st(16iso)late s B SnpleciaT lmiso2 late
S ns l T b 2 M o st(1iso
3) late s SB p 2ec,Bial
4 ,P
iso 19lates

B io v ar S
(4 )O x id atio n o n
T re h alo se
X y lo se
In o sito l
G a s pro d u ctio n
fro m n itra te
In N aC l m edia
1 .3 %
1 .5 %
1 .7 %
2 .0 %( ) isolate quantity+ positive, - negative, -+weak growth

Ren etal. (1981)
reported
that the mulberry strains they tested belonged tobiovar 1 although an acidic reaction of medium containing mannitol was observed after
4 weeks of incubation. In a more recent report , Xu et aL (1986) indicatedthat 30 out
of 31 mulberry isolates they detected showed acid production from maltose, lactose,cellobiose
and mannitol. So, biovar 5 may be the predominantstrain of P.
solanacearum on mulberry although other biovars may be present.
Significant
variationsof P. solanacearum as observed in oxidationontrehalose, xylose and inositol and in denitrificationand tolerance to salt. Most isolates
produced acid from trehalose but isolates of biovar 2 and isolates Bnl and Tm2 fromrace 1 did not. A majority of isolates tested oxidized xylose but biovar 5 isolates
frommulberry did not. All isolates
examined except
a tobacco strain (Tb2) showedoxidationabilityon inositol.
Biovar 2 differedfrom other biovars in its inability
toproduce N2 from nitrate. Most isolates were able to grow in 1.5% NaCl medium but
biovar 2 isolates and some isolates from mulberry could not (Table 4).
The isolatesof P. solanacearum tested showed clear differenceswhile
growing in litmus milk containing
cream. Most isolates of biovars 2 and 3 produced
a stronger acid reaction than isolates of biovars 4 and 5 which, in fact, caused analkaline reaction of this medium (in the lower layer) after 17 days incubation (Table
5). Generally
biovar 3 can strongly
oxidise lactose which is containedin a highconcentrationin milk. This biovar is a common strain widely distributing
in Asiancountries , which may be the reason why E. F. Smith designatedthe strain of P.
solanacearumin Asia as var. asiaticum. In litmus milk without cream, all 44 isolates
tested caused an alkaline reaction, changing the medium from light purple to a blue
colour.

1

115

Table 5.

Effect of P. solanacearum on litmus milk containing
N u m be r

B io v aro f iso la tes

U pp er laye r
lst d ay lO th d ay 17 th d ay 3 0th d ay

lst d ay l O th d ay 17 th d ay 3 0 th d ay

red

red

re d

p u rp le

re d

18

w h ite

red

red

re d

p u rp lere d c oa guredlatio n

red
p ale
d ish

17

w h iteprereduddrpishlep u rp le p u rplep u rplepblue
ligu hrptle, d ark b lu ed ark b lu e

w h itepreredudrpdi,lshere d

p u rplep u rple lig h t b lu ed ark b lu eof some isolates

A n tib io tic

w h iteC h lo ram p h e n ico l

10

red

P o 3 ,2 7 6
W S

preuddrpish
le p u rp le

P 6 ,O l ,Z l ,B 2

M 4 ,M 5

R ‑W SW S

pu rp lepu rp le lig h t b lu e

30

R

R ‑W SO le an do m y c in

15

R

S

P e n icillin
S trepto m y cin , zone of inhibition
R =resistant

10
10absent;RR

WS=weakly susceptible,

1 (T m 2 )Variation

in Susceptibility

redp aled ish

82 ,P o Iso
2 latePs I l ,0 3 , C l, P o l , T b l(u g/m l)

1 (P o l O )p u rp leredof P. solanacearum to antibiotics.

C o n ce n tratio n

V io m y c in

L o w er laye r

w h iteTable 6. Susceptibility

2 ‑1

cream.

S=susceptible,
zone of inhibitionindefinite

wide zone of inhibitionor small (<1 mm).

w h ite

red

red

re d

p u rp le

p u rp le

R ‑W SR(> 12mm);lig
b luhtep u rp le

pu rp lish

to Antibiotics

Susceptbility
to antibiotics
was examined with Bacto antibiotic
sensitivity
disks (Difco Labratories,
Detroit, MI 48232). All isolates
tested were resistanttopenicillin(10 ^g), viomycin(2^g) and chloramphenicol(5 Mg) and were susceptible
to streptomycin (10 ^g). Isolates from China differed from one another in susceptibility
to oleandomycin(15 ^g); isolates belonging
to biovars 3 and 4 were susceptiblebutother biovars were resistant or only weakly susceptible.
All mulberry isolates weresusceptible
to chloramphenicol(30 peg) (Table 6).

116

SerologicalSpecificity

In order to determine the serological
specificityof P. solanacearum, antisera
were prepared against six isolates representing
all major races and biovars. Antigens
consistedof glycoproteinsextracted from bacterialcell walls. Thirty isolates of P
solanacearumfrom 13 different hosts from different regions were used to determine
serologicalsecificity
by cross absorption and micro agglutination.
Based on resultsof the Ouchterlony
double diffusion techniques,
5 different
serotypes were grouped.
The specificity
in some instances was related to host and geographicalorigin of thedifferent strains. While serotype I was complex and reacted with isolates from at leastnine different
hosts, isolates from the potato, mulberry or banana appeared to belong
to serotype III, II or V, respectively(Table 7).
Xu etal. (1986) studied serology of a number of strains from mulberry. Basedupon the significant
differences
in serological
specificity
the authors suggested 4
serotypes for 20 mulberry isolates.
A number of hybridoma cell lines secreting monoclonal antibodies
against P.
solanacearumwrereestablishedby fusing Sp2/0- Agl4 myeloma cells with spleencytes
from Balb/c mice immunized with representative
strains of P. solanacearum (Wangetal, 1989; Xie etal., 1989). The glycoprotein
extracts were used as immunogens andas antigens
in indirect
ELISA for screening specifichybridomas.
Monoclonal
antibodies(McAbs) produced by hybridomas were separately examined for specificity
to different
strains of P. solanacearum and to representative
species or pathovars ofother phytopathogenic
bacteria. Five McAbs that didn't reacted with other plant
Table 7.

Precipitation
Ouchterlony

reaction of glycoproteins
double diffusion assay.

from some isolates

A gof P. solanaceamm using theA s a g ain st g ly co p ro tein fro m strain

N o ofiso lates

R a ce
B iov a r
(p ath o ty p e)

P 14

10 V )PI

1 (IV )

O s
O 1

1 (D
10 0

M 5
M 4
Po3

5

K 60
J 84 1 8F
B2

1
2
1(ll)

P6

1 (T V )2+ =complete identity

H o st o ri g in

P 14

p ea n u t

2+

2+

2+

p ea n u t

2+

2+

2+

o liv eo liv e

2+

2+
2+

2+
2+

5

m u lb e rrym u lb e rry
p o tato

+

+

2+
2+

1

to m a tob an a n asw eet p otato

+
+
+

‑
+
+

‑
+
+

4

4

03

identity;

Po3

K 60

J 5 4 18 F

- =no reaction;

S e ro typ ereco m m e n d edID2+

p ea n u t; + = partial

M 5

‑
‑
+

fl
2+
+
+

+
+

TV
V
N D

ID

N D

ID = indefinite;

ND =not determined.

117

pathogenswere selected for detectionof specificity
to 36 different
isolatesof P.
solanacearumfrom different sources. Results demonstrated that 2 McAbs (McAbl and2) showed obvious strain specificity,whereas other 3 McAbs (McAb 3, 4 and 7) had
a wide spectrum in reaction with different
strains (Table 8).
Based on data we have so far it is difficult
to conclude the relationship

Table 8. Reaction of monoclonal antibodies
of P. solanacearum using ELISA.
P . soIso
la nlates
a ce ar um

R ac e(McAbs)with representative

isolates

M cA b l

M cA b 2

M cA b 3

M cA b 4

M cA b 7

2+

2+

4+
4+

3+
2+

3+
4+

3+
4+

3+

3+
2+

4+
4+

2+
2+

3+
4+

3+
4+
4+

2+

3+
3+
3+

1+

4+
5+

3+
4+

4+
4+

Tm4
Tm 6
Tm 7

2+

4+
4+
3+

3+
3+

4+
1+
2+

Tm 9
c ¥
C2

1+

4+
4+
5+

4+
3+
4+

4+
3+
4+

4+
5+

4+
4+

4+
3+

4+
4+
3+
3+

3+
4+
2+
2+

3+
3+
2+
2+

4+
4+
5+

3+
3+
3+

2+
1+
3+

5+
4+
4+

4+
4+
3+

3+
4+
3+

4+
4+
3+
5+

3+
3+
2+
4+

3+
3+
1+
4+

P o1
P o3
P o7
P o14
P o22
P o37

1+
1+

3+

P o41
P o45
82
Tm 1
Tm 2

1+

2+

2+

C3
P I
P6
P7
M 2
M 5

2+

M 6
B2
B4
Z2
Z3
O2

1+

1+

O 3
SS I
Sn 1
E 1

2+

2+

Pe 1
Bn1

2+
2+

2+

5+
4+

4+
3+

3+
3+

1+

5+

4+

4+

T b2

1 Absorbance
0.21-0.50(3+),

at 490nm:

0.00-0.05

0.51-1.00(4+).

(-), 0.06-0.10(1+),

0.1 1-0.20(2+),

118

Table 9. Rf and molecular weight of antigen glycoprotein
P.solanacearum determined by SDS-PAGE.
PO 3

B2

from some isolates

P 14

of

Im l

M 5

R fl

M W 2

R f

M W

R f

M W

R f

M W

R f

M W

0 .13

95000

0 .1 39500 00 .1 39 5 0 00

0 .13

9500 0

0 .13

95000

0 .2 4 4
0 .4 67300 0
43000

0 .44

4900 00 .4 643 0000 .4 2500 000 .6 2 5

2 50 0 0

0 .5 630000

0 .6 2 525000

00.6.5563

32 04 0 00 00

Relativeband speed,

Molecular weight

betweenserologicalproperties
and other characteristics
in P. solanacearum.
Glycoproteinsextracted
from some isolatesof P solanacearum wereanalyzed by SDS- polyacrymide
gel electrophoresis.
Results indicatedthat there exist
different
proteins while some commonproteins,with the same molecular weight, were
found among different
isolatesof P. solanacearum (Table 9).
Discussion

Significant
diversityin pathogenic,
physiobiochemical,
serologicalandother characteristics
have been found in strainsof P. solanacearum in China. Most
recently
obvious variations
in motility
and productionof extracellular
proteins
in P.
solanacearum were also observed in our laboratory.
But, it is still not known what theessential
variationsin the nature or evolutionof this important,widespreadorganismare. In recent years, molecularmethods including
RFLP analysis
and sequencing
of
nucleotides
in the 16s rRNA have been developed
for studying
the differentiationofmicroorganisms.
Progress achieved so far will provide
a basis for further studies of thediversityand the relationshipbetween strains of P. solanacearum.
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Zhang, T. S. and C. L. Zhang. 1992. Study on Kenaf hemp bacterial
wilt disease. Acta Phytophylacica
Sinica 19(2):123-126(in Chinese).
Zhen, G. B. and H. Z. Fan. 1962. Identificationof the pathogen
causing bacterial
wilt of sweet potato.
Acta Phytophylacica
Sinica 1: 243-253(in Chinese).

Diversity
Southeastof Pseudomonassolanacearumin Japan and
Asia

KENICHI TSUCHIYA and MITSUO HORITA
National Instituteof Agrobiological
Resources2-1-2, Kannondai, Tsukuba, 305 Japan

Introduction
Bacterial wilt caused by PseudomonassolanacearumE. F. Smith is a disease
widely distributed
in tropical,
and some warmtemperate regions of the world, and amajor constraint
to productionof many crop plants. Several reports have drawn
attention
to the predominance and diverse host range of this pathogen in Southeast
Asia, and differences in bacteriologicalproperties of P. solanacearum from Japan andother countries have been recognised.
This paper is a review of the present status of the research concerning thediversityof P. solanacearum and fundamental aspects of the pathogen
and itsphylogenic
relationships
as revealed by modern techniques of molecular biology, as
well as, host range and geographicaldistribution
in Japan and Southeast Asia.
DiseaseSymptoms

There are numerous commonnames for the diseases
caused by P.
solanacearumdepending on host and locality.
The most commonsymptom on hosts
attacked is wilting, which generally occurs more rapidly in younger plants. Infection
is systemic in the vascular system, producing a wilt in parts or the whole plant. Other
symptoms which may occur, with or without wilting, include browning of vascular
tissue, bacterial exudate from cut vessels, stunting and chlorosis of plants.
The symptoms of the disease on major hosts are;
Tomato: A rapid wilting of the foliage occurs which is particularly
noticeable
duringthe warmest part of the day. Occasionally,
plants are stunted and leaflets and leaf
stalks may curl downwards. To compensate for vascular plugging, infected plants often
form adventitious
roots on the lower stems. A milky exudate is also apparent if the endof the cut stem is placed in water.
Ginger: Wilting and yellowing of the lower leaves occurs, extending upwards until
all the leaves are affected. The stem becomes water-soaked and readily breaks awayfrom the rhizome. Affected rhizomes are generally
darker than normal and whenrhizomes are cut and a little pressure applied, a milky exudate appears.
Potato: A sudden wilting of leaves occurs without preliminary
yellowing.
In affected
tubers, a light- brown breakdown of the water- conducting tissues will be seen and122milky fluid may be squeezed from this discoloured
area.
Tobacco: Symptoms are generally first noticed near the budding stage when leaves
wilt and turn yellow prematurely. Sometimes only leaves on one side of the plant are
affected.In
many cases, only one half of a leaf may show symptoms.
Banana: On young, rapidly growing plants, the youngest three leaves turn pale green
or yellow and collapse near the junctionof the lamina and the petiole. The mostcharacteristic
symptoms occur on young suckers, which have been cut back once andbegun regrowth, become blackened, stunted and may be twisted.
Properties

and Detectionof the Bacterium

P. solanacearum is a non- fluorescent pseudomonad belonging
to rRNA
group II (Palleloni
et al.,1913).
P. solanacearum is a heterogeneous species and has
been divided into either races based on pathogenic specialization,or biovars based on
physiologicalproperties.
Differences
between colony types and levels of virulence are related topresence, absence, or amount of an extracellularpolysaccaride
slime. Mutants of P.
solanacearumcan be detected readily when bacterial suspensions are streaked on anagar medium containing
2,3,5- triphenyltetrazolium
chloride.
Weakly virulent or
avirulent mutants form small butyrous colonies with a distinct dark red color. Two main
butyrous types and several subtypes can be detected.
The virulent wild type forms are irregularly-round,
fluidal, white colonies
which develop light pink centers. The development in culture of avirulent types varies
with storage conditions;the virulent type is maintained readily by storage in sterile
distilledwater. The bacterium has been proved not only to survive but also multiply
in sterile distilledwater (Wakimoto et al., 1982).
SubspecificClassification

Strains of P. solanacearum differ in host range, geographicaldistribution,
pathogenicity,
epidemiologicalrelationships,
and physiologicalproperties.
For the past
three decades a binary system has been in use reflecting two different approaches todifferentiation,
one placing emphasis on host affinity and establishmentof races, theother making of use of selected biochemical
properties
as the basis for separation into
biovars (Buddenhagen
and Kelman, 1964; He et al., 1983).
Three races have been described,
designatedaccording to the pathogenic
specialization; Race 1 affects tobacco, tomato, many solanaceous and other weeds andcertain diploidbananas; Race 2 causes bacterial
wilt of triploidbananas (Mokodisease)and Hellconia ; Race 3 affects potato and tomato.
According to another system five biovars (1, 2, 3, 4 and 5), were designatedon the basis of the ability
to use and/or oxidize three hexose alcohols and threedisaccharides(Hayward, 1994, Table 1). Although
the biovar characteristics
are in

123
Table

1.

Differentiationof biovars

Property

BiovarU t i l i z a t i o n o f t dmrs uaeo nhrl cnab il to os le .

ox ida t ion of+of Pseudomonas

1

Biovar‑2

Biovar3

Biovar+3l4

Biovar5+3

ml a lc t o s e

Based on the c edataand Doudoroffl l o b iofo s ePalleroni
G a sandfr oHayward(1994).
m n i tra te( dUncommon
e n i t r i f i cisolates
a t ion )give a positive
reaction.

Uncommon isolates2

solanacearum.

give

a negative(1971),He etal. (1983)

reaction.

themselves insufficient to provide a reliablebasis for taxonomic separation, it is clearthat biovar 3 and 4 are more nutritionallyversatile
than those of biovars 1 and 2(Palleroni
and Doudoroff, 1971), and biovar 1 and 2 are distinct from biovars 3, 4, and5 on the basis of DNA and RFLP analysis (Cook, Barlow and Sequeira, 1989).
The biovar system has been widely used and in general has proved to be
reproducible
in different laboratories.
He et al. (1983) obtained a series of isolates frommulberry (Morus alba L.) in China, and these were designatedbiovar 5. The
relationshipbetween host specializationand phenotype is most clear between race 3,
the potato race, and biovar 2. In general race 3 and biovar 2 are equivalent.
Biovar 2
has a limited host range and is found mainly on potato and sometimes on tomato anda few weed hosts.
Recently, Ozaki and Kimura (1992) has proposed another grouping system
for P. solanacearum isolates on the basis of pathogenic
specialization
to fourdifferential
Solarium species. Based on this system, P. solanacearum proved to begrouped into five pathogenic groups.
Geographical

Distribution

The geographical distributionof biovars suggests they have separate origins.
In general, biovar 1 is predominant in the Americas and biovar 3 in Asia. Biovar 2 has
a limited host range and is found mainly on potato and sometimes on tomato and a few
weed hosts ; many strains are adapted to growth and are pathogenic at low temperatures, and have a limited capacity for survival in fallow soil compared to biovar 3. The
distribution
ofbiovar 3 is part of a wider picture in which strains of P. solanacearum
biovars 3, 4, and 5 (race 1, 4, and 5) predominate
in Asia and much of Africa and124biovar 1 (race 1 and 2) in the America (Hayward, 1991).
This is consistent with the concept that pests and pathogens are most variable
near the center of origin of their primary hosts, and also with the proposal of Martin
et al. (1982) that the genetic diversity of /! solanacearum decreases with distance fromthe equator.
Host Range
The host range of P. solanacearum is exceptionallywide; there are many
hosts of economic importance, as well as many weed hosts. Several hundred species
representing 44 families of plants, including monocotyledons
as well as dicotyledons,
and many newly recognized
hosts (Hayward, 1994) have been recognizedsinceKelman (1953)
reviewed the subject.
In 1953 only 33 families
had hosts of P.
solanacearum and most hosts were in the Solanaceae,
Musaceae, Asteraceae andFabaceae families.
There are several apparent anomalies in the distributionof bacterial wilt oncertain hosts.
Relatively
few monocotyledonsare affected by bacterial
wilt. Out of 97
families of monocots, nine or 9.3% are reported to include hosts of P. solanacearum
such as the Cannaceae, Heliconiaceae,
Musaseae, Strelitziaceae
and Zingiberaceae.
The economicallymost important hosts are banana and plantain (Musa L. spp.) andginger {Zingiber officinale Roscoe), which is reported
to be severly affected bybacterial wilt in China, Indonesia, Malaysia, the Philippines
and Thailand (Hayward,
1994 ; Buddenhagen,1961).
The status and distributionof bacterial
wilts of banana, including
race 1strains of P. solanacearum affecting banana and abaca, race 2 on banana (mokodisease),blood disease of banana and other Musaceae in Indonesia,
and bugtok andtapurokdiseasesof banana in the southern
Philippines(Eden-Green
and
Sastraatmadja,
1990) are considered in detail by Eden-Green (1994).
Bacterial wilt of ginger is widespread and exceedingly
destructive
in several
countries including Indonesia, China, Malaysia, Philippines
and Thailand. All isolates
from naturally infected ginger have proved to be either biovar 3 or 4. There appear to
be strains of different virulence and pathogenic
specialization.
There are a few records of bacterial
wilt on bird- of- paradise {Strelizia
reginae Banks), the disease was reported in Shizuoka Prefecture, Japan and Taiwan aswell as in Hawaii (Quinon and Aragaki, 1963). In all instances on this host, the isolate
have proved to be biovar 3 and race 1.
Recently bacterial wilt has been reported on several leguminous hosts, suchas cowpea {Vigna sinensis Savi) in the Philippines,
Phaseolus vulgaris L. in Malaysia,
and winged bean {Psophocarpus tetragonolobus DC) in Malaysia and the Philippines
were described in southeast Asia. The pathogen is biovar 3 or 4.
Bacterial
wilt of cucumber has been recently reported in Okayama, Japan

125(Date et al., 1992). This is the first report of P. solanacearum on a Cucurbitaceae.
Bacterial wilt on strawberry (Fragaria L spp.) has been reported only from Japan andTaiwan, although strawberry is grown in many other countries where P. solanacearum
is present. Strawberry is regarded as a host tolerant to bacterial
wilt. In Japan the
disease is found in younger strawberry seedlings in nurseries, but is rarely manifest in
mature plants growing in the field, which neverthelesscan act as symptomless carriersof the disease (Kawaguchi et al, 1981). All isolates from strawberry have proved tobe either biovar 3 or biovar 4.
An important development has been the recognitionof several new tree andshrub hosts including examples of wilts on perennial fruit and nut crops, long known
as diseases of economic importance, but only recently diagnosed as being caused byP.solanacearum.
P. solanacearum biovar 3 has been describedon several woody perennial
hosts, including
cashew {Anacardium occidentale L.) in Indonesia(Shiomiet al,
1989), custard apple {Annona L. spp.) in Taiwan.
Bacterial
wilt of cassava {Manihot esculenta Crantz)
caused by P.
solanacearum biovar 3 and 4 has been reported only in Indonesia(Nishiyama
et<z/.,1980).
The botanicallyrelated weed host, Croton hirtus (Euphorbiaceae)
wascommonin areas showing the highest incidence of bacterial
wilt, may serve as primarysources of inoculum and as bridging hosts at times when cassava is not being cultivated(Machmud, 1986).
StrainDifferentiation

by RFLP Analysis

Nucleic acid probes offer a new approach for the identificationand detectionofP. solanacearum, without the need to test the host range. However, the developmentof a DNA probe specific for P. solanacearum is complicatedbecause this species
shows a high degree of genetic diversity
as mentioned above. Some nucleic acidprobes, which may not be specific, hybridize
to different
lengh restriction
fragments
in different strains. However, restriction fragment length polymorphism (RFLP) analysis
has been found useful to distinguishdifferent
types and to verify the race or biovar of
P.solanacearum (Cook et al., 1989, 1991).
From the calculationof similarity
coefficientsa phenogram for P.
solanacearumwas constructed (Fig. 1). The characteristic
most strongly correlated with
RFLP data was geographicalorigin of the strain. About 97% of the strains fromdivision
II are from the America, while 92% of the strains from division
I are from
Australia and Asia.
On the other hand, all the major branches predictedby cluster analysis of the
RFLP data correlate with either the race or biovar system of classification.
The primarybranch in Fig. 1 separates the species into biovar 3/4 group (divisionI) and the biovarl/2
group (divisionII).
The RFLP analysis of the 90 selected strains of P. solanacearum, differing

126

Fig.

1. Average linkage
clusteranalysisof 35 multilocus
genotypes.
Division
I: race 1 biovars
3,4 and 5. Division
II: biovar(race 1 and 2) and biovar
2 (race 3). (Cook et al.,
1989)

1

in geographicalorigin and isolated hosts collected from Japan and Southeast Asia wascarried out.
The RFLP patterns of the 26 of bacterial
strains, consistingof three 3 ginger
and 3 sesame strains from Thai, 5 cassava, 2 tomato, 1 ginger and a Croton hirtus
strain from Indonesia,
2 strains from China, and 5 strains from Japan as well as thetype strain from USA and 3 of blood disease bacteria of banana (BDB), a bacterium
related to P. solanacearum, were compared.
They were associated with 19 RFLP groups and a phenogram was constructedon the basis of similarity
coefficient
calculations
according to the formula of Nei andLi(1979).
The strains tested were divided into three major clusters (Fig. 2).
Cluster 1 comprised the Japanese race 1/biovars
1 to 4, and Chinese and all
strains from Southeast Asia.
Cluster 2 includes the type strain from the USA.
Cluster 3 included Japanese race 3 and BDB strains.
Average similarity
between Japanese race 1 and race 3 was 22.5%, while thatof strains among race 1 was 74.7%.
On the other hand, sixty four Japanese race 1 strains were divided into three

127

Fig. 2. Dendrogram displaying
the relationshipamong 26 strains
P. solanacearum and Blood disease bacteria (BDB).

ofmajor cluster groups ; Cluster 1 was subdivided
into 4 groups, and contained all strains
belong to biovars 1 and 4, and most strains of biovar 3.
Cluster 2 included all of biovar 2 and biovar 3 strains isolated from tobacco.
Cluster 3, on the other hand, comprised biovar 3 from Strelitzia reginae (Fig.
3).

Conclusions
The results mentioned above were concluded as follows;
Strains of Rsolanacearum distributed
widely in Japan and Southeast Asia
proved to be variable both in pathogenicity(races) and physiologicalproperties (biovars).
There are also several anomalies in the occurrence of bacterial wilt on certain hosts
such as strawberry, casava and cashew in some parts of the region.
Comparison of RFLP analysis of the strains from Japan and Southeast Asia
showed that the geographicalorigin was strongly correlated with RFLP data rather thanrace and biovars of the strains.

128

Fig. 3. Dendrogram displaying
the relationshipstrains of solanacearum.

P

closelywithof Japanese

race 1

Japanese
race 3 strains
were distinct
from those of race 1, and they
relatedto the type strain from the USA or BDB strains.
Clusteranalysisof the strains
belongingto Japanese
race 1 correlated
biovars
rather than with regional
origins.

were
well

References
Buddenhagen, I. W. 1961. Bacterial wilt of bananas: history and known distribution.
Tropical Agriculture
38:107-121.
Buddenhagen,I. W. and Kelman, A. 1964. Biologicaland physiologicalaspects of bacterial wilt
caused by Pseudomonassolanacearum. Ann.Rev. Phytopath. 2: 203-230.
Cook, D., Barlow, E. and Sequeira, L. 1989. Genetic diversityof Pseudomonas solanacearum'.
detectionof restriction
fragment length polymorphismsthat specify virulence and thehypersensitive
response. Molecular Plant-Microbe Interactions
2:1 13-121.
Cook, D., Barlow, E. and Sequeira, L. 1991. DNA probes as tools for the study of host- pathogen
evolution: the example of Pseudomonassolanacearum.In Advances in Molecular Genetics
of Plant- Microbe Interactions,Vol. 1, Hennecke, H. and Verma, D. P., eds., Kluwer,

129
Dordrecht, The Netherlands,
pp. 103-108.
Date, H., Ozaki, K., Shirakawa, T., Nasu, H., Hatamoto, M. and Okamoto, Y. 1992. Bacterial wilt of
cucumber plant grafted on pumpkin rootstock caused by Pseudomonas solanacearum «-Ann.
Phytopath.
Soc. Japan 58 : 220-227.
Eden- Green, S. J. and Sastraatmadja,
H. 1990. Blood disease present in Indonesia.
FAO Plant Protection
Bulletin
38 : 49-50.
Eden- Green, S. J. 1994. Diversity of Pseudomonas solanacearum and related bacteria in South East Asia:
New directionsfor Moko disease. In Bacterial
Wilt: The disease and its causative
agent,
Pseudomonas solanacearum^ Hayward, A. C. and Hartman, G. L., eds., CAB INTERNATIONAL, United Kingdam, pp. 25-34.
Goto, M., Shiramatsu,
T., Nozaki, K. and Kawaguchi, K. 1978. Studies on bacterial wilt of strawberrycaused by Pseudomonassolanacearum (Smith)
Smith. I. Strains of the pathogen
and disease
tolerance of strawberry plants. Ann. Phytopath.
Soc. Japan 44 : 270-276.
Hayward, A. C. 1991. Biology and epidemiologyof bacterial
wilt caused by Pseudomonas solanacearum.
Annu. Rev. Phytopathol.
29 : 65-87.
Hayward, A. C. 1994. The hosts of Pseudomonas solanacearum. In Bacterial Wilt: The disease and its
causative agent, Pseudomonassolanacearum^Hayward, A. C. and Hartman, G. L. , eds., CAB
INTERNATIONAL, United Kingdom, pp. 9-24.
He, L. Y., Sequeira, L. and Kelman, A. 1983. Characteristics
of strains of Pseudomonas solanacearum
from China. Plant Disease 67: 1357-1361.
Kawaguchi, K., Ohta, K. and Goto, M. 1981. Studies
on bacterial
wilt of strawberry
plants causedby Pseudomonas solanacearum 2- D- Glucogallin,
the antibacterial
subdstancedetectedin the tissues of strawberry plants. Ann. Phytopath.
Soc. Japan 47: 520-527.
Kelman, A. 1953. The bacterial
wilt caused by Pseudomonas solanacearum. North Carolina Agricultural
Experiment Station Technical
Bulletin
99: 194.
Machmud, M. 1986. Bacterial
wilt in Indonesia.
In Bacterial Wilt Disease in Asia and the South Pacific.
Persley, G. J., ed., ACIAR Proceedings
13, ACIAR, Canberra, pp. 32-34.
Martin, C, French, E. R. and Nydegger,
U. 1982. Strains of Pseudomonas solanacearum affecting
Solanaceae in the Americas. Plant Disease 66: 458-460.
Nei, M. and Li, W. 1979. Mathematicalmodel for studying
genetic variationin terms of restriction
endonucleases.
Proceedingsof the National Academy of Sciences, USA 76: 5269-5273.
Nishiyama,
K., Achmud, N. H., Wirtono, S. and Yamaguchi,
T. 1980. Causal agents of cassava
bacterial
wilt in Indonesia.
Contributions
CentralResearch Institutefor Agriculture,
Bogor 59: 19.
Ozaki, K. and Kimura, T. 1992. Crouping of Pseudomonas solanacearum on the basis of pathogenicity
to Solanum plants. The Bulletinof the Chugoku National Agricultural
Experiment
Station
10: 49-58 (in Japanese).
Palleroni,
N. J. and Doudoroff,
M. 1971. Phenotypiccharacterizationand deoxyribonucleic
acid
homologies
of Pseudomonas solanacearum. J. Bacteriol.
107: 690-696.
Palleroni, N. J., Kunisawa, R., Contopoulou,
R. and Doudoroff, M. 1973. Nucleic acid homologies
in thegenus Pseudomonas. Int. J. Syst. Bacteriol.
23: 333-339.
Quinon, V. L. and Aragaki, M. 1963. Bacterial
wilt of bird- of- paradise caused by Pseudomonas
solanacearum. Phytopathology
53: 1 115-1 116.
Shiomi, T., Mulya, K. and Oniki, M. 1989. Bacterial
wilt of cashew (Anacardium occidental)
caused by
Pseudomonassolanacearum in Indonesia.
Industrial
Crops Research Journal 2: 29-35.
Wakimoto, S., Utatsu, 1, Matsuo, N. and Hayashi, N. 1 982. Multiplicationof. Pseudomonas solanacearumin pure water. Ann. Phytopath.
Soc. Japan 48: 620-627.

131

Questionsand Answers Session 3

Q.
Dr. He and Dr.Tsuchiya , you have reported wide diversity among strains ofPseudomonas
solanacearum.Can you give ideas for possible approaches or strategies
that can be taken to control this disease?
Would plant breeding resistance help? (delaCruz)
A.
Bacterial
wilt caused by P. solanacearum can be reduced by integrated
control, especially
importantare cultural control measures. Also, breedingforresistance has been successfully
applied to control the disease on peanuts, tomato andother crops. Transgenic plants and bioengineering
microorganisms using antibacterial
protein genes are expected to provide new approaches in controlling
this destructive
disease. (He)A.
Development of resistant
plants would be the most desirable
method, if
possible.
Other methods, such as biologicalcontrol using antagonistic
flourescent
Pseudo- monads is another approach. Heating by solarization
has been used to control
some biovars of potato strain 3 in some regions of Japan. (Tsuchiya)Q.
I have heard that avirulent
commenton that Dr. He? (Kaku)

mutants have been used for biocontrol.

Could youA,
Wehave tried to use spontaneous avirulent mutants and avirulent mutants
derived from Tn5 mutagenesis of Pseudomonassolanacearumto control bacterial wiltof peanut and tomato. Trials in the greenhouse gave good results, but field trials did
not show the expected effectiveness.
This work is continuing.
(He)Q,
Dr. Tsuchiya, have you used RFLP analysis without using probes? We wereable to visualise
genes with multiple copies, by staining with ethidium
bromide. In
basidiomycetes,
especially,
mtDNA and rDNA could be observed clearly in this way.
(Yanagi)
A.
So far we have not tried that. Hopefully
we will try that method in the near
future. We would appreciate collaborationon that. Thank you. (Tsuchiya)Q,(Suzui)Dr. Tsuchiya,

why does Pseudomonas
solanacearummultiply

A.
This has not been clarified(Tsuchiya)

yet, despite

researchin pure water?

on this issue since

1953.

C.
Dr. Tsuchiya, I noticed that you did not include isolates from Malaysia inyour presentation. I would be happy to help you collect isolates from banana, tobacco,

132

tomato etc. from Malaysia.

(B. Salleh)A.
Thank you. I hope we can do this so our experiments(Tsuchiya)

can be more complete.

Q.
(Gamo)

for P. solanacearum?.

What are the criticalgenes controlling

pathogenicity

A.
So far critical genes controlling
pathogenicity
have not been determined.
However, there are some reports on the possible
relatednessof genes for HR(hypersensitive
reactions).
(Tsuchiya)
Q.
(Vaughan)What are the restrictions

A.

None. (Tsuchiya)

on the internal

movement of pathogens

in Japan?

TECHNICALREPORTS

Session 4

Plant

Pathogenic

Chairpersons
Hiroshi Yaegashi
Li-Yang He

Fungi

Diversityof Pathogenic

BAHARUDDIN SALLEH
School of Biological
Sciences,
1 1800 Penang MALAYSIA

Universiti

and Toxigenic
Sains Malaysia

Fusaria in Southeast

Asia

(USM)

Abstract
Our intensive studies since 1977 have shown that Fusarium spp. are associated with a wide
range of host plants and agricultural
products and naturally infested soils in Southeast Asia (SEA). They
are also carried by several imported plants and feeds. Some of them are responsible
for serious losses in
economically important crops. Only a few isolates, however, have been isolated from immunity-deficientanimals and humans. Some have also been shown to produce harmful mycotoxins. The need for accurate,
rapid and affordable
identification
services of such an important group of fungi by the SEAsian scientists
has been increasing. To date, more than 3,500 Fusarium accessions, mostly associated with diseased plants,
have been isolated, identified
into 20 species, and preserved at the Fusarium Culture Collection
Unit,
Universiti Sains Malaysia (USM). The Unit serves the region in identification,exchange and maintenancesiFusarium cultures, as well as providing training and references related to the genus. Some progress andfuture research in taxonomy, plant pathology
and toxigenicity
of fusaria in SEA are briefly discussed.

Introduction
Fusarium is an ubiquitous
genus of imperfect fungi widely distributedinsoils and organic substrates
the world over. However, Fusarium is wellknown for
causing serious losses to crops. In Southeast
Asia (SEA), Fusarium has become athreat to farmers by causing serious losses to their crops (Benigno & Quebral, 1977;
Giatgong,
1980; Lim, 1982; Salleh,
1994; Semangun, 1992; Singh, 1980). Some
species have been found to produce mycotoxins causing both animal and human
toxicoses (Joffe,
1986; Marasas et al., 1984; 1988; Salleh & Safinat, 1994; Savard etal., 1990). Still, a number of species can cause opportunistic
human infections (Salleh& Safinat, 1994; Salleh & Strange, 1988). Nevertheless,many species are commonsoil
saprophytes(Ho & Varghese, 1985; Lim, 1972a; b; Lim & Chew, 1970; Lim &Varghese, 1977; Nelson et al., 1983; Salleh & Zunaidah, 1984; Varghese, 1972).
In this paper the diversityof plant pathogenic
and toxigenic fusaria will be
discussed
in relation
to Brunei Darussalam, Indonesia,
Malaysia,
Philippines,
Singapore and Thailand. Publishedreports on pathogenic and toxigenic fusaria in other
SEAsiancountries
are very scarce and generally
unavailable.
Systematic
mycogeographicalsamplings remain to be done across the whole of Southeast Asia.
Fusariumspp. were isolated following standard methods only from diseaseassociatedsources i.e. naturally
diseased plants (Salleh& Sulaiman, 1984), soils of
infected plants (Salleh & Zunaidah, 1984), humans, animals, and contaminatedfeeds
and food (Zulkaflee& Salleh, 1989; 1990).
Some disease- associatedFusarium
cultures were sent by researchers
and institutions
in SEA for identification
andmaintenance. Single- spored isolates on potato sucrose agar (PSA), potato dextrose

136

agar (PDA) and carnation leaf agar (CLA) plates were identified
following
Booth(1971),
Burgess et al. (1994)
and Nelson et al. (1983)
and other recently publishedpapers describing
new Fusarium species. In order to maintain their virulence,theisolates were preserved in liquid nitrogen (Salleh,1989), sterile sandy loam soil (Salleh& Sulaiman, 1984), and silica gel (Windels etal, 1988; 1993). Extraction,
analysis andbioassay of mycotoxins produced by fusaria were carried out as described elsewhere(Salleh& Safinat, 1994; Salleh & Strange, 1988; Savard etal., 1990; Sazlina, 1994).
Diversity

and Culture

Collection

The History of Fusarium taxonomy has been discussed in detail by Nelson(1991).
It began with the descriptionof the genus by Link in 1809 which was based on
the presence of fusiform non- septate conidia borne on a stroma. The publicationof DieFusarien by Wollenweber and Reinking (1935)
became the foundationof the present
system of classification.
Later, several systems of classification
which were based onmorphology of the macroconidia and mode of production of conidia were introduced
in different
parts of the world. Some more recent systems of classifications
are socomplex they are nearly impossible
to follow. However, the genus Fusarium was
reduced to merely 9 species by Snyder & Hansen (1940, 1941, 1945, 1954). Currently,
the taxonomy of such an important genus is somewhat confusing and more than 1,500
species names can be found in the literature.
So far, the most comprehensive taxonomic
treatmentof tropical fusaria was conducted by Gordon (1960).
The fusaria isolated
from disease associated sources in SEA, are quite diverse in terms of species and host
preference (Salleh, 1994; Salleh & Mushitah 1991a; b). So far, at least 20 species havebeen identified
and accessioned (Table 1). However, some of the doubtful
Fusarium
species i.e. F anguioides, F arthrosporioides, F batatatis, F batatas, F bulbigenum,
F. coeroleum,F cubense, F incarnatum, F javanica, F javanicum, F neoceras, F
orthoceras, F rostratum,F sarchochroum,and F zonatumare commonly cited in theliterature
as occurring in SEA (Benigno & Quebral, 1977; Giatgong,
1980; Lim, 1982;
Semangun, 1992; Singh, 1980). The presence of F avenaceum,F culmorum,Fgraminearum, F nivale, F sporotrichioides and F subglutinans in SEA arequestionable
as these species have never been authentically
documented for the tropics.
For Malaysia
alone at least 12 species
i.e. F beomiforme, F bugnicourtii, F
camptoceras,F chlamydosporum,F compactum,F decemcellulare, F dimerum,F.
longipes, F nygamai, F. proliferatum, F polyphialidicum, and F scirpi, have beenreported since 1977. Most of the fusaria interceptedfrom agricultural
products in the
ASEAN countries have not been identified
to the species level (Tables 2 and 3).
Therefore, the need for a correct, quick and also affordable
identification
service forplant pathologists,
plant breeders, mycotoxicologists
and other applied mycologists
in this region is pressing. This need is further enhanced since most of the isolates sent
to the international
culture collection centres have been destroyed, mainly due to space
limitation.
In addition, the identification
fees are also unaffordable
to many institutions
inSEA.

Table1. Fusarium species isolated from disease-associatedsources and depositedat the Fusarium Culture
Collection
Unit School of Biological
Sciences, USM until December 1994

S p ec ies

S ectio n

P e rcen ta g e

S o u rc e

d im eru md ec em cellu la reE u p io nn o te s
S p icario id e s

0 .1
0 .5

S o il.
D u rian , eg g plan t, m ang o , so ila nd v an illa .

so la n i

M a rtie lla

2 4 .4tum id um
bitg n ico u rtii

M a rtie lla
M artiella

0 .4
0 .1

B ean sp ro ut, c assav a, eg g plan t, h u m an ,jack fru it, lad ies fin g er,
co rn , m a ng o , o il p alm , on io n, p ap ay a, p ep p er,p o ta to , rice, so il,
su g arcan e, w a ter m e lo n.
C oc o a, ru b be r an d soil.
M a ng ro v e, to b acc o a n d w aterm elo n.

o xy sp or um

E le g an s

2 4 .0

A spa rag u s, ba na na, c assava , ch illy ,C hin ese c abb a ge , cit ru s, co co n u t,
co ffe e, co tto n, d u rian , garlic, g ing er, k ang k u ng , lem o n g rass, lo ng
be an ,c o rn , m an g o , m elo n , o il p alm , on io n, o rch id, p a ssio n fru it,

0 .5

p hy sa lis, p o tato , p u m p k in , red b e an ,rad ish, rice, silk w o rm ,so il, starfn iit,straw be rry, su ga rc an e, to m a to , v anilla, w aterm e lon ,w ee d s a nd ya m .
A sp ara gu s, co rn, p ine ap ple and to b ac co .

L a te ritiu m

0 .2

E g g p la nt, lo n g b ea n, co rn, m an g ro v e, rice an d to ba cc o .

L ise ola
L ise o la

2 .0
6 .9

A sp ara gu s, b an an a, feed s, g arlic, c o rn , m a ng o , p in ea p ple , ric e an d so il.
A sp ara gu s, b an an a, chilly, c o co nu t, c orn , m an g o , o rc hid , ric e, so il,be om if o rm e

?

0 .1

ny g a m a i

?

1 5.1

A sp ara gu s, b an an a, ch illy, fire w o rm , grap es, m ang o , o rch id , ra tta n,

p o ly p h ia lid icum
p a llid o ro seu m?
A rth ro sp o rie lla

0 .1
16 .7

ric e, sap o dilla, so il, so rg hu m , sug a rc ane ,to ba cco , w ate rm elo n an d w h ea t
S o il.
A sp arag us, b an an a, cassav a, ce m p ak a, c hilly, Ch ine se ca bb ag e ,ca m p to ce ra s
A rth ro sp o rie lla
ch la m y do sp o rum S p o ro tric hiella

0 .2
0 .8

co co n u t, co ffe e, eg g p la nt, fi re w o rm ,g reen g ra m , in sects, lo n g b ean ,co rn, m ang o , m ang ro ve ,m elo n , m u lbe rry , n utm eg, o il p alm , o n io n ,p ap aya , pa prik a, pa ssio n fru it, p ea nu t, p u ru t lim e , ra d ish , ric e, sap o d illa,
so il, so rg h u m , soy a b ea n, sp inac h, sta rfru it, stra w b erry , su g arc an e, tea,
to b acc o, tom ato a nd w aterm e lo n .
A sp arag us, m a ng o , m u lb erry, rice a nd so il.
A sp aragu s, b an an a, bro ad be an ,h u m a ns, m a ng o , rice, so il, so rg h u m

g ra m in um
co n co lo r
scirp ieq u iseti

0 .1
0 .2
0 .2
4 .1

an d to ba cco
S o il and v an illa .
M ang ro ve ,rice a nd to b acc o .
A sp arag us, m an go , ri c e, so il a nd to b acco .
A sp arag u s, b an an a, bro ad be an ,b ro cco li, chilly ,c hin ese cab b ag e,

2 .0

co c on u t, lo ng b ean , m u lb erry, p ap ri k a, p ea nu t, ri c e, so il, so rg h u m ,so ya b ea n, su g arc ane , to ba cco , to m ato , v an illa a nd w ate rm elo n .
A sp arag u s, b an an a, bitter g o u rd , ca ssa va ,c uc u m b er, lo ng b ea n, m elo n ,oxy sp o rum
va r.r ed o len sla teritium

E leg an s

m o n ilif orm ep ro Uf era tu m

so rg hu m , su g arcan e, to b ac co ,v a nilla, w aterm e lo n an d w h ea t.

lon g ip es

R o seu m
G ib b o su m
G ib b o su m
G ib b o su m

G ib b o su mo ilp alm , rice , so il, soy a b ean , to b acc o a nd w a te rm elo n .

Undoubtedly,
the scope of microbial
collection
activities
to agriculture,industry and research in SEA is wide ranging. We (particularly
at the School of
Biological
Sciences, USM) have started to study the diversityand various aspects ofplant diseases caused by and mycotoxins produced by fusaria. The activities
aremanifested
through the establishmentof a Fusarium Culture Collection
Unit whichTable 2. Fusarium species interceptedfrom imported vegetables
into ASEAN countries (Source: Ganapathi& Chew, 1989;
Sittichai,
1989)

Table

3. Fusarium species isolated from leguminous
cover crops in Malaysia (Source: Ganapathi& Chew, 1989)

serves Malaysia and the region in preserving fusaria collected from various sources(e.g. plant, animal, and human diseases; food and feeds, including those interceptedatthe port of entries),
in identification,in providing
isolates for teaching, research anddevelopmental
purposes, in compiling
publishedreports related to the genus, anddirectoryof researchers actively engaged with Fusarium research so as to enable theUnit to serve as a resource centre. Through this, our understandingon the diversity,

139role and significancedisciplines
in SEA.
Plant

of fusaria

will be improved

for the benefitof various biological

Diseases

SEA is still largely an agricultural
region which produces a number of highvalue commodities and agricultural
products. Plant diseases caused by fusaria in SEA
are becoming more significant
since the introductionof intensive
and high yielding
production
systems and geneticallyuniform cultivars(Salleh,
1994; Waller &Brayford, 1990). However, publishedinformation on plant diseases caused by fusaria
in SEA is still rather sparse and patchy. About 75 % of fusaria accessionedby theInternational
Mycological
Institute (IMI) came from tropical countries and mostly wererelated to agriculturalproblems (Waller & Brayford, 1990). In SEA, severe losses havebeen incurred in some economicallyimportantcrops such as tobacco {Nicotina
tabacum\ slanting death caused by F oxysporumand F solani) (Azmi & Salleh, 1990;
Salleh & Azmi, 1988; 1989; Salleh et al., 1991; Zainal et aL, 1993), rice {Oryza sativa\
bakanae caused by F proliferatum and F nygamai) (Mew & Merca, 1992; Mew &Rosales,
1990;
Saad, 1986;
Salleh& Rosmayati,1988),
asparagus{Asparagus
officinalis\ wilt and rot caused by F oxysporumand F nygamai) (Melor et al. , 1992;
Nik Norulaini& Salleh, 1990; Salleh, 1990; Sapumohotti& Salleh, 1992),
pepper{Piper nigrum\ yellows or slow decline caused by F solani) (Kueh, 1979; Salleh,
1993a; Sarbini & Sumarli, 1985; Turner, 1971; Williams and Liu, 1976), watermelon{Citrullus lunatus\ vascular wilt caused by F oxysporumf. sp. niveum) (Salleh,
1993b;
Salleh & Sapumohotti, 1994), vanilla {Vanillaplanifolia\
stem rot caused by Fusarium
oxysporum)(Soetono, 1962; Tombe, 1985), banana {Musa spp. ; Panama wilt caused
by F oxysporumf. sp. cubense) (Liew et al., 1994; Yaakob, 1991),
passion fruit{Passiflora edulis\ vascular wilt caused by F oxysporumf. sp. passiflorae) (Semangun,
1989), sugarcane {Saccharum officinarum ; pokkah boeng caused by Fusarium spp.),corn {Zea mays\ stalk, ear and kernel rots caused by F moniliforme) (Lim & Pama,
1992; Quebral & Piamonte, 1988; Salleh 1994; Sitepu & Kasim, 1983), and pineapple{Ananas comosus; fruitlet core rot caused by F moniliforme). Although the presenceof the most devastating
oil palm disease, vascular wilt caused by F oxysporumf. sp.
elaeidis, has never been reported in SEA, serious studies have been conducted outside
the region to understand the aetiology,
symptomatology,
and both biotic and abiotic
factors affecting the development of the disease (Ho & Varghese, 1985; 1986; 1987;
1988; Ho etaL, 1985).
Fusaria have also been isolated from agriculturalconsigments imported into
SEA (Tables 2, 3 and 4). During seed health testing conducted by Mew et al. (1989)
in the Philippines,
F moniliforme accounted for 4% of contaminationon outgoing riceseeds while on incoming seeds it was 1.65%. Fusaria, together with 10 other genera offungi, were isolated from coconut samples sufferingfrom coconut wilt from Natuna
Islands, Indonesia(Sitepu& Kasim, 1983). Unfortunately,some planting materials,

140

Table 4. Fusarium species isolated from ornamental
imported into Malaysia (Source: Ganapathi
Chew, 1989)
S

peciesF. pallidoroseum
F. culmorum
F. momliforme
F. oxysporumF. solaniplants&

Sourceleaf blight of anthurium
soft rot of gerbera
leaf rot of Dendrobium sp.
soft rot ofcalla lily tuber
bud rot of mulberry

particularly
ornamental plants do not have to undergo proper post- entry quarantine
procedures in most port of entries in SEA. Unfortunately,some of these plants do carryseveral Fusarium species with a potential
for causing diseases (Salleh,1992). Thesematerials
are infrequently
screened at the importer's premises which are not always
equipped with the minimum facilities
required.
Mycotoxins
Several Fusariumspecies are able to produce mycotoxins e.g. trichothecenes,
butenolides,
moniliformin, zearalenon, fumonisins, and fusarins (Joffe, 1986; Marasas
etaL, 1984; Farber & Sanders, 1986; Savard etaL, 1990). These toxic compounds havebeen
implicatedin various
animaldisorderssuch
as fescuefoot,leukoencephalomalacia,
feed refusal, as well as skin lesions and various haemorrhagic,
emetic and estrogenic syndromes (Tanaka et al., 1986; Ueno, 1983; Vesonder et al ,1978) and human diseases such as aesophageal
cancer, alimentary toxic aleukia, andscabbygrain intoxication(Joffe,1986;
Marasas et al., 1984;
Snyder,1986).
Furthermore, Fusarium mycotoxins have also received much attention
as potential
nonconventional
warfare agents in SEA (Haig, 1982; Mirocha et aL, 1982; 1983;
Rosen & Rosen, 1982; Schiefer,
1982).
Althoughthere has been no clear evidenceof human or animalfusariotoxicoses
in SEA, it was shown that the occurrence of toxigenic
Fusarium
species in this region (Bahari etaL, 1989; Dharmaputra et al., 1993; Salleh & Strange,
1988; Widiastuti
etaL, 1985; Zulkaflee& Salleh, 1989; 1990; 1991) and other placesin the tropics (Joffe,
1986; Marasas et al., 1984) was widespread and that they may
pose a serious health hazard to human, animal and plant populations.
At least 10
Fusariumspecies in SEA are known to be toxigenic (Salleh& Strange, 1988). Strains
isolated mainly from corn in Indonesia, the Philippines
and Thailand produced fusarins
A, C and F as well as fumonisins Bi and B2 (Miller et al, 1993; Savard & Miller,
1992). Sporadic incidencesof human eye keratitis
was observed on patients admitted
to the hospitals
in SEA (Salleh et al., 1994). The most recently discovered fusariotoxin,

141

produced in rice culture by F chlamydosporumisolated from rice seeds in Malaysia,
wasidentified
as chlamydosporol(Savard et al. , 1990). F nygamai and F solani arecommonseeds contaminants and also frequently isolated from patients suffering fromeye keratitis and onchomycoses in Malaysia (Salleh,
1989; 1992). Fusariotoxicosesarelikely to be more prevalent in SEA, where storage conditions
for agricultural
products
are generally
unsatisfactoryand both temperatureand humidity
are frequently
conducive to extensive fungal growth.
Conclusion

and Future ResearchThis work reported here on Fusarium is only in its infancy with muchresearch and development still ahead. In SEA, pathogenic
and toxigenic
fusaria arequite diverse in terms of species and their hosts preference. However, more systematic
myco-geographic survey is required to further understand this diversity,
particularlyin the remote jungle and non- cultivatedsoils. The present Culture Collection
Unit
continues to serve the region in identification,exchange and depositionof Fusarium
cultures, in providing references and facilities
for sabbaticaland training. More criticalstudies on aetiology,
symptomatology,
distribution
and control of several Fusarium
species associated with plant disease in the SEA are needed. There are at least 10 very
destructive plant diseases caused by fusaria that need to be tackled collectively
in orderto formulate sustainable
control measures. These pathogens could react individuallyor as combined invaders. Still, a number of plant diseases require immediate attention
such as orchid "die- back" and vascular wilts of carnation, cucurbits, ginger, onions,sweet potato, cucumber, and tomato. Other plant diseases caused by fusaria of which
wehave very little knowledge are post-harvest
storage rots, cankers of woody plants
and dry rot of potatoes.
Plant quarantine enforcement officers should continue to be
vigilant to reduce the risk of introducing virulent and toxigenic fusaria, particularly
theoil palm vascular wilt pathogen, F oxysporumf. sp. elaeidis.
The agriculturalsituationin the tropics, including SEA, has changed since
newintensive production systems for increased yields were introduced along with new
geneticallyuniformed cultivars(Waller& Bray ford, 1990). Furthermore,
plantssubjected to stress as a result of disease or pest damages, poor soil fertility
and cultural
practices, as often can be found in SEA, are more susceptible
to Fusarium attacks. The
trend in agriculture
is towards monoculture, shorter and even no crop rotation, highinput of pesticides
and chemical fertilizers.
Generally,
the responsible
Fusarium spp.
on most of the crops in SEA are not new, but their increased significanceis a newphenomenon (Salleh, 1994).
To date, there has been no single clear-cut approach that could successfully
control plant diseases,
particularly
those diseases
caused by Fusarium. Throughexperience, the level of damage caused by these diseases is reduced by manipulationof various environmental
and biologicalfactors,
and in many cases by using a
combination of fungicides,
insecticides
and nematicides.
Crop improvement which
incorporates
aspects of plant disease resistance should be pursued vigorously.
The

142

applicationof soil solarization(Jimenez-Diaz,1991)
and biologicalcontrolof plant
diseasescaused by fusaria,
particularly
vascular wilts using nonvirulent
fusaria,shouldbe considered
for the benefitof farmers
in this region.
Mycotoxins
producedby fusaria should
also be given more attention
in SEA
since the presenceof toxigenic
speciesis widespread(particularly
in feeds)
and their
growth are enhanced
by both higher
temperatureand humidity.
Acknowledgements
I thank
the Director
Generalof National
Instituteof Agrobiological
Resources of Japan for his kind invitationand financial
supportto enable me to present
this paper. The Universiti
Sains Malaysia
and the Governmentof Malaysia
have been
generousfor providing
grantsin conducting
research
relatedto this topics.
I am
indebted
to all my post- and undergraduate
students,
research
officers,
laboratory
andtechnical
assistants,
particularly
Mr. Kamaruddin
Mydin and Miss Wan Faridah
Mydin
for their help.
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Pathogenic
Specialization
Causing Wilts of Cucurbitsof Fusarium oxysporum

FUMIO NAMIKI and KAZUFUMI NISHI
Kyushu National Agricultural Experiment Station

Introduction
The phytopathogenic
fungus Fusarium oxysporumSchlechtend.
: Fr. attacks
many important crops and causes variable symptoms, such as vein clearing, leaf epinasty,
wilting,chlorosis,
necrosis, and absicion. However, individual
pathogenic
strains
withinthe species have a limited host range. Strains with similar or identicalhostranges are assigned to intraspecific groups, called formae speciales (f. sp.). The formae
speciales are distinguishedby the ability of their members to cause a wilt disease on
a limited taxonomic range of host plants (Armstrong and Armstrong, 1981; Snyder andHansen, 1940). Some of the formae speciales are further divided into subgroups, named
races, based on pathogenicity
to a set of differential
cultivars within the same plant
species (Armstrong and Armstrong, 1981).
The forma specialis
concept proposed by Snyder and Hansen (1941) for F
oxysporumwas based on strict host selectivityof the strains. However, notable
exceptions to this rule have been reported. The infection spectra of F oxysporumare
known to be more complex than that described by Snyder and Hansen. For example,
F oxysporum
f. sp. vasinfectumattacks plants of more than one family. Later, this was
also found in other formae speciales,such as batatas, tracheiphilum and apii
(Armstrong and Armstrong, 1981)
This paper focuses on pathogenic specializationof F oxysporumcausing wiltsof cucurbit plants and on genetic relatedness
among their strains inferred from DNA
fingerprinting
with nuclear repetitive
DNA sequences.
Pathogenicityof the Cucurbit

InfectingFormae Speciales

The Cucurbitaceae family includes many important crops. In Japan, more than
10 cucurbit plant species and a number of their cultivars
are grown for foods,
cosmetics, and stocks for grafting (Matsuo, 1989). Corresponding
to these diverse plant
species and cultivars, pathogenic variation of F oxysporumon the cucurbit plants has
been categorized into six formae speciales: forma specialis
cucumerinumon cucumber(Cucumis satfvus L.) (Owen, 1956), forma specialis melonis on muskmelon ( Cucumis
melo L.) (Leach and Currence, 1938), forma specialis
lagenariae on bottle gourd\Lagenaria slceraria (Molina.)
Standley](Matsuo and Yamamoto, 1967), forma
specialisniveum on watermelon \Citrullus
lanatus (Thumb.) Matsum. et Nakai](Armstrong and Armstrong, 1981), forma specialis
luffae on loofah {Luffa cylindrica

wasRoem) (Kawai et a/., 1958),
and forma specialis
momordicae on balsam pear
{Momordica charantia L.) (Sun and Huang, 1983).
The formae speciales pathogenic to cucurbit plants are basically host specificand distinguishedby host species. However, exceptions to the initial concept of forma
specialis
have been reported in these pathogens(Kim et aL, 1993; Martyn and
McLaughlin, 1983; McMillan, 1986; Nomura, 1992). McMillan (1986) reported thatisolates of F. oxysporumwhich were obtained from wilted cucumber plants in the
Bahamas were also pathogenic to muskmelon and watermelon. Similar isolates weredetected by Kim et al. (1993). Martyn and McLaughlin (1983) found that some isolatesof the forma specialis niveum infected some summersquash cultivars. Nomura (1992)
showed that the forma specialis lagenariae
pathogenic not only to bottle gourd but
also to pumpkin and malabar gourd.
To revaluate host specificityof the cucurbit- infecting formae speciales, wecollected the F. oxysporum
strains causing wilts of cucurbit plants from various locationsin Japan, and examined for their pathogenicity
to 10 cucurbit plants (Namiki et al.,1992). Pathogenicityof the F. oxysporumstrains was assayed by a root dip method(Wellmann, 1939) using plant seedlings that had fully expanded leaves. The roots weredipped in a spore suspension (107 conidia per ml) for 15 seconds. The inoculated
seedlings
were transplanted
to plastic pots filled with sterilized
soil and placed in agreenhouse. External symptoms and vascular discoloration
were scored 28 days after
inoculation, and the pathogen was reisolated from vascular bundles of inoculated plants.
The host range of the Japanese strains of cucurbit-infecting
formae speciales isshown (Table 1). Strains of the formae speciales
cucumerinum and niveum were
pathogenic only to their original hosts, cucumber and watermelon, respectively.
These
formae speciales have been reported to contain pathogenic variants which infect cucurbit
plants other than original hosts (Kim et al, 1993; Martyn and McLaughlin, 1983;
McMillan, 1986). However, such variants were not detected in our samples.
Strains of the forma specialis melonis were only pathogenic to muskmelon among
cucurbit plants used. They, however, carried pathogenic variants different in pathogenicity

Table 1 Host range of cucurbit-infectingof Fusarium oxysporum

formae speciales

149

to oriental melon (Namiki et aL, 1993b). The details will be described later.
The forma specialis lagenariae contained three pathogenic variants that differedin pathogenicity
to pumpkin and malabar gourd in addition to the original host, bottle
gourd. These three variants were (i) pathogenic only to bottle gourd, (ii) highly virulent
to bottle gourd and weakly virulent to pumpkin and malabar gourd, and (iii) highly
virulent to all three plants (Namiki et aL, 1992).
The forma specialis momordicae, the causal agent of balsam pear wilt, was foundto cause disease also in bottle gourd, pumpkin, and malabar gourd, showing that the formae
speciales lagenariae and momordicae have commonhost plants (Namiki et ah, 1 992).
The formae speciales that attack related host plants may be closely related geneticallyandshare characteristics necessary for pathogenicity.
On the basis of the cross-infectivity
ofthese formae speciales, the genetic relationships
within and among three formae speciales
have been questioned.
Genetic Relatedness

Among The Cucurbit-Infecting

Formae Speciales

Knowledge of the genetic variability and its distribution within and among formae
speciales is a prerequisite to study their genetic relationship(Brutis h aL, 199 1 ; McDonald
et aL, 1989; Michelmore and Hulbert, 1 987). Over the past several years, genetic diversity
in R oxysporum has been examined using various genetic markers, such as vegetative
compatibility
grouping (VCG) (Leslie, 1 993), isozyme profiles (Bosland and Williams,
1 987), and restriction fragment length polymorphisms (RFLPs) in mitochondrial
andnuclear DNA (Jacobson and Gordon, 1990; Kim et aL, 1992; Kim et aL, 1993; Kistler
etal, 1987; KistleretaL,
1991; Mmicom et aL, 1987; Manicom et aL, 1990). Of theseprocedures, RFLP analysis has the advantage of potentiallydetectingnumerous
polymorphisms at the DNA level.
Recently, DNA fingerprinting
with nuclear repetitive DNA sequences has beenused to distinguish strains of/7! oxysporum belonging to different pathogenic variants orfrom different geographic localities(Bruns et aL, 1991; Kistler et aL, 1991). Thissensitive method was used to examine genetic diversity within the cucurbit-infecting
formae speciales and to determine genetic relatedness
among the formae speciales(Namiki et aL, 1994a). The genomic clones, named FOLR clones, were isolated from the
DNAlibrary oiF. oxysporumf. sp. lagenariae strain MAFF305 1 1 8, and used as probes
for DNA fingerprinting.
FOLR clones were identified to carry moderately repetitive
DNAsequences dispersed in fungal chromosomes. Total DNA of 5 0 strains representing
five cucurbit-infecting
formae speciales and 6 strains pathogenic to plants other thancucurbits was digested with EcoRY and hybridized with 32P-labeled FOLR DNA. Fiftytwo fingerprint types were detected among the 56 strains using all FOLR probes. The
similarity coefficient for all possible pairs of fingerprint types from their hybridization
profiles was estimated by the method of Nei and Li (1979).
A dendrogram wasconstructed fromthe similarity coefficient data by the unweighted pair group method witharithmetic average clustering (UPGMA) (Sneath and Sokal, 1973).
The dendrogram identified six genetic groups within the cucurbit-infecting
strains,

150

corresponding to the forma specialis classification,at the similarity
level of more than 75%(Fig. 1). Each population of the formae speciales lagenariae, cucumerinum, niveum,
and momordicae clustered into a single group. K oxysporum strains pathogenic to plants
other than cucurbits were distinguishedfrom one another and also from the strains of the
cucurbit-infecting
formae speciales on the dendrogram. Parsimony analysis also indicatedforma specialis-dependentgrouping. These results suggest that the cucurbit-infecting
formae speciales are intraspecificvariants distinguishable
at the DNA level and in their
host range.
The formae speciales lagenariae and momordicae have commonhost plants, bottle
gourd, pumpkin, and malabar gourd. However, the strains were easily distinguished,

Fig. 1. Dendrogram showing the levels of genetic relatedness
different formae speciales of F. oxysporum.

of 56 strains

from

et

151

corresponding to their forma specialis classification,
by FOLR DNA fingerprinting;eachpopulationof these formae speciales was grouped into a distinct genetic cluster byphylogenetic
analyses.
The forma specialis melonis population carried two genetic groups that weredistinguishable
from one another on the basis of DNA fingerprint profiles and phylogenetic
analyses. We found that the Japanese strains contained pathogenic variants different in
the infection spectra on Japanese cultivars of muskmelon and oriental melon (Namiki etal, 1992). The 16 strains of the forma specialis were assayed for pathogenicity
to twocultivars each ofmuskmelon and oriental melon (C melo L. var. makuwa). The assay
divided these strains into two groups that corresponded to the groups identified by FOLR
DNAfingerprinting. One group was pathogenic to both muskmelon and oriental melon,
and the other group was pathogenic only to muskmelon. Muskmelon and oriental melon
have different geographic origins and different histories of cultivation in Japan (Matsuo,1989).
Thus, genetic differences between these pathogenic
types could be due togeographic isolation of the pathogen population during their establishment
and prior totheir dispersal throught the world.
Recently it has been demonstrated that hypervariable
repetitive DNA in fungal
chromosomesmaybe useful for differentiationof strains belonging to a particular pathotype
or froma particular locale (Bruns et al, 1991; McDonald
al, 1989; Michelmore andHulbert, 1987). Kistler et al (1991)
used nuclear repetitive
DNA to infer the genetic
relationship amongthree crucifer-infecting
formae speciales ofF oxysporum.The analysis
indicated that a close relationshipexisted among members of the same formae specialis.
These result are similar to our results with FOLR probes to determine genetic relationships
amongthe cucurbit-infecting
formae speciales.
Kim et al (1993) determined the genetic divergence and the relatedness of fiveformae speciales within the cucurbit plants based on RFLPs of mitochondrialDNA. They
identified a close relationship between the different formae speciales and presented evidence
for genetic similarity.
Both cluster and parsimony analyses of the mitochondrial
DNA
RFLPs indicated that all of the F oxysporum formae speciales in cucurbits are closely
related and that in some cases, isolates of different formae speciales were geneticallymoresimilar than isolates of the same forma specialis. On the basis of the complexity of
infection spectra of the formae speciales on cucurbits and the RFLP analysis of theirmitochondrial DNA, Kim et al ( 1 993) hypothesizedthat the genetic differences between
the formae speciales were relatively small and that determinants for host specificity
could
be combined or lost in individual strains. Our results, based on FOLR DNA fingerprintingof nuclear DNA, are not consistent with this hypothesis.
Cluster and parsimony analyses
based on FOLR DNA fingerprinting
readily distinguishedthe formae speciales
suggesting that the cucurbit-infecting
formae speciales of F oxysporum are intraspecies
variants differing not only in pathogenicity
but also in nuclear DNA content andorganization. Kim et al (1993) used strains collected from the United States and a few
other countries, but we used only Japanese strains. It is necessary to measure the genetic
diversity and the relationship among the F. oxysporumstrains on cucurbits collected fromother countries by means of FOLR DNA fingerprinting
and to analyze RFLPs of

Fig. 2. Race classificationof pathogenic groups (Groups
strains oiF. oxysporwnf. sp. melonis.

I to ID) in the Japaneseand to infer genetic relatedness among the races (Namiki et ai, 1993b). Total DNA fromthe 4 1 Japanese strains was digested with EcoRV and hybridized
with FOLR probes.
These probes detected 36 fingerprint types among the 4 1 strains. A cluster analysis of thefingerprint
data was employed to calculate the similarity
coefficients
between thefingerprint types, and a dendrogram was constructed by using UPGMA. The dendrogram
identified four genetic groups. The fingerprint types detected in race l ,2y and race 0 (MOtype) were grouped into distinct single clusters. However, two different genetic groups
occurred in race 0 (M type). These results suggest that pathogenic variation and the otherfactors effect the DNA of the forma specialis melonis population.
Genetic Relatedness
Geographic
Originsamong Strainsof the Forma Specialis

Melonisof Different

We collected strains of the forma specialis
melonis from other countries tocompare pathogenic and genetic variability
among strains different in geographic origins(Namiki et aL, 1993a; Namiki et al, 1994b).
Strains from other countries that havealready been characterized
at race level were assayed for pathogenicity
to two Japanesecultivars of muskmelon and one Japanese cultivar of oriental melon (Table 2).
The U.S.A. race 0 strains were not pathogenic to these Japanese cultivars,
although the Japanese race 0 strains were pathogenic to one or two of the cultivars. The
U.S.A. and Mexican race 1 strains exhibited pathogenicity
to two muskmelon cultivars,
but not to an oriental melon cultivar. The U.S.A. and French race l,2y strains were
pathogenic to the three Japanese cultivars, similarly to the Japanese race l ,2y strains. The
genetic relationships
among the strains were examined by FOLR DNA fingerprinting.
Onthe basis of DNA fingerprint data, a dendrogram was constructed using UPGMA. The
U.S.A. race 0 strains were moreclosely related to the U.S.A. race 2 strains. Race 1 strains
fromU.S.A. and Mexico were not grouped into a single cluster though they had a similar
infection spectra to race differential
cultivars and also to Japanese cultivars. On the other
hand, race l,2y strains from U.S.A., France and Japan were grouped into a single cluster.
Although sample size might not be enough to provide a definitive conclusions, theseresults suggest that genetic variability
within the forma specialis results from the impact

154

Table 2 Pathogenicityof the races of the forma
specialis
melonis different in geographic
origins to the Japanese cultivars
R a ceO rig inra ce OU .S .A .

ra ce OJapan

ra c e O

Ja p a n

rac e lU .S .A .

ra ce lM e xic ora c e 2

U .S .A .

rac e l ,2 y

U .S .A .

ra ce l,2 y

F ra n c e

ra c e l ,2 yJapan

A m usS

S: Susceptible,

C u ltiva r
O hi
O gon 9SR

R: Resistantof coevolution with host plants and geographic isolation.
Molecular characterization of the FOLR sequences remains to be done to provide
more information. FOLR markers are expected to facilitate
the studies of population
dynamics ofF. oxysporumover time and space and of evolutionary dynamics correlated
host plants.
Conclusions
The infection spectra of the cucurbit-infecting
formae speciales have been foundto be complex; in some cases, a single forma specialis infects multiple genera or speciesof the family Cucurbitaceae and contains pathogenic variation; in other cases, different
formae speciales share the same host plants. In addition to pathogenicity tests, FOLR DNA
fingerprinting
provided a basis for evaluating the presented potential evolution ofpathogenic specializationin F. oxysporum, taking into account the impact of coevolution
with host plants and geographic isolation. Characterizationof pathogenic and genetic
diversity within the species F. oxysporum should provide more understanding of thiseconomically important species and contribute to more effective management of thediseases for which it is responsible.

155

Acknowledgments
Wethank T. Tsuge for useful discussions and critically reading this manuscript.
This work was supported by a grant-in-aid for special scientific research on agriculture,
forestry, and fisheries fromthe Ministry of Agriculture, Forestry and Fisheries of Japan.
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Mating
Types,
Isozyme Polymorphism
and Metalaxyl
Sensitivityof Phytophthora infestans Isolated
from Several
Countries in Asia
AKIRA OGOSHI
Faculty of Agriculture,

Hokkaido

University,

Kita-ku, Sapporo

060, Japan.

Abstract
Phytophthora infestans isolates were collected from potatoes and tomatoes in Korea, Indonesia,
India, Taiwan and Thailand from 1992 to 1994 and their mating type, isozyme polymorphism
andmetalaxyl sensitivity were compared to with Japanese isolates. All Korean isolates tested were A2 mating
type. In Indonesian, Thai and Japanese Al and A2 mating types were found. Isolates from India andTaiwan were only Al isolates were found. Isozymes of glucosephosphate
isomerase (Gpi-1),
peptidase(Pep-1) and malic enzyme (Me) were analyzed. Genotypes were different depending on countries andmatingtypes. New populations
were found among Korean, Indonesianand Japanese A2 isolates.
Metalaxyl resistant isolates were found in Korean, Indonesian and Japanese A2 isolates.

Introduction
1. Diseases by Phytophthora infestans.
Phytophthora infestans (Montagne) de Bary has a rather narrow host range.
Only three species are reported as host crops, potato (So/anum tuberosum L.), eggplant(S. melongenaL.) and tomato {Lycopersicon esculentum Mill.). However, the diseaseof eggplant is rarely reported.
The pathogen causes late blight on potato and tomato, and attacks not only
leaves but also stems and tubers or fruits. If the disease is not controlled
almost allplants are killed. Of about 40 kinds of disease on potato reported in Japan late blightis the most important disease.
Late blight of potato is the most serious disease ofpotato in the world. The late blight pathogen can be found wherever potato or tomato
is cultivatedand causes great yield loss.
2. Distributionof A2 mating type.
P.infestans is a species belonging to Pythiaceae, Peronosporales,
Oomycetes,
Mastigomycotina,
Eumycota. This species is heterothallic
and has Al and A2 mating
types. When both mating types are present, the sexual spores (oospores),are formed.
P.infestans is thought to have originatedin Mexico where both Al and A2
mating types are found. However, it had been believed for long time that only Al
mating type was to be found in other potato cultivatingareas.
In 1984 the A2 mating type was reported from Switzerland(Hohl and Iselin,1984).
After that the presence of A2 mating type was reported from Europe, Egypt,Israel, Brazil, North America and Asia.
The presence of A2 mating type lead to several problems in the control of158

this disease.

They are :1) the possibilityof oospore formation in the field,2) persistenceof primary inoculum (oospores)
in fields,
which means P. infestans becomes a soilborne pathogen in addition
to a tuber-borne
pathogen, and3) change of characters (e.g. race) of the pathogen can occur through sexual
recombination.
3. Research objectives
Wehave studied Japanese P. infestans for several years. Based on the resultsof studying
Japanese isolates of P. infestans it was decided to organize a project toextend the study to other Asian countries.
This project was approved by the Ministryof Education,
Science and Culture of Japan (Monbusho)
and a Grant- in-Aid wasgiven.
The objectivesof the project are to clarify :1) whether or not A2 mating type is present in Asian countries,2) whether Al and A2 mating types have been present for a long time or areof recent origin
3) whether populationsof Al and A2 mating types are differs among Asian
countries, and
4) whether or not metalaxyl resistant isolates are present.
For this project a team was organized, which consisted of 12 researchers from
6 countries.
They were:
Lee, Wang Hyu (Chunbok National University,
Korea),
Euis Suryaningsih(Lembang Horticultural
Research Institute,
Indonesia),
Singh, Udai P. (Banaras Hindu University,
India),
Chang, Tun-tschu (Taiwan Forestry Research Institute,
Taiwan),Chiradej
Chamswarng (Kasetsart
University,
Thailand),
Pipob
Lumyong and Somkiat Suwonakenee (Chiang
Mai University,
Thailand),
Kato, M. (Hokkaido National Agricultural
Experiment Station, Japan), andKobayashi, K., Kondo, N., Akino, S. and Ogoshi, A. (Hokkaido University,
Japan).
P. infestans isolates were collected from June to July in 1992 in Korea, from
November to December in 1992 in Indonesia, from January to March in 1993 in India,
from May to June in 1993 in Taiwan and from December, 1993 to January, 1994 in
Thailand.
Studieson Phytophthora

infestans in Asian Countries

1. A2 mating type and its distribution.
In 1987 we found isolates of A2 mating type in Hokkaido, the north islandof Japan (Mosa et al., 1989). This was the first report of this mating type in Japan.

159

From 1988 to 1990 we surveyed R infestans in all Japan and could find A2
isolates in all districts.
A2 mating types were more commonly found than Al mating
type. In southwest part of Japan almost all isolates were A2 mating type.
Althoughwe do not know exactly when A2 mating type isolates
came toJapan, it seems that this occurred in the early 1980s so that by the late 1980s A2mating type became dominant. Frequency of isolation of A2 mating type is increasing
year by year (Table 1).
Wecould find A2 isolates in Korea, Indonesia and Thailand but not in India
and Taiwan, where only Al mating type could be found. All our isolates from Korea
are A2 mating type. A2 is dominant in Korea and Japan, although Lee and So (1993)
found a few Al isolates.
Recently Al mating type from China and Philippineswasreported (Koh et aL, 1994).
2. Colony types.
Japanese isolates of P. infestans can be divided into two types on the basisof colony appearance on potato tuber slices (Kato et al., 1992).
We call themSporangial
type (S-type)
and Mycelial
type (M-type).
S-typehas abundant
sporangiophores
and sporangia (conidia)
and M-type has abundant aerial mycelia andfew sporangia.
The relationshipbetween mating type and colony type is shown (Table 2).
All S-type
isolatesare Al and all M-type are A2. Korean A2 isolates are also
Mycelial
type and Indian Al isolates are Sporangial
type. We have not yet studiedcolony appearance of isolates from other countries.
Table 1. Mating
D istric t

Types of Phytophthora
Nisoo .lao tefinfestans Isolates

in Japan found between 1987-1990.

A I 1 9 8 7A 2

A I 1 9 8 8A 2

A I 1 9 8 9A 2

A I 199 0A 2

26

18

46

45

2 22

29

18 2

20

86

H o k k aid d o

6 10

T oh ok u

13 0

8

2

3

11

K a n to

78

9

13

1

46

C hub u

45

1

5

3

26

K in k i

36

1

3

2

25

C hug ok u

45

2

4

1

37

S h ik o k u

34

0

6

2

13

0

13

K yu syu

1 68

0

20

0

11 8

0

30

T o ta l

1 146

39

99

57

498

52

33 3

26

42

42

160

Table 2. Relationship
Pathogenicity

among Colony Type, Mating Type, andof Isolates of Phytophthora infestans inJapan.
Isolate

C typ
olony
e

M typ
atine g

D N 70 1‑71 0

SA l

C O 7 01‑7 04

SA l

C O 7 05‑70 6

MA2

C O 7 07 ‑70 8

SA l

C O 7 09 ‑71 3

MA2

C O 7 14SA l

C O 7 15

M

A2

C O 7 17SA I

C O 7 18 ‑72 0

MA 2

K K 70 1‑7 03

MA2

K K 70 4

SA l

K K 70 5‑7 06

MA 2

T K 7 01SA l

T K 7 02

MA 2

T K 7 03SA l

T K 7 04‑71 3

MA 2

T K 7 14SA l

K S 70 1‑7 02

MA 2

H O 7 0 1‑702

SA l

H O 7 03 ‑712

MA 2

H S 70 1‑7 02

S

A I

S M 7 0 1‑705

M

A 2

S: Sporangial
R: Resistant,type, M: Mycelial
S: Sensitive

R el actio
v ariety
n of

R

R

type

3. Virulence.
Wetested isolates
for virulence on a susceptible(r) (Danshakuimo)
and aresistant(1) (Rishiri)
potato varieties.
Al isolates
are able to attack only thesusceptible
variety, A2 isolates are able to attacked both varieties(Table 2).
There are eight possiblecombinationsof colony type, mating type andvirulence, that is, S-Al- R, S-Al- S, S-A2- R, S-A2- S, M-Al- R, M-Al- S, M-A2-R
and M-A2- S. However, we found only S-Al-R and M-A2-S (Table 3). Al isolates
are Sporangial
type and cannot attack Rl-variety,while A2 isolates is Mycelial
type

161

Table 3. Combinationof Colony Type - Mating Type

-Virulence
S ‑A l ‑R

and Number of Isolates.
26

M ‑A l ‑R

S ‑A l ‑ S

M ‑A l ‑S

S ‑A 2 ‑ R

M ‑A 2 ‑R

S ‑A 2 ‑ S

M ‑A 2 ‑S

4 4

and can attack Rl-variety.
Recently, we have found a few exceptions to this general
pattern in Japanese isolates.
The virulence of isolates from other countries have notyet been studied.
4. Isozyme polymorphism.
Recently isozymes have been used to analyse pathogen populations
and this
methodology
has been applied to P. infestans, too (Andrivon et al.,1994;
Daggett eta/.,l993;
Koh et a/.,1994; Mosa et a/.,1993; Shattock et a/.,l990;
Sujkowski et a/.,1994;
Tooley et a/.,1989;
Tooley et a/.,l993).
Of many enzymes glucosephosphate
isomerase-1(Gpi- 1) and peptidase1 (Pep- 1) are used for P. infestans, because
polymorphism
exists for these isozymes. We found malic enzyme (Me) is also
polymorphic,
we are therefore analysing for this isozyme as well.
In starch gel electrophoresisof enzyme proteins
allelic
variantsaredesignatedaccording to relative mobility.
One allele, generally the most common,isarbitarily
designated100. Based on this allele, other alleles are then assigned anumerical value according to their relative mobilities.
A summary of isozyme analysis of isolates from Korea, Indonesia,
India,
Taiwan and Japan is shown (Table 4). Isolates from Thailand are currently being
analysed. Gpi-1 genotypes 100/100 and 86/100 were detected.
Al isolates has both
genotypes, while A2 isolates has 100/100 only. These genotypes are said to belong tothe old population.
Analysis of Pep- 1 isozymes showed the following
genotypes,
92/100 and 100/100 for Al and 96/96 for A2 mating types. Genotype 96/96 is said tobelong to the new population which means a newly migrated population.
That is, A2
isolates in Korea, Indonesia and Japan are newly migrated populations.
Genotypes
90/90, 90/100 and 100/100 were detected in Me isozymes. All isolates having 90 are
A2 mating type and 90 also belongs to the new population.
Koh et a/.(1994)
reported
that Chinese, Korean and Philippines
Al isolates had the same genotypes as Japanese
Al isolates.
In summaryAl mating type of these countries is the original (old) population
and A2 mating type is a recently introduced (new) population.
Two new genotypes 90 of Gpi- 1 and 83 of Pep- 1 has been found only in
Mexico and Europe (Table 5). Another new genotype 96 is found in Europe and Asia.
It is surprising that this 96 genotype has not been reported in Mexico, as it is thought

162

Table 4. Genotypes of Isozymes of Phytophthora infestans Isolates
in Asian Countries.
C o u n try

M a tin g ty p e

G p i‑1

P ep ‑1

M e

K o re a

A 2

1 0 0 /1 0 0

9 6/9 6

9 0/1 0 0

A 2

1 0 0 /1 0 0

In d on es ia

A 2

10 0 /1 0 0

9 6/9 6

9 0 /10 0

In d ia

A 1

8 6/1 0 0

92 /10 0

1 0 0 /10 0

T aiw an

A 1

8 6/1 0 0

9 2 /10 0

1 0 0 /1 0 0

Jap anA 1

8 6/1 0 0

1 0 0 /10 0

1 0 0 /1 0 0

A 1

8 6/1 0 0

1 0 0 /10 0

1 0 0 /1 0 0

A 1

1 0 0/1 0 0

9 2 /10 0

1 0 0 /1 0 0

A 2

1 0 0/1 0 0

9 6 /9 6

9 0 /1 0 0

A 2

1 0 0/1 0 0

9 6 /9 6

9 0 /9 0

Original

population:

The recently

86, 100 for Gpi-1, 92, 100 for Pep-1, 100 for Me.

introduced

population:

90 for Gpi-1,

83, 96 for Pep-1,

for Me.

Table 5. Worldwide

Distribution

Isomerase-1

of Genotypes

and Peptidase-1

of Phytophthora infestans.

G p i‑1
C o u n try

86 1 90

100of Glucosephosphate

P ep ‑ 1
83

92

96 10 0M e x ico
P eru
U SA
C anadaIre lan d
U K
T h e N eth e rlan d so

G e rm an y
P o la n d
In d ia
In d o n e sia
P h ilip p in e s
T a iw a n
C h in a
K o re a
Ja p a n

Molecular weight (kDa)
2 Mexico has 83, 98, 111, 122 and 130 for Gpi-1,

too.

90

163

that the new population must have migrated from Mexico (Fry et al.,1993).
As isozymes of Me has been little studied except our study, we cannot yet
make comparisons. Further studies is needed of the Me isozyme.
5. Metalaxyl sensitivity.
In Japan metalaxyl has been used to control potato late blight since 1987.
There were no metalaxyl resistant isolates in P. infestans isolates in 1987 (Ogoshi
ettf/.,1990).
However, in 1989 several resistant isolates were found and the percentageof resistantisolates had increased to 29% by 1991 (Table 6). All highly resistantisolates(ED50>10ppm)
belong to A2 mating type,while
only a few isolates of Al
mating type are intermediately
resistant(ED50>0.1<1). The reason is not known,
though there may be a relationshipbetween mating type and metalaxyl resistance.
Resistant isolates were found in A2 isolates from Korea and Indonesia, but
not found in Al isolates
from India, Taiwan and Thailand
and A2 isolates
fromThailand.
A few isolates
showing partial resistance were found in Al isolates from
Taiwan (Table 7).
Table 6. Increase of Metalaxyl Resistant Isolates in Japan.
A I

A 2

Y e ar19 8 7

23

0

0

198 8

38

0

0

198 9

4 0

454

19 9 0

4 2

208

55

2 9 (10 % )33

18

2 1 (2 9 % )199 1

S: Sensitive,

0

38
97

0

9 (2 % )I: Intermediate,

R: Resistant

Table 7. Sensitivityof Phytophthora

infestans Isolates

C o u n tr y

Mty pa tein giN s uo ml a tbees r teo fs t e d

K o re a

A 2

1 6

In d ia

A 1

12

12

T a iw a n

A 1

42

39

I n d o n e s ia

A 2

T h a il a n d

Ja p a n< 0 .1

0 .1 <

A 1

2 1

2 1

A 2

2 1

2 1

A 1

45

33

10

A 2

294

212

19

to Metalaxyl.
E1 D< '5 0

10<

0

11

39

17

10 0 <

164

Currently
resistantisolates
have been reported
from Ireland,
The
Netherlands, Israel, Greece, England, Wales, Japan, Poland, Germany, India, Mexico,
Canada, USA, France, Korea and Indonesia.
III. Conclusions
and Prospect.
As potato is one of the most important food crops and the late blight is themost important disease on potato, the disease has been the subject of extensive studies.
The recent spread of A2 mating types to many countries and several new genotypes are
newdimensions to this disease.
Further research to characterize
populationsof P. infestans is needed in thefollowing areas.
1) Mating type (Al, A2 or homothallic)2) Colony type (Sporangial
type or Mycelial type)3) Isozyme (allozyme)
polymorphism4) Virulence (race)5) Metalaxyl(and other fungicides)
sensitivity
6) Ploidy (haploid,
diploid,
triploid,
tetrapoidor aneuploid)7) MitochodrialDNA8) Nuclear DNA
9) Double stranded RNA
10) Fertility
Although
we have studied and are studying
some of above items, there
remain many items that we need to study to understand the differences
between Asian
populations
and populations
from other regions.
The ploidy of isolates is another important area of research. Ploidy of isolates
has been studied and hap- , di- , tri- , tetra- , and aneuploids
were reported (Gu eta/.,1993;
Therrien et a/.,1989;
Therrien et a/.,1993;
Tooley et a/.,l989;
Tooley eta/.,1993).
However, we have no data of ploidy of isolates Asian countries except
Japanese (Table 8).
Although analyses of nuclear DNA and mitochodrial
DNA are used for moredetail study of populations(Drenth et a/.,l993;
Goodwin et a/.,1992;
Goodwin eta/.,l992;
S.B. Lee et a/.,1993;
Moeller et a/.,1993;
Panabieres et al.,1989;
Whittaker
et a/.,1994),
we still have insufficient
data on these traits.
We are studying P infestans in several Asian countires but there are big
vacant areas for studying P. infestans, China and eastern Russia. We have to study
Chinese and eastern Russian isolates in relation to the above mentioned items, andundertake comparative analysis.
Through these studies we can understand not only Japanese populations
but
also P. infestans worldwide. This will help us to understand how P infestans migrates
and how to prevent its spread.

165
Table 8. Ploidyof Isolates

C ou ntry

M typatine gPhytophthora
2ninfestans.
P loid3n y

4nM exico
U SA

A l

P eru

A l

B razil
IrelandA l

U K
T h e N eth erlan ds

A l
A 2

Sp ainP o lan d

A l
A 2

E g ypt
IsraelJap anA l
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Mosa, A. A., M. Kato, N. Sato, K. Kobayashi
and A. Ogoshi. 1989. Occurrence of the A2 matingtype of Phytophthora infestans on potato in Japan. Phytopath.
Soc. Japan 55: 615-620.
Mosa, A. A., K. Kobayashi
and A. Ogoshi. 1993. Isozyme polymorphismand segregationin isolates of
Phytophthora infestans from Japan. Plant Pathology
42: 26-34.
Ogoshi,A., A. A. Mosa, K. Kobayashi,
M. Kato and N. Sato. 1990. Distributionof mating typesof Phytophthora
infestans and occurrence of metalaxyl
resistantisolatesin Japan.
In Abst. of Fourth International
Congress (IMC-4),
p. 293.
Panabieres,F., A. Marais, F. Trentin, P. Bonnet and P. Ricci. 1989. Repetitive
DNA polymorphism
analysis as a tool for identifing
Phytophthora species. Phytopathology
79: 1 105-1109.
Shattock,
R. C, D. S. Shaw, A. M. Fyfe, J. R. Dunn, K. H. Loney and J. A. Shattock. 1990. Phenotypesof Phytophthora
infestans collected
in England and Wales from 1985 to 1988: mating type,response to metalaxyl
and isoenzyme analysis. Plant Pathology
39: 242-248.
Sujilowski,
L. S., S. B. Goodwin, A. T. Dyer and W. E. Fry. 1994. Increased
genotypic
diversity
via migrationand possible
occurrence of sexual reproductionof Phytophthora
infestansin Poland. Phytopathology
84:201-207.
Therrien,
C. D., D. L. Ritch, L. C. Davidse, L. Spielman
and B. K. Jespers. 1989. Nuclear DNA
content,
mating type and metalaxyl
sensitivityof eighty- three isolatesof Phytophthora
infestans from The Netherlands.
Mycol. Res. 92: 140-146.
Therrien,
C. D., D. L. Ritch, L. S. Sujkowski,
L. Spielman,
W. E. Fry, S. S. Daggett,
J. Sim and
P. W. Tooley. 1993. Phytophthora
infestans in Poland from 1987- 1989; Nuclear DNA
content, mating type distributuin
and response to metalaxyl. J. Phytopathology
139: 68-80.
Tooley,P. W., C. D. Therrien and D. L. Ritch. 1989. Mating type, race composition,
nuclear DNA
content, and isozyme analysis of Peruvian isolatesof Phytophthora infestans. Phytopathology
79:478-481.
Tooley, P. W., C. D. Therrien, J. H. Sim, E. OSullivan
and L. J. Dowley. 1993. Mating type, nuclear DNA
content and isozyme genotypes of Irish isolatesof Phytophthora infestans. Mycol. Res. 97:
1131-1134.
Whittaker, S. L., S. J. Assinder and D. S. Shaw. 1994. Inheritanceof mitochondrial
DNA in Phytophthora
infestans. Mycol. Res. 98: 569-575.

167

Questionsand Answers Session 4

Q,
Have you ever collected
non- pathogenic
fusaria as microbial
resources for possible biologicalcontrol against Fusarium wilt? (Tsuchiya)

genetic

A.
We have been able to collect non- virulent isolates of F oxysporumfrom
healthyplants in the field infectedby virulent F oxysporum.One example iswatermelon. A few non-virulent isolates of F oxysporumwere assessed in the field
to control the disease. A glasshouse experiment has shown that these non- virulent
isolates could significantly
reduce disease severity.
(B. Salleh)
Q.
Does head rot of wheat caused by Fusarium graminearumoccur in Southeast
Asia? If yes, how important is it? (He)A.
We do not grow wheat in Southeast
graminearumyet in the region. In a few instances,on imported wheat seeds. (B. Salleh)

Asia. We have not come across F
we managed to detect this pathogen

Q.
You mentioned that f.sp. of/: oxysporumfrom tobacco shows wide diversity
even though these isolates were found only in Peninsula Malaysia. Did you determine
the VCG for these isolates?
If so, what was the diversity observed?
(Nagao)A.
Wehave not done any VCG work on this pathogen
banana, watermelon and asparagus fusaria. (B. Salleh)Q,
condition

What is the reason for the highthat favors a high level of mutation

yet. We have done so on

mutabilityof Fusarium}in this genus?
(Ohmasa)Is thereany

A.
Wedo not know the exact reason why Fusarium readily mutates. Normallyfusaria are highly mutable in rich media e.g. potato dextrose agar or potato sucrose
agar. (B, Salleh)
Q.
You mentioned that the medium on which you grow Fusarium is important.
What do you think of the use of SNA medium instead of carnation medium or PSA?
Do you believe that the use of different media may lead to confusion in identification?
(Smith)A.
One can use any media, as long as they are nutritionallypoor, foridentificationof Fusarium spp. People in different
laboratories
have been using
different nutrient poor media such as SNA, water melon agar, carnation leaf agar. Some

168

laboratories
It is better

still continue to use rich media e.g. PSA or PDA, but this is not advisable.
to stick to one media for the purpose of species identification.
(B, Salleh)

Q.
Have you tried freeze drying as a storage method?
in variability
in survival. Have you an opinion?
(Smith)This method has resulted

A.
There are pros and cons to ever method of preservation.
Historicallywestarted our fusaria in liquid nitrogen. Throughout the world, I think lyophilizationismore popular. However, I have seen many good laboratories
only keep their culturesin a low temperature freezer (-70 °C). (B. Salleh)Q.
handling

Considering the toxigenic nature of Fusarium how do you assess the risk in
the organism in the laboratory?
(Smith)A,
Not all Fusarium isolates are toxigenic.
and handle fusaria with care. (B, Salleh)
Q.

Is the pepper

pathogen

However, we take this issue seriously

Fusarium oxysporumor F so/ami

A.
Similar to the South American form, the slow decline or yellowing of pepper
in Southeast Asia is caused by F solani. The most important disease of pepper though
is Phytophthorapalmivora in Southeast Asia. A combination of good cultural practices
e.g. field hygiene, good drainage, suitable shade plants and balanced fertilizationcanfrevent F solani infecting pepper. So far, no pepper variety has been identified
to beresistant to F solani in Southeast Asia. (B. Salleh)
Q,
Is there any correlation
pathogenicity?
(Ohmasa)

between pulse

field

electrophoresis

patternandA.
There is no correlation
between electrophoretic
karyotyping
andpathogenicity
of tested cucurbit infecting
formae speciales.
Electrophoretic
patternsof chromosome-sized DNA were diverse among strains. (Namiki)C.
I would like to suggest you study VCG on all forma speciales
to compliment your finger printing data.
A.
infectingThank you for your suggestion.
formae speciales.
(Namiki)

I want to analyseof cucurbits

VCGs on the cucurbit-

Q.
Are there any VCGs in Fusarium oxysporummelonis? I speculate
is no correlation between VCGs and races. However, I would like to analyse
the forma specalis melonis strains. (Namiki)that there
VCGs onQ.
Why does the Al mating strain still persist
disappearedalmost completely from western parts?

in northern(Matsumoto)Japan, whereas it hasA.
I don't know the reason. However, Al mating type has not disappeared
fromwestern Japan completely.
We can find Al isolates sometimes from tomato plants.
(Ogoshi)TECHNICALREPORTS

Session5

Mushrooms

Chairpersons
Masatake Ohmasa
Sonoe Yanagi

Distributionof BiologicalSpeciesERI HASEGAWA
Forestry and Forest Products Research Institute
P.O.Box 16, Tsukuba Norin Kenkyu Danchi-nai,

Ibarakiof Genus Armillaria in Japan

305, Japan

Introduction
The genus Armillaria (Fr. ex Fr) Staude is distributed
from tropicaltotemperate regions, is found on various hosts, and sometimes causes serious root rot
damage to forest and orchard trees (Hood et al., 1991; Kile et al., 1991; Watling et al.,1991). Studies in Europe, North America, and Australia have revealed that the genus
includes more than ten intersterility
groups, biological
species.
The genus is diverse
having a host range, worldwide distribution
and various levels of pathogenicity(Gregory etal , 1991; Guillaumin
etal., 1991; Kile etal., 1991).
Several biological
species of Armillaria have been reported in Japan, but
pathogenicity,
host range and distributionof these biological
species is still unclear(Cha etal, 1994; Mohammed etal., 1994; Suzuki etal, 1993). If Armillaria
root rotis caused by more than one species, prevention and control may differ. Therefore, thecorrect identificationof the species in the genus Armillaria is necessary to protect
forests. In this study, Armillaria isolates
in Japan were identified
by their mating
behavior.
Materials

and Methods

Because young plantationsof conifers are the major victim of Armillaria
root
rot in Japan, 22 isolates were obtained from 7 coniferous host species (Table 1). The
hosts are Abies sachalinensis (Fr. Schmidt)
Masters, Abies veitchii Lindley,
Chamaecyparisobtusa (Sieb. et Zucc.) Endlicher, Cryptomeriajaponica (L. f.) D. Don,
Picea abies Karsten, Pinus densiflora Sieb. et Zucc. and Pinuspalustris Mill. All ofthese exceptAbies veitc/iiiweie, trees of cultivation.
P. abies and P palustris are exotic
species in Japan. Source of isolation and state of hosts are given (Table 1). Isolates arelisted according to the location, roughly from northeast to southwest (Fig. 1).
Unfortunately,
the standard set of tester strains for all biological
species inJapan has not been establishedyet, so isolatesof European and North American
biological
species were used as testers for mating tests to identify
Japanese isolates.
European testers (5 biological
species, 25 isolates in total) and North American testers(8 biological
species, 28 isolates in total) were used in this study. In Europe, biologicaland taxonomic species are synonymous, so European biological
species are called bytheir Latin names in this study (Guillaumin
et al., 1991). North American BiologicalSpecies (NABS) are called by their Roman numerals in this study because some of thebiologcal
species in North America need further study to be describedwith Latin

174

Table 1. Hosts, locations
Is o la te

S o u rc e l

and resultsof mating tests of Armillaria

H o stS ta te o f H o s tisolates

L o c a tio n 2collected

in Japan.

c o m p a tib le w ith 3

8 9 ‑0 3 ‑ l ls

A b ie s s a c h a lin e m iso ld st u m p

1

A . o s to y a e

9 1 ‑0 1 ‑0 1s

A b ie s s a c h a lin e n s iso ld stu m p

2

A . o s to y a e

93 ‑32m

C ry p to m e r ia j a p o n ic a

d e a d tre e

8 8 ‑0 1 ‑ 0 1

s

P in u s d e n s ifl o r a

d e a d tr e e 8

4A . o s to y a e

9 3‑1 3f

C h a m a e cy p a ris o b tu s a

d e a d tr e e

5

A . c ep is tip e s

9 2‑1 9f

C h a m a e cy p a r is o b tu s ao ld s tu m p

6

A . c e p is tip e s

90 ‑10 ‑0 7s

P ic e a a b ie slitt e r

7

A . c e p is tip e s

9 2‑0 5r

A b ie s v e itc h i

d e a d tr e e

8

A . c e p is tip e s

90 ‑0 7f

A b ie s v e itc h ii

d e a d tr e e

9A . g a llic a

A b ie s v e itc h ii

o ld lo g

108 9 ‑10V II

9 0 ‑0 3m

C h a m a e cy p a r is o b tu s a

d e a d tr e ell

A . c e p is tip e s

9 3 ‑4 2f

C h a m a e cy p a r is o b tu s ao ld s tu m p

12

A . m e lle a

A ‑ 10m

P in u s p a lu s tr is

?

13

A

9 2 ‑4 1m

C h a m a e cy p a r is o b tu s a

w e ak e n e d tr e e

14

A . m e lle a

9 2 ‑3 7m

C h a m a e cy p a r is o b tu s a

d e a d tr e e

15

A . o s to y a e

9 2 ‑0 2

m e Ile aV I

A b ie s v e itc h ii

o ld lo g

16

9 3 ‑0 2m

C h a m a e cy p a r is o b tu s a

d e a d tr e e

17

V II

9 4 ‑0 1m

C h a m a e cy p a r is o b tu s a

d e a d tr e e 8

18

A . m e lle a

A ‑12

h

C h a m a e cy p a ri s o b tu s a

d e a d tr e e *19

A . m e lle a

V I

A S ‑1m

C h a m a e cy p a r is o b tu s a

d e a d tr e e

20

A . m e lle a

V I

8 9 ‑0 7m

C h a m a e cy p a r is o b tu s a

d e a d tr e e

21

A . m e lle a

A S ‑2m

C h a m a e cy p a r is o b tu s a

d e a d tr e e *22

A . m e lle a

V I

s: single spore, m: mycelial mat, f: fruitbody tissue, r: rhizomorph, h: host tissue.
Point numbers on the map of Figure 1.
Species names represent European biological
species, and Roman numerals represent North
American BiologicalSpecies (NABS).
aLess than 1 year after death. b More than 1 year after death.

names (Guillaumin
et a/., 1991).
The isolates were incubated on PDA (1.5% Eiken Chemical)
at 25°C for 1
month. Small cubes of agar, approximately
3 mm3, were cut from the margins ofmycelia of each isolate. The cubes of a tester and a Japanese isolate were placed 3 mmapart on malt extract agar (2% Difco malt extract, 1% agar) and incubated at 25 °C for
1 month. All the combinations between testers and their Japanese isolates were tested.
Each combination
was replicated
2 times. Mating
reactionswere scoredmacroscopically,
when mating reaction occurs, the appearance of mycelia of singlespore isolates change from fluffy to crustose (Guillaumin
et al., 1991). The reason that
this criterion was used is that the karyotype of most of the Armillaria species changes
from dikaryotic
to monokaryotic
diploidsoon after the mating reaction occurs(Guillaumin
et al., 1991). Thus, it is difficult
to observe dikaryotic
hyphae and clamp
connections microscopicallyas the evidence of a mating reaction.

175

Point

numbers are in table

Fig. 1. Location

Resultsof collection

1.

of Armillaria

isolatesin Japan.

and Discussion

Results of mating tests are shown (Table 1). 21 of 22 Japanese isolates werecompatible with at least one tester of European or North American biological
species.
They are A. ostoyae (Romagnesi)
Herink, A cepistipes Velenovsky, A gallica
Marxmuller et Romagnesi, A mellea (Vahl ex Fr.) Kummer, NABS I, V, VI and VII.
NABS I,VI and VII are regarded as probably conspecificwith European A. ostoyae, A

to
A.

176

mellea and A gallica respectively,
and NABS V (A. sinapina Berube et Dessureault)
is reported to be partiallyinterfertile
with A. cepistipes (Guillaumin
et al , 1991). The
present results are consistent with these findings.
Japanese isolates were separated into
5 groups according
to their compatibility
with the testers:
A. ostoyaell group, A.
cepistipesfW group, A. mellea/W group, A. gallica/Vll
group and 1 isolate whichshowed no mating reaction with the testers.
Geographicaldistribution
seems to be different
among the groups. Isolates
inA ostoyae/Zgroup were collected from the northeasternpart of Japan, whereas thosein A. mellea/W group were collectedfrom the southwestern
part. The number ofisolates
examined in this study is not enough to determine the distributionof eachgroup, but it can be said that the former group tends to be found in cooler areas and thelatter in warmer areas.
Differencein host species is also distinct
among the groups. Isolates
belongingmellea/VZwere^ collected almost exclusively
from C. obtusa, one of themost important forest plantation
species in Japan. Furthermore, isolates of this groupwere obtained from a weakened tree and trees less than 1 year after death in all but twocases. Isolationfrom hosts right after their death suggests a high possibility
that theisolatescaused the mortality.
Consequently,
the A. mellea/W group may be anaggressive pathogen on C, obtusa.
Three isolates
out of 5 in the A. ostoyae/I group and 2 out of 5 in the A.
cepistipesfW group were obtained from trees less than 1 year after death. These groups
may include isolates more saprophytic than pathogenic together with pathogenic
ones,
and seem to have a wider host range than the A. mellea/WI group, at least with respect
to conifers. Isolates of the A. gallica/W‡U group were found only from trees more than
1 year after death in natural forest of Abies veitchii, suggestingthat it is more
saprophytic
than pathogenic.
In studies
in Europe and North America, A. ostoyae and NABS I wereregardedas moderately
or highly virulent
on conifers, andA. mellea and NABS VI
were supposed to be aggressive pathogens on hardwood species (Gregory
et al , 1991).
The results of the present study suggested
that isolates
compatible
with A. mellea
and/or NABS VI may be aggressive pathogens on C. obtusa. This is in agreement withseveral authors who have suggested the possibilityof aggressive
pathogenicityof A.
mellea on conifers from the results of inoculation
tests (Klein-Gebbinck
et al.,1993;
Mallett and Hiratsuka, 1988; Morrison, 1989).
A gallica andA. cepistipes are said to he weakly or non-pathogenic
on bothconifers and hardwoods, and NABS V shows weak pathogenicity
on lodgepole pines
in Canada (Gregory et al., 1991). A recent study showed that interfertility
between A.
cepistipes and NABS V is much lower than intrafertility
in each group, a result whichreflects
differences
in morphology,
distribution
and habit (Berube
et al., 1994).
Therefore, Japanese isolates compatible withal cepistipes and/or NABS V need further
study to make the relationships
between these groups clear.

and

177

Acknowledgments
I am pleased to thank Dr. K. Korhonen, of the Finnish
Forest ResearchInstitute,
Vantaa, Finland and Dr. Y. Hiratsuka, of the Northern Forestry Center,
Edmonton, Canada who sent me European and North American tester strains
respectively.
I am also grateful to all the people who helped me to collect Armillaria
isolates throughout
Japan.
References
Berube,J. A., M. Dessureault,
S. Berthelay
and J. J. Guillaumin.
1994. Inter fertility
betweenArmillaria
cepistipes
A. sinapina. In Proceedingsof the 8th international
conference on root and buttrots, Johansson, M. and J. Stenlid, eds., 1993 August 9-16, Wik, Sweden and Haikko, Finland,International
Union for Forestry Research Organizations,pp. 489-497.
Cha, J. Y, J. M. Sung and T. Igarashi.
1994. Biological
species and morphologicalcharacteristics
ofArmillaria mellea complex in Hokkaido:
A. sinapina and two new species,^,jezoensis andA. singula. Mycoscience35: 39-47.
Hood, I. A., D. B. Red fern and G. A. Kile. 1991.Armillaria
in planted
hosts. In Armillaria root disease,
Shaw, C. G. m and G. A. Kile, eds., Agricultural
Handbook No. 621, USDA, Forest Service,
Washington,
D. C, USA, pp. 122-149.
Gregory,
S. C, J. Rishbeth
and C. G. Shaw III. 1991. Pathogenicity
and virulence.
In Armillaria
root disease, Shaw, C. G. Ill and G. A. Kile, eds., Agricultural
Handbook No. 621, USDA,
Forest Service, Washington,
D. C, USA, pp. 76-87.
Guillaumin, J. J., J. B. Anderson and K. Korhonen. 1991. Life cycle, inter fertility,and biological
species.
InArmillaria
root disease, Shaw, C. G. Ill and G. A. Kile, eds., Agricultural
Handbook No.
621, USDA, Forest Service, Washington,
D. C, USA, pp. 10-20.
Kile, G. A., G. I. McDonald and J. W. Byler. 1991. Ecology and disease in natural forests.InArmillaria
root disease. Shaw, C. G. Ill and G. A. Kile, eds., Agricultural
Handbook No. 621, USDA,
Forest Service, Washington,
D. C, USA, pp. 102-121.
Klein-Gebbinck,
H.W., P. V. Blenis and Y. Hiratsuka.
1993. Fireweed (Epilobium angustifolium)
as apossible inoculum reservoir for root- rotting Armillaria
species. Plant Pathology
42: 132-136.
Mallett,
K. and Y. Hiratsuka.
1988. Inoculation
studiesof lodgepole
pine with Alberta isolatesof theArmillaria mellea complex. Canadian J. Forest Research 18: 292-296.
Mohammed, C, J.J. Guillaumin
and S. Berthelay.
1994. Armillaria species identified
in China and Japan.
MycologicalResearch 98: 607-613.
Morrison,
D. J. 1989. Pathogenicity
of Armillaria species is related
to rhizomorph
growth habit.
In Proceedingsof the 7th international
conference on root and butt rots, Morrison, D. J., ed.,1988 August 9-16, Vernon and Victoria,
Canada, International
Union for Forestry Research
Organizations,pp. 584-589.
Suzuki,K., K. Fukuda, Y. Shiga, N. Matsushita
and T. Terashita.
1994. Identificationof Japanese
biological
species of Armillaria by isozyme patterns. In Proceedingsof the 8th international
conference on root and butt rots, Johansson,
M. and J. Stenlid,
eds., 1993 August 9-16, Wik,
Sweden and Haikko, Finland,International
Union for Forestry Research Organizations,pp.
376-382.
Watling,R., G. A. Kile and H. H. Burdsall,Jr. 1991. Nomenclature,
taxonomy, and identification.
InArmillaria
root disease, Shaw, C. G. Ill and G. A. Kile, eds., Agricultural
Handbook No.
621, USDA, Forest Service, Washington,
D. C, USA, pp. 1-9.

Distributionof the Ectomycorrhizal
Fungus Tricholoma
matsutakeand the Related Species and Some Characteristics
of
Their Isolates
KOJI IWASE
Biological
Environment InstituteKansai Environmental Engineering
8-4 Ujimatafuri,
Uji 61 1, Japan

Center Co., Ltd.

Tricholomamatsutake and the related

species

Tricholoma matsutake (S. Ito & Imai) Singer is an ectomycorrhizal
fungus,being symbioticallyassociated mainly with Pinus densiflora. Other host trees belong
to the genera Pinus, Tsuga,Pseudotsuga, and Picea. The pileus surface of a fruit-bodyof this fungus is covered with brown scales and fibers (Fig. 1). Gills are white, but
often becoming brownish with age. The stipe is white above the ring, but lower parts
below the ring are covered with brown scales and fibers like a pileus surface. The
distinctive
character of this mushroom is its fragrant aroma, and the chemicalcomponents of this aroma have been determined to be mainly trans-methyl cinnamate
and 1- octene-3- ol (Yajima et a/., 1981).
The area covered with mycelia and their
mycorrhizae is called "Shiro" in Japanese.
"Shiro" exists in the layer A-B in the soil,
and expands by 10- 15 cm a year (Ogawa, 1975). Fruit-bodies
occur in positionsatthe edge of the "Shiro". This species is the most valuable edible mushroom in Japan,
and the large quantities of fruit- bodies including those of the related species are imported
fromvarious foreign countries, such as China, North and South Korea, Canada, U.S.A.,
and Morocco.
Species related to T. matsutake are those with a similar morphology of theirfruit- bodies. They are all ectomycorrhizal
species, and some of them have a fragrantaroma. At least three species are found in Japan and another three or more in othercountries.
Tricholomaponderosum(Peck) Singer forms mycorrhizae with some speciesof Pseudotsuga, Tsuga,and Pinus. The fruit- body of this fungus is white in color (Fig.
2), therefore
this mushroom is commonly called white "Matsutake".
Rather large
quantities of this mushroom are imported to Japan from Canada, U.S.A., and recently
from Mexico.
The species Tricholoma caligatum (Viv.) Ricken seems to be an indeterminate
species, and at least three types are recognized from a the literature.
One of these types(T caligatum 1) is distributedin North Africa and South Europe, and the fruit-bodyresembles T matsutake very much (Fig. 3) with strong aroma, but it has a somewhat
dark brown color. This fungus symbioticallyassociates with some species of Cedrus

180

Figs. 1- 7. Fruit- bodies

of Tricholoma matsutake and the related

ponderosum.
Fig. 3. Z caligatum.bakamatsutake. Fig. 7. Z zelleri.

1.

Fig.

species.

4. T. fulvocastaneum.

Fig.

1. 77 matsutake.

Fig. 5. Z robustum.

Fig. 2. Z
Fig.

6. Z

and Pinus. This type of mushroom is imported to Japan mainly from Morocco in lateNovember. The second type (T. caligatum 2) is found in the west of North America,
and the fruit- body appearance is just like a small sized T. matsutake. This fungus
forms mycorrhizae with various conifers (Fischer and Bessette, 1992).
The third type
{T. caligatum 3) is found in the east of North America, and seems to be closely related
to T. bakamatsutake, or it may be T. bakamatsutake itself because it is associatedwithoak trees and has a fragrant aroma (Fischer and Bessette, 1992).
Tricholoma fulvocastaneum Hongo is symbioticallyassociatedwith some181

speciesof Quercus and Castanopsis. Fruit- body occurs a little earlier
than T.
matsutake, therefore this species is sometimes called "Samatsu", literallymeans earlyoccurring "Matsutake". The fruit- body appearance clearly resembles T. matsutake, but
this fungus has weak or no aroma, and stipe base is somewhat slender (Fig. 4).
Tricholoma robustum (Alb. et Schw. : Fr.) Ricken sensu Imazeki forms
mycorrhiza with P. densiflora, the same as T. matsutake, in Japan. Fruit-bodies
areformed later in the season than T matsutake. The fruit-bodyis rather small (Fig. 5),
and has no aroma. The "Shiro" is rather close to soil surface compared with T.
matsutake (Ogawa, 1981).
Tricholoma bakamatsutake Hongo forms mycorrhizae with some species ofQuercus and Pasania (Ogawa, 1978).
Fruit- bodies are somewhat smaller than T.
matsutake (Fig. 6) and they have a very strong aroma.
Tricholoma zelleri (=Armillaria zelleri Stunts and Smith) forms mycorrhizaewith some species of Pinus. A fruit-body (pileus)
color is brown to yellowish
brown
or olive (Fig. 7).
Distributionof T. matsutake and the related

species

In Japan, at least four "Matsutake" related species are distributed,
T matsutake,T robustum, T. fulvocastaneum, and T. bakamatsutake. Tricholoma matsutake isfound across the whole of Japan except for Okinawa (Fig. 8). This mushroom is very
popular in Japan and has high commercial value in the market. The center of its
distribution
lies toward the southwest of Japan, especially
the west of the Kansai district.
Tfulvocastaneum is confined to the southwest of Japan (Fig. 9). The host of
this fungus is evergreen trees such as Castanopsis, which is distributedin this area.
This fungus has not been found outside of Japan. It is possible,
however, that thisfungus will be found in areas where Castanopsis is distributed,
such as China,
includingTaiwan.
T robustum has a limited distribution
compared with T. matsutake (Fig. 10),
but the main host tree is the same Japanese red pine as T matsutake. Since thismushroom has a bad taste and no aroma, it is not a target for mushroom hunting inJapan. Therefore, it seems that this fungus has about the same distribution
area as T
matsutake.
Tricholoma bakamatsutake is similarly
distributed
throughout
Japan as T.
matsutake (Fig. ll).
In 1974, this fungus or closely
relatedone was found in
Castanopsis forest in the mountain of Papua New Guinea (Otani, 1976).
It seems
highly likely that this fungus will be found in other countries.
T.ponderosum is only found in North America (Fig. 12), mainly along thewest coast. Both T caligatum types 2 and 3 are distributedin North America (Fig. 12),mainly in the west and the east area, respectively.
But, detailed
aspects are unknown.
T zelleri is also found in North America (Fig. 12). T caligatum type 1 (Fig. 12) isfound in southern Euope and North Africa.

Tricholoma matsutake

Tricholoma fulwocastaneum

Tricholoma robustum

Tricholoma foakamatsutake

Figs. 8- ll. Distribution
map of Tricholoma matsutake and the related species in Japan.
matsutake. Fie. 9. Z fulvocastaneum. Fie. 10. T. robustum. Fie. ll. T. bakamatsutake:

Fig.

8. Z

Fig. 12. Distribution

Nuclear distribution

map of Tricholoma matsutake and the relatedof hyphae of Isolates

from fruit-bodyspeciesin the world.

tissues

The life cycle of ordinary basidlomycete
mushrooms is shown (Fig. 13). In
general, basidiospore
contains a single nucleus, and it germinates to form monokaryon.
Monokaryon is characterizedby consistingof uninucleatedcells.
When twocompatible
monokaryons come in contact with each other, then anastomosis occurs
and each nucleus migrates into reciprocal
monokaryotic hyphae. Finally, a dikaryon
is established*
This mycelium consists of binucleatedcells, and a specific structure,a called clamp connection, is observed at each septum. Under appropriate
conditions,a fruit- body is formed. Within specific cells called basidium, two compatible nuclei
are fused for the first time, and then meiosis occurs immediately.
Usually 4

Fig. 13. Life cycleof the basidiomycetes.

T

184

basidiosporesare formed on top of the basidium.
In "Matsutake" related species, isolation
for mycelial culture from fruit-bodytissues was achieved when fresh gill tissue was explanted onto conventionalagar
medium, whereas using inner part of pileus or stipe tissue as explants was not
successful.
Mycelial growth of isolates was very slow. The hyphae have no clamp
connections at each septum, and septa are hardly observed (Fig. 14). Nuclei and septa
were stained with two different fluorescentdyes in order to count nuclei number percell. Nuclei were stained with ethidium bromide and septa were stained with fluorescentbrightener,
and then hypha was observed under epifluorescent
microscope (Iwase etal, 1988; Iwase, 1990).
Septa were visualized
by the observationof hyphae with UV
setting (Fig. 15), whereas nuclei were visulaizedwith G setting (Fig. 16). Comparisonof Fig.15 and 16 shows that hyphae are dikaryotic.
When the hyphae of isolates of all"Matsutake"related species were double stained as above and observed underepifluorescent
microscope, it was shown that they are all dikaryotic
hyphae (Iwase,1990).
There is no difference in this characteristics
among "Matsutake" related species.

Fig. 14. Hyphaeof isolates

from fruit-bodytissuesofmatsutake.

185

Fig. 15. Epifluorescencemicroscopy of hyphae of isolates from fruit-bodytissues of T. robustum. Hyphae
were stained with ethidium
bromide and fluorescentbrightener,
and observed under epifluorescencemicroscope excited by UV setting. Septa are shown as line within hyphae.

Fig. 16. Epifluorescencemicroscopy of hyphae of isolates from fruit-body tissues of Z robustum. Hyphae
were stained with ethidium bromide and fluorescentbrightener,
and observed under epifluorescencemicroscope excited by G setting. Nuclei are shown as bright circle within hyphae.

186

Gluconic

acid synthesis

When isolated mycelia of T. robustum are grown in synthetic liquid medium
containing
glucose as a carbon source, culture medium graduallybecame acidified
during mycelial growth (Iwase, 1992a).
A pH value of the culture filtrate decreased
from the initial 6.0 to 3.8 at 42 days culture. When culture filtrate was analysed by
HPLC, the acidic substance produced was detected and identified
to be gluconic acid
(Iwase, 1992a). Enzymatic analysis also indicatedthat the responsible
substance was
gluconic
acid. In cell free extracts, high activityof glucose oxidase, an enzyme
responsible
for the productionof gluconic acid from glucose, was observed when
gluconic acid was produced.
Gluconic acid productivity
was compared among "Matsutake" related species.
Mycelia of T. robustum produced the most gluconic acid among species tested, and T.
bakamatsutake was the second highest.
Other species
were weak gluconic acidproducers (Iwase, 1992a).
The organic acids produced by ectomycorrhizal
fungi are important because
they can complex with or precipitate
metals, increasing phosphate dissolution
in soils(Cromack et aL, 1979).
The phosphate
dissolved
would be available
to bothsymbionts(Harleyand Smith, 1983). There have been several reports concerning the
production of oxalic acid by certain species of ectomycorrhizal
fungi. However, thisis the first report of gluconic acid production.
Inductionof basidiospore

germinationby conspecificmycelium

Experiments
concerning basidiospore
germinationin T. robustum wereconducted. Very few basidiospores
germinated on conventional agar medium. When
mycelium of T. robustum was coinoculatedwith basidiospores,
germinationwas
induced, at first in the vicinity of a growing colony of T, robustum, but graduallyitseffect extended to the whole surface of the medium (Iwase, 1992b).
Taxon specificinductionof basidiospore
germination
has already been reported in Leccinum (Fries,1979),
but the chemicals
which induce germination
could not be identified
in
Leccinum.
Addition of gluconic acid ranged from 0.01% to 1.0% remarkably promoted
basidiospore
germination in T. robustum (Iwase, 1992b).
This finding indicatesthat
one of the germinationinducing substances produced by mycelium of T. robustum is
gluconic acid.
Basidiospore
germinationinduced by conspecificmycelium is exclusively
observed in T. robustum among "Matsutake" related species tested. The ecologicalsignificanceof taxon specific basidiospore
germinationinduction mentioned above
would be explained
as follows (Fig. 17).
There is an establishedcolony of T.
robustum, and from the mycelia of this colony, basidiospore
germinationinducing
substancessuch as gluconic
acid are secreted into the surrounding
soil.
When
basidiospore
from other colonies settle near to the colony, spore will react to induce
germination.
Germinated hypha will cross with hyphae of the establishedcolony.

187

Germinationinducing
substances[Colonyof Tricholoma

robustum|Fig. 17. Basidiospores
alternative role as gene transporterto the establishedcolony. Basidiospore
germination is induced by germination inducing substances secreted from mycelium of establishedcolony.
Germinated hyphae cross with the hyphae of establishedcolony, and this results in the acquisitionof new
genetic characters for the establishedcolony.

Crossing will result in the acquisitionof new genetic characters for the establishedcolony. Usually, it is considered that basidiospores
have a role as a resting propagulefor the formation of new colonies, but in some species as T. robustturn,they might havean alternativerole as gene transporter to the establishedcolony.
References
Cromack, K. Jr., Sollins, P., Graustein, W. C, Speidel,K., Todd, A. W., Spycher, G., Li, C. Y., and Todd,
R. L. 1979. Calcium oxalate accumulationand soil weatheringin mats of hypogeous
fungusHysterangium crassum. Soil. Biol. Biochem. ll : 463-468.
Fischer, D. W. and Bessette, A. E. 1992. Edible wild mushrooms of North America. Universityof Texas
Press, Austin, p. 254.
Fries, N. 1979. The taxon specificspore germination
reaction in Leccinum. Trans. Br. Mycol. Soc. 73:
337-341.
Harley, J. L. and Smith, S. E. 1983. Productionof metabolites
by ectomycorrhizal
fungi. In Mycorrhizal
symbiosis,
Academic Press, London, pp. 170-182.
Iwase, K. 1990. Nuclear distribution
in hyphal cells of isolates
from fruit-bodies
and single basidiosporesin ectomycorrhizal
fungus Tricholoma robustum and allied species: Visualizationof nuclei andsepta by a double staining method. Mem.Fac. Sci. Kyoto Univ. Ser. Biol. 14: 117-127.
Iwase, K. 1992a. Gluconic acid synthesisby the ectomycorrhizal
fungus Tricholoma robustum. Can. J.
Bot. 70: 84-88.
Iwase, K. 1992b. Inductionof basidiospore
germination
by gluconic
acid in the ectomycorrhizal
fungusTricholoma robustum. Can. J. Bot. 70: 1234-1238.
Iwase, K., Matsui, S., Taniguchi, T., and Obayashi, A. 1988. Identificationof the cell type of hyphae from
cultures of single basidiosporesand fruit- body tissues of the mycorrhizal
fungus Tricholoma
robustum by double staining of nuclei and septa. Trans. Mycol. Soc. Japan 29: 383-390.

188
Ogawa, M. 1975. Microbial
ecology of mycorrhizal
fungus, Tricholoma matsutake Ito et Imai (Sing.)
inPine forest I. Fungal colony (Shiro')of Tricholoma matsutake. Bull. For. Exp. Sta. 272: 79-121.
Ogawa, M. 1978. Biologyof Matsutake mushrooms. Tsukijishokan,
Tokyo, p. 326.
Ogawa, M. 1981. Microbial
ecologyof 'Shiro' in Tricholoma matsutake and its allied
species
X.
Tricholoma robustum in Pinus densiflora and P. pumila forests and T zelleri in P. contorta
forests.
Trans. Mycol. Soc. Japan 22: 231-245.
Otani, Y. 1976. Tricholoma bakamatsutake Hongo collected
in New Guinea. Trans. Mycol. Soc. Japan
17:363-365.
Yajima, I., Yanai, T., Nakamura, M., Sakakibara,
H., and Hayashi,
K. 1981. Volatilefalavor compoundsof Matsutake- Tricholoma matsutake (Ito et Imai) Sing. Agric. Biol. Chem. 45: 373-380.

Molecular

Genetic Analysis

of Diversity

in Agaricus bisporus

BRITT A. BUNYARD and DANIEL J. ROYSE
Mushroom Research Lab, Department of Plant Pathology,
College of Agriculture,
The Pennsylvania State University,
University Park, PA 16802 USA
Abstract
Agaricus bisporus (syn. A. brunnescens) is an important commercially- produced mushroom,accounting for more than 35% of the total world production of cultivatedmushrooms. Although much is
known about A. bisporus^ little information
exists about the evolutionary
relationships
and diversityofspecies within the genus Agaricus. We have begun using recently developed methods of genetic analysis
for genotypic classificationo>iAgaricus bisporus. The 26S and 5S ribosomal RNA genes and the intergenic
region between the 26S and 5S rRNA genes of the ribosomal DNA repeat region were amplified using thepolymerase chain reaction and then digested with 10 restriction
enzymes. Restriction
fragment length
polymorphisms were found among the 21 putative species of Agaricus investigatedand used to develop
a phylogenetic tree of the evolutionary
history of Agaricus bisporus\ The phylogenetic
tree clustered mostisolates of Agaricus closely to A. bisporus^ suggesting that most species in the genus are closely related.
However, the genus seems to be highly polymorphic.
In several cases there is greater intra- (than inter-)
species dissimilarity.
Similarly,
a phylogenetic
tree for more than 100 isolates of Agaricus bisporus
suggests more than twelve genotypic classes exist among isolates in the Pennsylvania
State University
Mushroom Culture Collectio.

Introduction
The white button mushroom, Agaricus bisporus (Lange) Imbach (syn. A.
brunnescens Peck; Malloch, 1976) is the most important commerciallycultivatedmushroom species in the world (Loftus et aL, 1988). According to the most current
data, world productionof Agaricus is in excess of 1 million metric tons annually(Chang and Miles, 1991). This species accounts for over 35% of the total world
productionof cultivatedmushrooms. Since records of mushroom production began
in 1967, the U.S. output of Agaricus has increased steadily.
Total production peakedin 1992- 3 at 176.5 thousand metric tons (USDA, 1994) (Fig. 1); U.S. production
accounts for about one quarter of the total world output. The 1993-4 crop in the U.S.
was valued at US$692 million (USDA, 1994).
Although much is known about A. bisporus, several aspects remain unclear,especiallythose concerning its genetic life history (Royer and Horgen, 1991; Castle
etaL, 1988, 1987; Spear etaL, 1983; Royse and May, 1982a; Royse and May, 1982b;
Elliott,
1972; Raper et aL, 1912; Jiri, 1967; Pelham, 1967; Evans, 1959; Kligman,1943). Fundamental processes such as the segregation
and assortment of genes duringmeiosis remain poorly defined (Summerbell
et aL, 1989; Royse and May, 1982a). In
addition, the evolutionary
history and taxonomic placement of A. bisporus within the
Agaricales
remains uncertain(Kerrigan
et ai., 1993b;
Singer,1986).
Althoughaddressed in great length by Kerrigan (1990), many questions still exist concerning theevolutionary
origin ofA. bisporus, the relationshipof cultivars to wild populationsofFigure1. Total

U.S. productionof Agaricus

bisporusfrom 1967-94(USDA,

1994).

this species, and its position
within the Agaricales.
Agaricus bisporus is considered an atypical member of the Basidiomycetes.
Basidiomycetous
fungi are typicallyclassified
as homothallic
or heterothallic.
Homothallic
fungi have the ability
to produce fruit bodies, though only one type of
nucleus is found in the mycelium. Heterothallism
is a condition where two different
nuclear types (with different
incompatibility
factors) are required for fruiting.
The
latter can result from the hyphal fusion of two different
individuals
or from being
passed on within heterokaryotic
spores, a condition known as secondary homothallism(Summerbell etal., 1989; Loftus et al., 1988; Elliott,
1972; Raper et al. , 1972; Miller,
1971). The smaller proportion of basidiospores
which are homoallelicat the matingtype locus produce self- sterile hyphae. These are capable of nuclear exchange withother homokaryons and, therefore, are used in breeding experiments. Royse and May(1982a)have shown that nearly 90% of single- spore progeny are heteroallelic.
Similarly,
Summerbell et al. (1989) found 95.6% of individuals
to be heterokaryons
with RFLP phenotypes identicalto those of their parental-type
heterokaryons.
Commercial isolates of^i bisporus have very little genetic heterogeneity.
This
condition
may have resulted from the developmentof all (Western)commercial
isolates from a few wild European ecotypes (Kerrigan
et a/., 1993a; Royse and May,
1982a).
Because of the lack of variability,
new genetic resources will have to comefrom wild populations in the future (Anderson, 1993). As the natural sources for these
populations
become more scarce, it is urgent that efforts are directed towards thelocation of remaining sources before they are lost (Anderson, 1993). Kerrigan et al.
(1993a)demonstrated
that one of the best known sources for new genetic resources
may come from the western part of the USA (California).
Populations
examined bytheir study were comprised of wildA bisporus isolates, as well as, commercial escapes(Kerrigan et al., 1993a).
In addition,evidence for hybridizationof the two was found(Kerrigan et al., 1993a). Being able to locate and assess the genetic makeup of such

191

wild populations
future.
Applications

will become increasinglyof Methods

for Molecularimportant
Genetic

for breeding

A. bisporus in the

Analysis

SingleSpore Sterility- To develop strains of A. bisporus having desirableor
improved traits, traditionally,
selections
were made from single spore or multispore
cultures (for review, see Fritsche, 1991). Homokaryons are necessary when making
crosses and these have proven difficult to produce (Loftus et aL, 1988). Homokaryons
were obtained historicallyby random spore analyses, i.e., by assaying for the one
consistent feature indicativeof the homokaryotic
state-lack of the ability to produce
fruiting bodies (Summerbell et aL, 1989; Elliott and Wood, 1978; Raper et al, 1972).
Homokaryons also were obtained through micromanipulationof basidiospores
fromrare four- spored basidia(Elliott,
1972; Miller,
1971; Miller and Kananen, 1971).
Later, allozyme analysis was used to detect homokaryons as well as to confirm crosses
between homokaryons (May and Royse, 1982a; Royse and May, 1982a).
Allozyme
Analysis- - Allozymes are enzymes having different
electrophoretic
mobility due to allelic differences in a single gene. Allozyme analysis is useful because
phenotypic
differences
in banding patterns between individuals
correlate directlytogenotypic
differences.
Therefore,
allozyme analysis
can identify
unambiguous
codominant genotypes useful in conducting genetic studies of fungi (Royse et aL, 1993).
Researchers have used allozyme analysis (also referred to as multilocus
enzyme electrophoresis)
for identificationof genotypicclasses and to follow the
segregationof allozyme encoding alleles during meiosis (Royse et aL , 1983a; Spear
etaL, 1983; Royse and May, 1982a) in commercial mushrooms. Genetic studies withallozymes have been conducted on more than 30 crop species (Abler et aL, 1991).
Inaddition,allozymes have been shown to be useful as genetic markers for identifying
and localizing
quantitative
trait loci in a number of crop species (Abler et aL, 1991;
Weller et aL, 1988; Stuber et aL, 1987; Tanksley et a/., 1982). Spear et aL (1983)
created the first map of linkage groups in A. bisporus using allozymes. However, thelack of numerous markers resulted in a very limited map. Recently, Kerrigan et aL(1991)
have added to this map of linkage groups using RFLP and RAPD markers. It
is important to note that gene linkage maps for A. bisporus, as yet, do not link markers
to any agronomicallyimportant characteristics.
Restriction
Fragment Length Polymorphism(RFLP) Analysis- - RFLP analyses
also involve detectionof genotypic
differencesusing electrophoresis.
Different
fragment length polymorphisms result in bands migrating at different rates through anelectrophoretic(agarose) gel. Restriction fragments having different lengths/migration
rates are generated using restriction
endonucleases
to digest total whole DNA or
specific regions within the genomic (nuclear) or mitochondrialDNA. These enzymes
cut DNAat specific, palindromic sites along the DNA sequence. The resulting bands
are visualized by Southern hybridizationor ethidium bromide staining.
RFLP analyses have been used to define genotypesof heterokaryotic
and

192

homokaryotic
strains and to confirm crosses among several isolates ofA. bisporus(Summerbell et a/., 1989; Castle et a/. , 1988; 1987). Conclusions about reproductive
events are scarce due to the small number of known genetic markers availablefor
analysis of the species (Kerrigan,
1993b; Kerrigan, 1990; Castle et a/. , 1987), as wellas the lack of an extensive genetic map of linkage groups.
Other methods of genetic analysis, used to explore the relationships
amongand between fungal taxa have been discussed (Anderson, 1993; Royse et a/., 1993).
These modern methods include DNA-DNA hybridization(Appels and Honeycutt,1986), RAPD analysis (Williams et a/., 1991), DNA sequencing (Fincham, 1985), andelectrophoretic
karyotyping(Skinner
et a/. , 1991).
Ribosomal DNA Analysis- - Recently, researchers conducting phylogenetic
studieshave begun to use the variation found in the sequences of the ribosomal RNA genes(rDNA) and the stretches of DNA between these genes, known as internal transcribedspacers (ITS) and nontranscribedintergenic
regions (IGR). The ribosome is theorganelle of the eukaryotic cell responsible
for the translationof mRNA (the transcriptof DNA) into the polypeptide
sequences that comprise proteins. The ribosome is madeof protein and ribosomal RNA, containing 80 to 90% of the total cellular RNA. To
achieve its high level of translational
efficiency,
rDNA exists as a tandomly repeatedarray of the three largest genes coding for rRNAs and are separated by transcribedandnontranscribedspacers. The number of repeated units of rDNA within the genome oforganisms ranges from one or two in prokaryotes
to thousands in some eukaryotes.
The eukaryotic ribosome has a sedimentation
coefficientof 80S, but is madeof two subunits having sedimentation
coefficientsof 60S and 40S. The rRNA genes
are initiallytranscribed
as a single precusor RNA molecule. This process occurs at thenucleolus by RNA polymerase I. The precursor RNA is then processed yielding
maturerRNA molecules of the following sizes: 5S, 5.8S, 18S and 28S. The order of the genesubunits appears to be universallyconserved with the exception of the 5S gene (Bruns
et a/, 1991; Lewin, 1980), which may or may not be present within the repeat,
depending on the species. For some Basidiomycetes,the 5S has been reported to exist
in the opposite
orientation(Cassidy
and Pukkila, 1987; Pukkila and Cassidy, 1986).
The 18S molecule is present in the 40S ribosomal subunit. The 5S, 5.8S and 28S
rDNAs make up the 60S ribosomal subunit. However, the 5S rDNA is transcribedindependently
by RNA polymerase III and may be highly variable
in sequence.
Ribosomal RNA genes and their associatedspacers have been used extensivelyinphylogenetic
studies (Bunyard et a/., 1994; Hibbett and Vilgalys, 1991; Wheeler andHoneycutt, 1988; De Wachter eta/., 1985; Ohama eta/ , 1984; Fox eta/., 1980) anda large amount of sequence data exists from taxonomicallydiverse organisms(Erdmann <?/ #/., 1985).
The ITS lie between the 5.8S and 28S rRNA genes while the IGRs flank the
5S rRNA gene (Jorgensen and Cluster, 1988; Appels and Honeycutt, 1987; Hillis andDavis, 1986; Long and Dawid, 1980). The ITS and IGR regions may make up only
20% of the primary transcript in lower eukaryotes but up to 50% in higher eukaryotes
such as mammals.Because they are not conserved, these regions evolve more rapidly

193

than the rDNA regions they flank and, therefore,
are highly variable (Schaal et aL,
1987; Suzuki et aL, 1987; Williams et aL, 1987; Flavell et aL, 1986; Saghai-Maroof
et aL, 1984; Arnheim, 1983). Due to their highly variable
nature, the noncoding(nongenic)
regions may be suitable for phylogenetic
studies of many closely relatedorganisms, including fungi.
RFLP analysis
has been shown to be an effective
method to determine
differences
within the rDNA of many fungal groups. Although
direct sequencing
provides more information, RFLP analysis is less time consuming and allows for the
analysisof more isolates. Many researchers have used RFLP analysis of rDNA toprovide new information about evolutionary
relationships
of many different levels offungal taxa, including
family (Bunyard et aL, 1995a), genus (Bunyard et aL, 1995b;
Johansen etaL, 1992), species (Bunyard, et aL, 1994b; Anderson and Stasovski,
1992;
Martin, 1990; Kohn et aL, 1988; Raeder and Broda, 1984), and some strains (Bunyard
et aL, 1994a; Laguerre et aL, 1994; Ward and Akrofi, 1994; Vilgalys
and Gonzales,
1990). RFLP analysis of PCR- amplified
rDNA has also resulted in the constructionof restriction
site maps of fungal species (Nicholson
et aL, 1995).
Our protocols involve the production of mycelium and subsequent processing
and analysis of DNA as follows: Mycelium of Agaricus was grown in liquid potato
dextrose broth supplemented
with yeast extract. The mycelium was filtered and air
dried prior to DNA extraction. Fungal DNA was extracted by the method of Zolan andPukkila(1986).
For amplification
by polymerase
chain reaction (PCR; Mullis andFaloona, 1987), DNA was diluted in water to optimal concentrations
for each isolate.
Dilute DNA was combined with dNTPs (dATP, dCTP, dGTP, dTTP), a buffer
containing
Mg+ , each of two custom-made primers, DNA polymerase, and water togive a final volume of 50.0 iA per each reaction tube. The mixture was overlaid witha drop of mineral oil and subjected
to typical PCR cycling parameters of heating andcooling. Products of PCR amplification
were detected by agarose gel electrophoresisand visualized with ethidium bromide.
Knownfungal ribosomal DNA primers found to be conserved among fungal
taxa were used to PCR-amplify
the 3 ' and 5' halves of the 26S ribosomal RNA geneand the IGR- 1/5S rDNA for analysis of species of Agaricus and the IGR-1 and IGR-2
for analysis of genotypes ofAgaricus bisporus. Primer sequences (Table I) were basedon the known sequence of the rDNA repeat from Saccharomycescerevisiae (Georgiev
et aL, 1981). DNA amplified from the 26S, 5S rRNA genes, IGR-1 and IGR-2 (Fig.
2) was digested using restriction
enzymes and analyzed by gel electrophoresis
for thepresence of RFLPs between the species of Agaricus. Restriction
enzymes used in our
lab were the four-base cutters Hha I, Alu I, Hpa II, BstU I, Hae III, Rsa I, Taq I, and
NdeII; the five- base cutter Hini I; and the six- base cutter Hpa I. Restriction
fragments
were visualized
by gel electrophoresisand staining with ethidium bromide as above.
The restriction fragments were scored for each isolate by comparing the fragment sizewith a 123 bp X DNA ladder (Figs. 3 and 4).
Phylogenies
were inferred from data collected by RFLP analysis of the 26S
rDNA and the IGR- 1/5S rDNA. RESTSITE, a computer program written by M. Nei andTable I. Primer name, primer sequence and location and direction of primer extension used for PCR
amplificationof the 26S rRNA gene (Fig. 2) of Agaricus spp.

* Within S. cerevisiae rRNAa Vilgalys, Pers. Commb Walker and Doolittle,
1982
c Georgiev, Nikolaev and Hadjiolovd Georgiev, Nikolaev and Hadjiolov

et al., 1981
et al, 1981

Figure 2. Primer locations for amplificationof the IGR-1 , IGR-2, and 5' and 3* ends of the 26S rRNA geneof Agaricus spp. Complementary strand synthesis in the 5' to 3' direction(shown by arrows).

J. C. Miller (1990), was used to determine the amount of variation between isolates and,thus, gauge the evolutionary
divergence between isolates.
The degree of genetic
divergence between two DNA sequences was correlated with the proportionof DNAfragments that they share. RESTSITE is based on the method of Nei and Li (1979) tocompare electrophoretic
patterns between isolates
to estimate d, the number ofsubstitutions
per site (Nei, 1987). RESTSITE computes /^-hat values from the numberof restriction fragments of OTUs (operationaltaxonomic unit) X and Y, the number offragments commonto both, and the number of bases recognized by the restriction enzyme(four, five, six, etc.). The estimatednumber of substitutions
per site, d- hat, wascalculatedby an iterative method (Nei, 1987) from the T^-hat values.
The data (d- hat) from the pair- wise comparisons (Table II) were analyzed usingd-hatTable II. Distancematrix ofvalues, calculatedby the method of Nei ( 1 987) and based on restriction

phenotypes

for the 26S and IGR- 1/5 S rDNA of Agaricus spp. isolates.

the computer program MEGA(Kumar et al., 1993), a software package developed forthe purposes of sequence distance analysis
and reconstructionof phylogenetic
relationships.
The resulting phylogenetic
trees were assembled using the Neighborjoining (NJ) Method (Saitou and Nei, 1987) for the species within the genus Agaricus
and for genotypes of A. bisporus.
A single product 1.4 kb in size resulted from PCR amplificationof genomic
DNAusing the primers LROR and LR7 for the 5' end of the 26S rDNA gene. Likewise,
a 1.5 kb product was amplified for all isolates using the primers ALR7R and LR12 forthe y end of the 26S rDNA gene. The 5S/IGR- 1 product, amplified
using primers
LR12R and M-l, varied from 1.4 kb to 1.7 kb, depending on the isolate.
A number of RFLPs were seen upon digestion of the PCR- amplified DNA using
the previously mentioned restriction
endonucleases. For example, using the restriction
enzymeMspI, a 610 bp band was seen in most isolates, but absent from many others(Figs. 3 and 4). In addition, two isolates possessed unique RFLPs (Figs. 3 and 4). With

Figure 3. Restriction
analysis ofPCR-amplified
5' end of the 26S rRNA gene of Agaricus spp. digested
with
Msp I. Lanes Al-1 and A2-1 are 123 A DNA run as a size marker. LanesAl-2 through Al-20
include the isolates: WC145, MW14, WC213, MW8, WC21 1, WC83, WC344, WC714, WC71,
WC280, WC212, MW17, WC413, WC253, WC345, WC346,WC136,WC133,
MW10. Lanes
A2-2 through A2-20 are: WC415, WC278, WC19? WC416-26, WC416, WC76, WC418, WC106,
WC1 1, WC277, WC414, ^^0129^4,
WC28, WC210, WC209, MC348, MC404, MC310.
Figure 4. Restriction analysis ofPCR-amplified
5' end of the 26S rRNA gene of Agaricus spp. digested with
Msp I. Lanes Bl-1, Bl-13, B2-1, and B2-13 are 123 A DNA run as a size marker, lanes Bl-2
through Bl-12 were: WC773, MC428, WC277, MC13, WC774, MC430, WC782, WC781?
WC779, WC776, MC429. Lanes B2-2 through B2-12 were: WC784, MC406, WC778, WC780,
WC777-A, WC775, WC777-B, MC436, WG772, WC785, control.

197

MspI, WC773 had a 260 bp band, not found in any other isolate. Likewise, a 370 bp
band was unique to WC776.
The 5' end of the 26S gene showed more variability
than the 3' end. In^fact,many restriction
digests of the 3' end showed no RFLPs. The most variation among
restriction
sites was seen with the 5S/IGR-1 product. Analysis of restriction
data fromthis region alone, revealed a divergence of 16.5% between A. caroli and all the otherspecies of Agaricus, This species represented the greatest divergence from all other
Agaricus isolates examined.
Analysis of the 5S/IGR- 1 confirmed differences
between isolates that the 26S
gene analysis was unable to resolve. Many individuals
were found to be identical
basedon RFLP analysis of the 3 ' and 5' ends of the 26S gene. Three isolates of A. bisporus(MC310, MC348, and MC404) were found to be identical following analysis of all three
regions. Many isolates of A. augustus also were found to be nearly identical.
A phylogenetic
tree (Fig. 5) was contructed showing the amount of difference
among all the isolates
examined, using the Neighbor- joining
method. The NJ tree
clustered most isolates of Agaricus closely to A bisporus, suggesting that most species
in the genus are closely related. However, the genus seems to be highly polymorphic,
as several cases are shown where there is greater intra- (than inter- ) species dissimilarity(Fig. 5). Similarly,
the NJ tree, constructed for all the data pooled by species, also clusters
most putative species of Agaricus very closely toA. bisporus (Fig. 6). The most distantly
related species,^, caroli, differed fromall other species by more than 16.5%. The next
most distantly related species were A. chionodermus and A. excellens, which clusteredloosely to one another (Fig. 6). The phylogenetic
tree (Fig. 7) constructedfrom RFLP
analysis of IGR- 1 and IGR- 2 for all isolates of Agaricus bisporus suggests more thantwelve genotypicclasses exist among isolates in the Pennsylvania
State University
Mushroom Culture Collection.
Outlook
For commercial and wild mushroom isolates, researchers have used allozyme
analysis to identify genotypic classes among commercial and wild mushroom isolates,
including^,
bisporus (Royse and May, 1989; Royse et a/., 1983b; May and Royse,
1982b; Royse and May, 1982b), Morchetla spp. (Royse and May, 1990), Lentinula
edodes (Royse and May, 1987), and Volvariella volvacea (Royse et al., 1987). In
future studies, RFLP analysis of genes in a gene subunit or nongenic regions of rDNA
may lead to further elucidationof evolutionary
relationships.
Using modern methods
such as RFLP analysis to investigate
the subunits (18S, 5.8S, 28S, and 5S) or ITS/IGR
regions of ribosomal DNA's may allow for measurements as well as comparisons of
the phylogenetic
relationshipsof organisms (Sogin,1990) over a wide range oftaxonomic levels (Medlin et a/., 1988; Woese and Olsen, 1986). The nuclear small
subunit rDNA sequences (18S- like) are useful for studying distantly
related organisms
as they are believed
to evolve more slowly than mitochondrial
rDNA (White et a/.,1990). Thus the 18S- like rDNA may be more suitable for the study of more closely

198

Figure

5. Phylogenetic
relationshipsof Agaricus spp. isolates used in this study. The phylogenetic
tree isbased on data resulting from RFLP analysis of the 3' and 5' halves of the 26S rRNA gene and theIGR-1/5S rDNA region using the computer program RESTSITE (Nei and Miller, 1990). The
phylogenetic
tree was constructed using the Neighbor-joining
Method (Saitou and Nei, 1 987)
option within the computer program MEGA (Kumar et al, 1993). Branch lengths and scale barcorrespond to evolutionary distances (% X 100) assigned by MEGA, measured by the number ofnucleotide substitutions
between sequences.

Figure 6. Evolutionary positions of Agaricus species determined using RFLP analysis of the 3 ' and 5 ' halvesof the 26S rRNA gene and the IGR-1/5S rDNA region. The phylogenetic
tree is based on the datafrom Figure 1 , pooled by species using the computer program RESTSITE (Nei and Miller, 1990).
The phylogenetic tree was constructed using the Neighbor-joining
Method (Saitou and Nei, 1 987)
option within the computer program MEGA (Kumar et al.9 1993). Branch lengths and scale barcorrespond to evolutionary distances (% X 100) assigned by MEGA, measured by the number ofnucleotide substitutions
between sequences.

Figure7. Genotypic classes of Agaricus bisporus isolates used in this study. The NJ phylogenetic
tree isbased on data resulting from RFLP analysis of the IGR-1 and IGR-2 from the rDNA tandem repeat
using the computer program RESTSITE (Nei and Miller, 1 990).

201

related organisms (ie. at the family or genus level). The internal transcribed spacer region
and intergenic
spacers of the nuclear rDNA repeated regions are believed to evolve
fastest, therefore, are best suited for very closely related individuals(ie. within a genus
or population)(White et al., 1990).
The future of research with Agaricus and other microorganisms will be an
exciting one. As our knowledge of the genetic makeup, and of techniques to explorethat genetic makeup, continues to increase, great advances in the breeding of Agaricus
bisporus should continue to occur. Newly- developed techniques
for rapid genetic
analysis
will allow the detectionof untapped genetic resources from natural, wild
populations.
Location and preservationof this undiscovered
genetic diversity
is ofutmost importance nowconsidering the ever-increasingrate of habitat loss.
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207

QuestionsQ.

and Answers Session 5
Are biological

speciesof Armillariainterfertile?

(Matsumoto)A,
Some are, for example, A. cepistipes in Europe is partiallyinterfertile
withtwo biological
species in North America, and almost fully interfertile
with onebiological
species there. The three North American biological
species are completelyintersterilewith each other. (Hasegawa)Q.
Are there interspecies
sterility
genes which determine
between biological
species? (Matsumoto)genetic

relationships

A.
The intraspecificmating system is controlledby 2 mating genes, but genes
which determine the intersterility
between biological
species have not been found as
far as I am aware. (Hasegawa)
Q,

Which is better

to use Armillaria

or Armillariellaand why?

A.
Armillariella was used by Singer after Armillaria was established.
Watlingetal.(l982)
in Trans. Br. Mycol. Soc. pointed out that the description
associated with
Armillariella
was insufficient
to be considered
a new taxon. After their paper
Armillaria is used. (Hasegawa)Q.
species?

Howvalid are compatibility(dela Cruz)

tests in studying

the distributionof Armillaria

A.
From the stand point of preventionof root rots an understandingof thecharacteristicsof these pathogens
is very important.
If the pathogen
is different
between northern and southern Armillaria and the characteristicsof these pathogens
well differentiatedfrom each other, the methods for their control may also differ.
(Hasegawa)Q.
Are all Armillaria
Basidiomycetes?
(Yanagi)

diploid?

Is no dikaryotic

stage

present

as in otherA,
Single spore isolates are haploid in most species. Soon after mating occurs,
the dikaryotic
phase changes to monokaryotic diploid. A few species are homothallic
and their single spore isolates
have the same appearance that tissue isolates
have.
(Hasegawa)Q.
species

What is suggested
from other regions?

by the compatibility(Yanagi)of Japanese

specieswith so many

208

A,
One reason is that there is no standard set of Japanese testers of biologicalstrains. Another is that relationships
between Japanese isolates and European or North
American testers may reveal how the biological
species came to Japan, and how they
spread in the world. (Hasegawa)Q.
Do you have information
about the distributionof T. bakamatsutake) T.
bakamatsutake is also ectomycorrhizal,
having slow growth and no clamp connections.
So, what is the reason that you suggested it may be commercially important in the near
future? (Yanagi)
A.
Outside of Japan mainly in Europe and North America, this species could beclassified
to T. caliatum. Therefore, this fungus has not been found outside Japan
except for New Guinea.
In Japan, T.ponderosumfromCanada and U.S.A. and T.bakamatsutake from
Morocco already have commercial value in the market. T. bakamatsutake has somecharactersfavorable
to Japanese tastes, I suppose this fungus will have somecommercial value when it appears in the market. Slow mycelial growth and no clamp
connections are biologicalcharacteristics,
and this would be the reason this species isdifficultto use as experimental
material.
(Iwase)Q.
To obtain protoplasts
is an alternative
method to obtain homokaryons insecondary homothallic
species such as Agaricus bisporus. Have you ever tried this?
If so, was it successful?
(Matsumoto)A.
techniqueNo, I have not attempted to create protoplasts,
but it is possible.
has been used by others at Penn State and other institutes.
(Bunyard)

Q.
How do you distinguish(Yanagi)A.
hyphae.

mono- and di- karyons

Thein Agaricus bisporus1}

It is difficult
to tell the difference
between homo- and hetero- karyotic
They can be distinguishedby DNA or allozyme analysis.
(Bunyard)

Q,
Is there any prefered method for promoting the germinationbasidiosporesin Agaricus bisporus in order to obtain homokaryoticof homokaryotic
strains?
(Iwase)A.
There is no way to promote, selectively,
the germinationof homokaryotic
basidiospores.
A higher proportionof homokaryons can be obtained by selecting
slower growing single spore germlings. The creation of homokaryotic
mycelium ispossible through methods such as protoplast
generation,etc. (Bunyard)

GENERAL DISCUSSION

Chairpersons
Tomio Yoshida
Duncan A. Vaughan

211

General Discussion
Recommendationsof the workshop partcipants

1. General
Microbial Diversity 21(a) sets out a well thought out general action statement.
However, the workshop felt that this could be more clearly targeted.
With governments worldwide focussed on biodiversity
as an important
global issue, particularly
since the Convention on Biodiversity
has been ratified bymany countries, now is a time to develop some targets to achieve, and projects forgovernments to support financially.
The workshop recommended the developmentof such target oriented
projects.
2. Funding
Funding microbial
conservation
involves five separate but related issues,collecting,evaluation,
use/application,
preservation/maintenance,
and infrastucturedevelopment.
Especially rapidly developing
countries with much biodiversity
needs to besupported in development of their culture collections.
Specific projects should be agreed and funded properly.
It is necessary for
projects
to be provided with sufficientfunding to achieve worthwhile objectives.
Although long term funding can never be guaranteed, provision for this must be madeeither by core funding or meeting needs through income. The workshop recommended
centralised
coordinated funding for specific target oriented projects.
3. Training/Expertise
As has been recognised by other meetings of microbial scientists
there is a
decline in the number of microbial systematists.
The workshop recommended thedevelopment of a program to remedy the situation by producing the needed capabilities
through a carefully developed curricula and syllabuses
combining traditionalandmodern approaches.
The workshop recommended support for further informationexchangeamongyoung scientists including international
exchange of researchers.
4. Preservation
technology
The workshop recognised
the need for optimizationof preservation
technology.
The workshop recommended targeting cryopreservation
protocols for
additional
studies.
5. Targetingstudiesof particularspeciesor microbial

groups

212

The workshop recommended that culture collections should have clear focus
and targets. The workshop recommended that culture collections
should be expandedwith particularemphasis to detect unknown and useful organisms and those species
that have not yet been cultured.
6. Information
systems and exchange
The workshop recommended enhanced exchange of information.
The
workshop recommended that data storage should concentrate on one system e.g. World
Data Center for Microorganisms, rather than sponsoring new projects.
The workshop recommended that an enhanced effort to evaluate
microorganisms was necessary and that the lag time between evaluation and input of
evaluation data into an internationallyaccessable database should be shortened.
The workshop recognised the need to set up a world network of collections,with transfer of informationand member collections
meeting set standards.
The
workshop recommended that an agreed strategy should be formulated and acceptedby all members of the world network.
7. Internationalisationand sharing mechanisms
The workshop recommended increased international
responsibilities
for microbial conservation and evaluation.

cooperation

to share

8. Workshops
The workshop recognised
the value of workshops.
For several specificmicrobial groups, such as Fusarium, good international
networks already exist. The
personal contacts developed during workshops can be valuable in stimulating
more
international
cooperation
therefore
the workshop recommended that a further
workshop to plan future strategy should be arranged.
(a) Hawksworth, D. L. and R. R. Colwell.
amongst microorganisms and its relevance.

1992. Microbial Diversity
21: biodiversity
Biodiversity
and conservation 1 : 221-226.

CLOSING REMARKS

Masahiro

Nakagahra

215

Closing

Remarks

MASAHIRO NAKAGAHRA
Genetic Resources Coordinator,

NIAR

Wehave nowreached the end of all the sessions of this workshop. On behalfof the organizing committee, I should say a few words.
First of all, I would like to thank all the chairpersons,speakers and otherparticipants
for their valuable contributions.
I would like to express my thanks to thecooperating institutes,
the National Agriculture Research Center, the National Instituteof Agro- Environmental
Sciences, the Japan International
Research Center for
Agricultural
Sciences, the National Food Research Institute and the Forestry and Forest
Products Research Institute,in Tsukuba, for helping
to organize this workshop
successfully.
I am also grateful to the Agriculture,
Forestry and Fisheries ResearchCouncil for providing
full financial
support for this workshop.
The MAFF International
Workshops on Genetic Resources aim to promoteresearch, exchange and collaborationon the development of technologies
and global
strategies
for the conservation and use of genetic resources in national programs andthe international
research institutes.
I am concerned that, compared to plant genetic resources, we do not have asufficiently
strong network involved in the global conservation and, more importantly,
evaluation of microbial genetic resources. Weexpend great efforts on studying a few
commonfungi but how much effort are we placing on seeking useful products frommicroorganisms- very many of which are still not even documented, as Dr. Smith
discussed
yesterday.
I believe
we are in need of a major partnership
betweengovernment researchers and industry to understand better the microbial world for
mankinds benefit.
yThese last two days we have had papers which have, in some cases, only beenloosely connected around a central theme. A two day workshop, of course, cannot dojustice to the topic of soil microorganisms
and the conservation of microorganisms.
However, it is a small contribution
to the topic and , I believe, that perhaps more thanthe excellent papers that we have enjoyed hearing, it has been the person to person
contacts of scientists
with similar interests that has been most important. Tomorrowthese interactions
can continue in a less formal atmosphere and I hope the program
prepared will be enjoyed by all.
Besides, the above, I am very happy to hear that recommendations will comeas a result of this workshop. It will be incorporated
into the proceedings which shouldbe publishedin the near future.
On behalf of all members of the organizing committee, I wish to express our
sincere thanks to all of you who have so actively participatedin this workshop to makeit such a success. I thank you all for your active participationand cooperation.
I
would like to extend my best wishes to all of you for your work in your countries andorganizations.
I and all the staff members of Tsukuba institutions
are looking forward

216

to seeing you again in the near future.
It is now my duty and honor to declare

the Indoor-workshop

Thank you very much for your kind cooperation.

closed.

LIST OF PARTICIPANTS

220

AKAMA, Keiko
Forest Biology
MAFF, Japan

Division,

Forestryand Forest

ANDO, Shotaro (Secretary
Environmental

Staff)
Resources

Division,

JIRCAS,

AOKI, Takayuki(Secretary
Dept. of Genetic

Staff)
Resources

I, NIAR,

Products

MAFF, Japan

ASAWA, Kazutaka
Bio-resources
Technology
Division,
Research Institute,
MAFF, Japan

CHAU, N. T.
Postharvest
Technology
CHIBANA,

dela

Takashi(Secretary
Staff)
Dept. of Genetic
Resources

CRUZ, R. E. (Session
1)
Biotech,UPLB College,

EBASHI,

Shinji(Secretary
Administration

ENOKIDA,EZAKI,

Ryuzo
TsukubaTakeshiNational

FUJIMAKI,

Hiroshi
Director,

Staff)
Department,

Forestry

Forestryand Forest

Mushroom Research
State University,
USA

Technology
Division,
Institute,Vietnam)

NFRI,

II, NIAR,

Laguna,NIAR,(Opening
General,for Seeds

The Philippines

MAFF, Japan

Address)
NIAR, MAFF, Japan

Products

MAFF, Japan (Postharvest

MAFF, Japanand Seedlings,

and Fisheries

Lab., 3 15 Buckhout

Res. Labs. SANKYO Co. Ltd., Japan

CenterInstitute,

MAFF, Japan

AOKI, Toshimitsu(Secretary
Staff)
Liaisonand Coordination
Division,
Agriculture,
Research Council
Secretariat,
MAFF, Japan

BUNYARD, Britt (Session
5)
Department of Plant Pathology,
Laboratory,
The Pennsylvania

Research

MAFF, Japan221

FUKUOKA, Shuuichi
Dept. of Genetic

Resources

I, NIAR,

MAFF, JapanFURUKAWA, Tsutomu (Secretary
Staff)
Dept. of Genetic Resources I, NIAR,

MAFF, Japan

GAMO, Takuma
Director,

NIAR,

Dept. of Molecular

Biology,

MAFF, JapanHAGIWARA, Hiroshi(Secretary
Staff)
Dept. of Plant Protection,
NARC, MAFF, Japan
HAKAMADA, Tetsuji
Shizuoka
Pref.

Forestry& Forest

Products

5)
Division,

Forestryand Forest

Products

Research

Institute,

Division,

Forestryand Forest

Products

Research

Institute,

CAAS,

People's

Republicof China

HASEGAWA, Eri (Session
Forest Biology
MAFF, JapanHATTORI,

Tsutomu
Forest Biology
MAFF, Japan

HE, Li-Yuan

HIRAYAE,HIROKI,

MikiyaNationalof Plant

Institute

Mitsuo (Secretary
Dept. of Genetic

Protection,

Institute,Japan

MAFF, Japanfor Environmental

Studies,

Staff)
Resources

MAFF, Japan

II, NIAR,

Environment

Agency,FusakoFaculty

INABA,

3)
InstituteKazuyuki (Secretary
Staff)
Dept. of Genetic Resources II, NIAR,

HORITA,

IMAI,(SessionSKLBPI,

Researchof Horticulture,

Chiba

University,

Tadaoki(Organizing
Committee)
Director,
Dept. of Plant Protection,

ISHIKAWA,IWANAMI,

NARC, MAFF, Japan

Masaya (Secretary
Staff)
Dept. of Genetic Resources II, NIAR,
SetsuoDept. of Research

Planning

Japan

MAFF, Japan& Coordination,

NIAR,

MAFF, Japan

Japan

222

IWASE,Koji

IZAIKE,(Session
5)
Biological
Environment
Center Co., Ltd., Japan

Yoshiaki
Dept.

(Secretaryof Genetic

Staff)
Resources

KADOTA, Ikuo
Dept. of Environmental
MAFF, Japan
KAKU, Hisatoshi
Dept.

(Secretaryof Genetic

KANEKO, Shigeru
Forest Biology
MAFF, Japan
KATO, Kuraji
National
KATSUTA,

Center

KIDA, Shigeki

KansaiII, NIAR,

Biology,of Microbiology,

Japan

and Forest

Products

Forestry

for Seeds

and Seedlings,

NIAES,

Research

MAFF, Japan

I, NIAR,

MAFF, Japan(Welcome
Address)
Research Councillor(Deputy
Director
Fisheries
Research
CouncilSecretariat,

Division

General),Agriculture,
MAFF, Japan

Forestryof Microbiology,

NIAES,

KODAMA, Kentaro
Tsukuba Res. Labs. SANKYO Co. Ltd., Japan
KOIWA, Toshiyuki
Forestryand Forest
KUNIHIRO,

KUSUNOKI,

Products

Research

Yasufumi(Secretary
Staff)
Dept. of Genetic
Resources II, NIAR,
ManabuInstitute,

MAFF, Japan

I, NIAR,

Ryusuke (Secretary
Staff)
Dept. of Environmental
Biology,
MAFF, Japan

Engineering

MAFF, Japan

MAFF,

Division,

Staff)
Resources

Environmental

Division

Staff, Session
1)
Resources
I, NIAR,

Masumi (Secretary
Staff)
Dept. of Genetic
Resources

KAWADA, Masae (Secretary
Dept. of Genetic

KIMURA,

Institute,

Institute,

MAFF,

MAFF, Japan

Japan

and

223

Forest Biology
MAFF, Japan

Division,

LADHA, J. K. (Session
2)
Soil Microbiology
The Philippines

Forestryand Forest

Section,International

Rice

LUMYONG, Saisamorn
Dept. of Biology,

Facultyof Science,

Chiang

MAEDA, Miki
Dept. of Genetic

Resources

II, NIAR,

MANABE, Masaru
Director,(Organizing
Committee)
AppliedMicrobiology

MATSUMOTO, Naoyuki (Secretary
Dept. of Environmental
MAFF, JapanMIKAMI,MINAMI,MINOBE,MIZUNO,

Institute,

Research

Institute(IRRI),Mai University,

Division,

NFRI,

Divisionof Microbiology,

MAFF, JapanNIAES,

National
Institute
MITI, Japan

Kastuyuki
Director,(Organizing
Environmental

JIRCAS,

Committee)
Resources

Division,

Yuzo (Organizing
Director,
Dept.

Committee)of Genetic
Resources

Shun-Ichiro
Forest Biology
MAFF, Japan

Division,

Forestry

I, NIAR,and Forest

Susumu (Secretary
Staff)
Dept. of Genetic
Resources II, NIAR,
Shoji
Director,

BiologicalAkifumiDept. of Environmental
MAFF, Japan

Resources

Biology,Thailand

MAFF, Japan

AppliedMicrobiology
Department,
and Human-Technology(NIBH),MIYASHITA,MIZUNO,

Research

Eiichi
Director,
Bioscience

MIYASHITA,MIYAZAKI,

Staff)
Biology,

Products

of

MAFF, Japan

MAFF, Japan

Products

Research

Institute,

MAFF, Japan

Division,

Division

JIRCAS,

MAFF, Japanof Microbiology,

Tadayoshi
Tsukuba Res. Labs. SANKYO Co. Ltd., JapanNIAES,

224

MOCHIZUKI,
Masahiro
Facultyof Horticulture,
MORIWAKI,

Joji
Dept. of Environmental
MAFF, JapanNAGAO, Hideyuki
Facultyof Horticulture,

Chiba

University,

Japan

Biology,

Division

Chiba

University,of Microbiology,

NIAES,Japan

NAGATA, Toru (Organizing
Committee)
Director,
Dept. of Research Planning& Coordination,Japan
NAITO, Shigeo
Tohoku National
Agricultural
Experimental
Station,

1MIAR,

MAFF,

MAFF, Japan

NAKAGAHRA, Masahiro (Organizing
Committee)
Genetic Resources Coordinator,
NIAR, MAFF, Japan
NAKASE, Takashi
Director,
Physical(Organizing
Committee,
KeynoteJapan Collectionof Microorganisms
and Chemical
Research (RIKEN),

NAKASHIMA, Kazuo
Biological
Resources

Division,

JIRCAS,

NAKAYAMA, Hiroki (secretary
Staff)
Dept. of Genetic Resources I, NIAR,NAMIKI,

Fumio (Session
4)
Kyusyu National
Agricultural

NIRASAWA,NISHIJIMA,NISHIMURA,NISHIYAMA,

Address)(JCM),
Japan

MAFF, Japan

MAFF, Japan

Experiment

Station,Keijiro(Secretary
Staff)
Dept. of Genetic Resources I, NIAR,

MAFF, Japan

Takuya (Secretary
Staff)
Dept. of Genetic Resources I, NIAR,

MAFF, Japan

Marie (Secretary
Staff)
Dept. of Genetic Resources I, NIAR,

MAFF, Japan

KoushiDept. of Environmental
MAFF, Japan

Biology,

The Institute

DivisionMAFF,of Microbiology,

Japan

NIAES,of

225

NODA, Takahito
Crop ProductionJapan
NOGUCHI,Junko (Secretary
Dept. of Genetic

NUNEZ, Maria P.
Forest Biology
MAFF, JapanOCHIAI,and Postharvest

Staff)
Resources

Division,

Technology

II, NIAR,

Forestry

Hirokazu(Secretary
Staff)
Dept. of Genetic Resources

Division,

Products

Akira (Session
4)
Faculty of Agriculture,

OHKUBO, Horoto
Dept. of Environmental
MAFF, Japan

Hokkaido

University,

Japan

Biology,

Divisionof Microbiology,

OHMASA, Masatake (Secretary
Staff)
Bio-resources
Technology
Division,
Research Institute,
MAFF, JapanOISHI,OITA, Shigeru
Biological

Function

Division,

OKABE, Ikuko
Dept. of Environmental
MAFF, JapanOKAZAKI,

Takao
Tsukuba

NFRI,

Biology,and Forest

II, NIAR,

Masaomi (Secretary
Dept. of Genetic

Staff)
Resources

Institute,

Forestry

NIAES,

Products

MAFF, Japan

MAFF, Japan

Divisionof Microbiology,

Res. Labs. SANKYO Co. Ltd., Japan

OKUNO, Kazutoshi(Secretary
Staff)
Dept. of Genetic Resources
ONIKI,

Forestry

Takao (Organizing
Committee)
Director,
Dept. of Genetic Resources

Research

MAFF, Japan

OGAWA, Kazuki (Organizing
Committee)
Director,
Liaisonand Coordination
Division,
Agriculture,
Fisheries
Research Council Secretariat,
MAFF, Japan
OGOSHI,

MAFF,

MAFF, Japanand Forest

I, NIAR,JIRCAS,I, NIAR,

MAFF, Japan

II, NIAR,

MAFF, JapanNIAES,and226

PISAN,

Sirithorn
Director,

Dept.

of Molecular

Biology,

NIAR,

MAFF, Japan

Dept.

of Natural

Resources,

NIAES,

MAFF, Japan

RAN, Michio
Director,
RIFAI,

Mien A. (Session
1)
Herbarium
Bogoriense

SALLEH,

Puslitbang

B. Bin (Session
4)
School of Biological

SASAKI,

Sachio (Secretary
Dept. of Genetic

Sciences,

Universtiti

Staff)
Resources

SASTRAATMADJA,
Dudi
LIPI R&D Centre

Biologi-LIPI,

Sains

II, NIAR,

for Biology,

Indonesia

Malaysia,

MAFF, Japan

Indonesia

SATO, Kyoko (Secretary
Staff)
Liaisonand Coordination
Division,
Agriculture,
Research Council
Secretariat,
MAFF, JapanSAWADA, Hiroyuki(Session3)
Dept. of Environmental
MAFF, JapanSHIINA,

Tsugio (Secretary
Dept. of Genetic

SHIRATA,

SMITH,SONE,SUZUI,David (Keynote
International
Teruo
Faculty

TAKAHASHI,
Dept.
TAKEHARA,Division

I, NIAR,

Staff)
Planning& Coordination,

Address)
Mycologicalof Agriculture,

Takahito(Organizing
SDS-BIOTECH,

Biology,

Staff)
Resources

Kazuto (Secretary
Dept. of ResearchInstitute

Hokkaido

Forestryof Microbiology,

University,

NIAR,

UK

Japan

Committee)
Japan

Protection,

and Fisheries

NIAES,

MAFF, Japan(IMI),

Hideaki(Secretary
Staff)
of Genetic
Resources I, NIAR,

ToshiakiDept. of Plant

Malaysia

MAFF, Japan

NARC, MAFF, Japan

MAFF, Japan

227

TAKEYA,

MasaruDept. of Genetic

TAMANG, J. P.
AppliedIndia)TAMURA, Hirotada
Director,
Institute,
TANAKA, Osamu
Biological

Microbiology

MotoakiFacultyI, NIAR,

Division,

Function

MAFF, Japan

NFRI,(Organizing
Committee)
Forest Biology
Division,
MAFF, JapanTANGCHAM, B. (Session
Soil Microbiologyof Agriculture,
TOJO,

Resources

Forestry

TSUGE,TSUNODA,

TSUSHIMA,

UMEHARA,VAUGHAN,

Fellow,

Products

MAFF,

Japan

Research
ThailandGroup,

Divisionof Soil

Science,of Osaka Prefecture,Japan

Research2)of Agriculture,(Secretaryof Genetic

and Forest

NFRI,

University

Kenichi(Secretary
Staff,
Dept. of Genetic
Resources

MichiyoDept.

Japan (UNU

Division,

TOMOOKA, Norihiko(Secretary
Staff)
Dept. of Genetic
Resources
I, NIAR,
TSUCHIYA,

MAFF,

Staff)
Resources

Mitsutoshi
Bio-resources
Technology
Research Institute,
MAFF,
Seiya
Dept. of Environmental
MAFF, Japan

Department

MAFF, Japan

Session 3)
II, NIAR, MAFF, Japan

II, NIAR,

MAFF, Japan

Division,Japan

Forestry

Biology,

Division

Masamichi(Secretary
Staff)
Dept. of Genetic
Resources I, NIAR,

and Forestof Microbiology,

Products

NIAES,

MAFF, Japan

Duncan A. (Secretary
Staff, General
Discussion)
Dept. of Genetic
Resources
I, NIAR, MAFF, JapanWATANABE, Makoto M.
National
Institutefor Environmental

Studies,

Environment

Agency,

Japan

228

YAEGASHI,

Hiroshi(Organizing
Dept. of Environmental
MAFF, JapanYAMAOKA, Masakazu
National
InstituteJapan

Committee)
Biology,
Divisionof Bioscienceof Microbiology,

and Human-Technology

YAMAMOTO, Akio (Organizing
Committee)
Liaisonand Coordination
Division,
Agriculture,
Research Council Secretariat,
MAFF, JapanYAMANAKA, Takashi
Forest Biology
MAFF, JapanYANAGI,

Division,

Forestry

Sonoe (Secretary
Staff)
Biological
Function
Division,

YANAGISAWA,
Dept.

and Forest

Forestry

Products

NFRI,

MAFF, Japan

Chie (Secretary
Staff)
of Genetic Resources II, NIAR,

MAFF, Japan

YOKOYAMA, Tadashi (Secretary
Staff, Session 2)
Dept. of Genetic Resources I, NIAR, MAFF, JapanYOSHIDA,Tomio (Organizing
Faculty of Horticulture,

Committee,
GeneralChiba University,

Discussion)Japan

NIAES,(NIBH),AIST,

and Fisheries

Research

Institute,

Editors
Editor in Chief
Managing Editors

C onsulting

Editors

Kato, Kunihiko
Duncan A. Vaughan
Oniki, Masaomi
Horita, Mitsuo
Shirata, Kazuto
Seko, Hidefumi
Nakagahra, Masahiro
Minobe, Yuzo
Oishi, Takao

PublishedbyMarch, 1996
Research Council
National Institute2-1-2 Kannondai,
ISBN4-9900110-6-6

Secretariatof MAFF andofAgrobiological
Resources
Tsukuba, Ibaraki 305, Japan(N. I, A. R.)