Biological control system

This invention relates to a method of biologically controlling plant diseases caused by sclerotia of sporidesmium susceptible plant pathogenic fungi.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a system for the biological control of plant 
pathogenic fungi in soil and more particularly to the use of the 
mycoparasite, Sporidesmium sclerotivorum, to control and destroy or 
eradicate the sclerotia of plant pathogenic fungi susceptible to 
Sporidesmium such as those of the genus Sclerotinia and Sclerotium 
cepivorum. The invention also relates to a novel method of producing the 
mycoparasite, Sporidesmium sclerotivorum. 
2. Description of the Prior Art 
Although the concept of biological control of plant disease microorganisms 
has been known for a number of years, there is no known relevant art 
regarding the mycoparasite, Sporidesmium sclerotivorum, because it has 
only recently been discovered. 
A subculture of this mycoparasite can be obtained from the permanent 
collection of the Northern Regional Research Center, Science and Education 
Administration, U.S. Department of Agriculture, Peoria, Ill., U.S.A., 
61604. Its Accession No. in this repository is NRRL 11437. As of the 
filing date of this application, progeny of the subject strain will be 
made available to anyone who requests the same. A taxonomic description of 
the mycoparasite is found in Mycotaxon 7, 275-282, 1978, which is herein 
incorporated by reference. 
Coniothyrium minitans is a well known mycoparasite of Sclerotinia spp. and 
Sclerotium spp. and under controlled conditions has shown promising 
results in reducing the inoculum potential of these fungi. However, a 
field trial in soil naturally infested with S. sclerotiorum was only 
partially successful. 
BRIEF SUMMARY OF THE INVENTION 
An object of this invention is to provide a simple, economically sound, 
non-polluting means of controlling plant disease. 
Another object is to provide a means of increasing yields of agricultural 
products by controlling and eradicating soil-borne plant pathogenic fungi. 
A further object is to provide means of increasing plant health and yields 
of agricultural products while reducing or eliminating the use of 
chemicals for disease control. 
A still further object is to provide a means of biologically controlling 
the sclerotia of the plant pathogenic fungi of the genus Sclerotinia and 
Sclerotium cepivorum, the causative agents of lettuce drop, bean white 
mold, peanut blight, onion white rot, and other serious plant diseases of 
food crops and other plants such as sunflower, clover and alfalfa. 
Still another object is to provide a novel method of producing the 
mycoparasite, Sporidesmium sclerotivorum. 
According to this invention the above objects are accomplished by a novel 
method of producing the mycoparasite, Sporidesmium sclerotivorum, wherein 
a sterilized culture medium that will support the growth of sclerotial 
producing fungus is inoculated with a species of fungus susceptible to 
Sporidesmium, the inoculated medium incubated at a temperature and for 
length of time effective for the production of sclerotia, and the 
sclerotia collected and added to fine quartz sand or other inert media. A 
culture of S. sclerotivorum is then added to and thoroughly mixed with the 
sand-sclerotia mixture and incubated at a temperature and for a length of 
time effective for the growth and production of Sporidesmium. 
The above objects are further accomplished by a method wherein an effective 
sclerotia destroying amount of an inoculum of the so-produced 
mycoparasite, Sporidesmium sclerotivorum, is added to and mixed with soil 
to at least that depth at which Sporidesmium susceptible sclerotia reside 
and allowed to develop in the soil for that length of time required for 
the Sporidesmium to effectively infect, consume and destroy at least 90% 
of the Sporidesmium susceptible pathogenic sclerotia in the soil. 
DESCRIPTION OF THE INVENTION 
The control of soilborne plant diseases is the greatest unresolved problem 
of plant pathology. Conventional breeding for resistance has provided 
effective relief from very few of these diseases. Crop rotations, although 
sometimes effective, are becoming less and less practical under today's 
economic pressures. The use of chemical pesticides is frequently 
economically impractical or ecologically unacceptable. Therefore, finding 
how to produce the recently discovered mycoparasite, Sporidesmium 
sclerotivorum, and how to use it to achieve biological control of plant 
disease is a very important discovery. 
The recently discovered mycoparasite of plant pathogens such as the genus 
Sclerotinia and Sclerotium cepivorum, the causative agents of lettuce 
drop, bean white mold, peanut blight, onion white rot, and other serious 
plant diseases of food crops and other plants, destructively infects 
living sclerotia of the plant pathogens in natural soils in which it is 
introduced. The genus Sclerotinia includes three species: S. minor, S. 
trifolium, and S. sclerotiorum. Complete destruction of sclerotia by the 
mycoparasite, Sporidesmium sclerotivorum, can be effected within ten 
weeks. The mycoparasite has the unusual ability to grow through soil from 
the host sclerotium to another and produce great numbers of conidia 
throughout the soil. 
Certain plant pathogenic fungi as, for example, the Sclerotinia species and 
Sclerotium cepivorum cause severe economic losses throughout the United 
States and other countries on many vegetable crops including beans, 
lettuce, celery, potatoes, and tomatoes; on several oil crops including 
mint, peanut, and sunflower; and on forage legumes including clover and 
alfalfa. For most of these diseases there are no satisfactory chemical 
control measures available, nor are there genetically resistant plant 
varieties. 
Sclerotia are the principal survival structures of certain soilborne plant 
pathogenic fungi and constitute an important link in the epidemiology of 
plant diseases caused by several plant pathogens. These hard, resistant 
fungal structures survive desiccation in soil and can remain viable for 
years and act directly as sources of infection. 
We attempted to culture Sporidesmium sclerotivorum on ordinary culture 
media with no success. After much experimentation and many failures, we 
found that a medium prepared from comminuted sclerotia of S. minor in 
sterile water agar permitted germination of conidia of the fungus. Later, 
we obtained an axenic culture of the fungus from such a single germinated 
conidium. We tried to culture the isolate on many different potential 
substrates, such as sugars, amino acids, and vitamins, as well as on 
complex media that support the growth of many fastidious microorganisms, 
but were not successful. However, we did achieve continued growth of the 
isolate on media prepared from sclerotia of S. minor and on living 
sclerotia. Supplementation of sclerotial agar with a variety of extracts 
and compounds had either no observable effect or in some cases inhibited 
germination. The addition of the following substances in 0.1% 
concentrations to sclerotial agar did not further stimulate germination or 
growth: glucose, sucrose, trehalose, mannitol, chitin, melanin, calcium 
lignosulfonate, and yeast extract. Sterile soil extract (10 g loam stirred 
vigorously in distilled water for 1 hour, filtered, and sterilized by 
membrane filtration or by autoclaving) added to sclerotial agar before 
solidification also did not stimulate growth greater than that occurring 
on sclerotial agar alone. 
Other media which did not support either germination or growth included the 
following: water agar, potato dextrose agar, 0.1 and 1% yeast extract, V-8 
juice agar, and trypticasesoy agar. Autoclaved culture filtrates of S. 
minor grown for 14 days from potato dextrose broth (PDB), and moist 
autoclaved soil from which the isolate was originally obtained, also did 
not permit germination or growth. It did grow, but very poorly, on corn 
meal agar. 
We discovered that unlike other mycoparasites such as Coniothyrium minitans 
Camp., S. sclerotivorum invades only the sclerotia of Sclerotinia species 
and Sclerotium cepivorum and not the hyphae. We prepared agar media from 
comminuted washed hyphae mats containing sclerotia harvested from PDB 
cultures of S. minor 14 days old and found that it supported germination 
and growth of macroconidia and growth of the isolate. However, on a 
similar medium prepared from mats 7 days old which had not yet formed 
sclerotia, we obtained neither germination nor growth. This discovery 
indicates that sclerotia but not Sclerotinia hyphae contain an essential 
growth promoting substance for S. sclerotivorum. We found further evidence 
of this when we accomplished a gnotobiotic culture of S. sclerotivorum in 
vivo by the addition of small pieces of sclerotial agar bearing hyphae and 
conidia of the isolate to aseptically washed mycelial mats of S. minor 
with sclerotia from 4-week-old PDB cultures. After 3 weeks at 25.degree. 
C. many of the sclerotia were obviously infected and hyphae of the 
mycoparasite had spread throughout the culture forming new, typical 
macroconidia. The Sclerotinia hyphae were not invaded by S. sclerotivorum. 
We found that when we mixed sclerotia of S. minor into a natural soil and 
then artificially infested the mixture with a sand-sclerotia culture of 
Sporidesmium sclerotivorum, the sclerotia were rapidly infected by the 
mycoparasite. Survival of the sclerotia, as measured by their recovery on 
soil sieves, dropped dramatically after five weeks and almost to zero in 
ten weeks. The numbers of sclerotia in control soil to which the 
mycoparasite had not been added remained at about the original level. We 
obtained equivalent results when we repeated the experiment in different 
natural soils including soils in which the mycoparasite was introduced, 
that is, soils in which it was not a natural inhabitant. 
In our search for ways to grow and utilize the newly discovered 
mycoparasite as a biocontrol agent for sclerotial plant pathogens we found 
that Sporidesmium sclerotivorum has several distinctive qualities that 
make it a potentially-important biocontrol agent for sclerotial plant 
pathogens. Development of S. sclerotivorum is favored by living, rather 
than dead, sclerotia. The mycoparasite convincingly attacks sclerotia in 
natural soil in which it is introduced, as well as in soils in which it is 
a natural inhabitant. It spreads over and through soil from one sclerotium 
to another producing abundant conidia along its hyphae which serve as new 
infective units for further mycoparasitism of sclerotia. It forms 
thick-walled chlamydospores, which may function as propagules for 
long-term survival. These distinctive characteristics are lacking in most 
other mycoparasites of sclerotial fungi. 
The method of propagation of Sporidesmium and the method of its use differs 
significantly from previously known concepts of mycoparasite-biological 
control systems. Sporidesmium is cultured on living sclerotia of the plant 
pathogen for which biological control is sought. Other mycoparasites 
proposed for biological control of Sclerotinia and Sclerotium species are 
cultivated on sterilized grain, molasses or other sterilized organic 
material. Sporidesmium is grown in moist quartz sand or other inert media. 
It does not derive any nutrients from the media; all of its nutrients are 
derived from sclerotia which is added to the sand. Other fungi suggested 
for biological control derive nutrients from the grain or other organic 
media on which they are grown. 
The novelty of the present invention is attested to by the fact that there 
is only one antifungal biological control agent registered with the 
Environmental Protection Agency in the United States. This biological 
control system concerns the inoculation of pine stumps with Peniophora 
gigantea against infections of Fomes annosus, Ann. Appl. Biol. 52, 63-77, 
1963. 
The use of the living host material (sclerotia) as the substrate for 
propagation and dissemination of the mycoparasite is completely new and 
has several advantages over other methods: 
(a) It is not necessary to sterilize or maintain sterility in the mixtures 
during propagation. Living sclerotia have a natural resistance to invasion 
by most common soil microorganisms; thus, only certain mycoparasites, such 
as Sporidesmium, can invade the sclerotia and thereby propagate themselves 
without hinderance from contaminating microorganisms. On the other hand, 
sterilized organic materials such as cereal grains are subject to attack 
by a great many common bacteria and fungi, which may interfere in the 
cultivation of the mycoparasite unless special precautions are taken to 
exclude them. 
(b) The use of living host sclerotia in the propagating medium insures that 
the mycoparasitic behavior of the fungus is maintained, since it can 
obtain nutrients for growth only from the host. In contrast, mycoparasites 
propagated on sterilized organic matter may possibly lose or decrease 
their parasitic activity. 
(c) The delivery of the mycoparasite in such infected sclerotia mixtures to 
soil aids in the infection of sclerotia naturally present in the soil by 
serving as food bases from which the mycoparasite can expand. The presence 
of these specialized food bases (infected sclerotia) coupled with the 
distinctive, spreading behavior of Sporidesmium in soil is beneficial in 
hastening the mycoparasitism and biological control.

The invention is illustrated by the following examples: 
Preparation of Sporidesmium Inoculum 
In general, the method consists of culturing Sporidesmium sclerotivorum in 
living sclerotia of a species of fungus which is susceptible to 
Sporidesmium as, for example, the Sclerotinia species and Sclerotium 
cepivorum in moist quartz sand or in other inert supporting media, at a 
temperature of 15.degree. to 25.degree. C. The preferred method of culture 
at present is the following: 
(a) Sclerotia of S. minor are first cultured on moist, sterilized 
animal-feed oats in sterile glass containers. Oats and water in the ratio 
of 50:60 by weight are sterilized by autoclaving at 121.degree. C. for 30 
minutes. An inoculum of S. minor is introduced aseptically into the oats 
and the preparation is then incubated at room temperature 
(20.degree.-25.degree. C.) for 4 weeks or longer. The medium for producing 
the sclerotia is not critical, a variety of materials may be used. 
(b) Sclerotia are separated from the oats and collected by passing the 
mixture through appropriate-sized soil sieves, preferably with the aid of 
running water. 
(c) Collected sclerotia are added to fine quartz sand at the rate of 1% to 
2% by weight and water is added to achieve a favorable moisture level 
(about 13% by weight with fine grained quartz sand). Not essential, but 
preferred because it improves the growing medium somewhat, is the addition 
of powdered calcium carbonate, 0.01 to 0.7% by weight, to maintain a 
favorable pH and to ensure longer viability of the mycoparasite. 
(d) Sporidesmium sclerotivorum is added as a seed inoculum to the 
sclerotia-sand mixture in any suitable container such as a beaker or a 
plastic flat, from either a pure culture of mycoparasite (spore 
suspension) or preferably by the addition of about 1% (larger amounts may 
also be used) by weight of a sand-sclerotia culture of Sporidesmium. The 
mixture is thoroughly mixed and incubated at room temperature 
(20.degree.-25.degree. C.) for 7 weeks or longer. 
(e) Sporidesmium sclerotivorum grows and develops in the mixture by 
invading sclerotia of the plant pathogen and spreads by extension of 
hyphae over the sand particles, producing a great many spores or 
macroconidia over the surface, and in the depths of the sand. The numbers 
of the macroconidia, each of which is a potential sclerotium-infecting 
unit, can be quantitated by making counts of representative samples of the 
mixture by conventional microbiological microscope procedures. These 
mixtures usually develop 100,000 to 500,000 macroconidia per gram of 
sand-sclerotia material. 
Use of Sporidesmium in the Biological Control of Plant Pathogens and Plant 
Disease 
In general, the procedure consists of adding an inoculum of Sporidesmium 
sclerotivorum furnished in a sand-sclerotial mixture to soil which 
contains sclerotia of the plant pathogen which, for laboratory 
experiments, is artificially added to field soil containing sclerotia of 
the plant pathogen produced under natural conditions. The material is 
mixed into the soil and the mycoparasitism is permitted to develop in 
fallow soil for at least 10 weeks, preferably at beneficial soil 
temperatures of 15.degree.-25.degree. C., a soil pH of 5.0 to 8.0 and 
moisture levels of 70% of field capacity or higher. Under the conditions 
we used in our field experiments we obtained good results by allowing the 
mycoparasite to develop in the soil for 20 weeks. However, there is no 
reason why this time period cannot be shortened by altering the 
conditions. Macroconidia of Sporidesmium germinate in the soil immediately 
adjacent to individual sclerotia, the germ tubes elongate and, by 
mechanisms unknown, penetrate the sclerotia. Sclerotia thus infected, 
gradually become consumed by the mycoparasite and eventually become 
completely destroyed. The beneficial result of this destructive process is 
the decrease, or elimination, of sclerotia in the soil which can cause 
plant disease. 
Two examples of the use of Sporidesmium are given below: 
LABORATORY EXPERIMENT 
A natural slit loam was moistened to 70% of field capacity and artificially 
infested with sclerotia of Sclerotinia minor at the rate of 1% by weight 
and added to beakers in 150 g quantities. Four replicate beakers of the 
soil were treated with Sporidesmium by the addition of 1.5 g of moist, 
sand-sclerotia cultures of the mycoparasite followed by mixing. Four 
replicate beakers of the soil were kept as controls for comparison. Soil 
temperature and pH were about 25.degree. C. and 6:0, respectively. At 
weekly intervals, 5 gram samples of the soils were removed and the 
sclerotia from the samples were screened out and counted. The retrieved 
sclerotia were placed on moist filter paper in petri dishes for 2 weeks at 
25.degree. C. to determine the numbers which had become infected by the 
mycoparasite. In five weeks, 100% of the sclerotia were infected and by 
ten weeks, survival of the sclerotia was reduced almost to zero. Another 
experiment with an entirely different soil type produced practically 
indentical results. 
FIELD EXPERIMENT 
Field plots, 10 feet square, were established at Beltsville, Maryland, in a 
field that contained a natural population of the lettuce drop pathogen, 
Sclerotinia minor. The numbers of sclerotia in the plots were determined 
to vary from 12 to 37 sclerotia/100 g of soil. 
Five replicate plots each were treated with Sporidesmium sclerotivorum by 
the addition of weighed portions of sand-sclerotia cultures to the soil 
surface. The soil and Sporidesmium inoculum were rototilled to a depth of 
six inches. The pH of the soil was about 6.0 and the ambient atmospheric 
temperature ranged from about 10.degree. C. to about 30.degree. C. The 
experiment was started in late spring of 1978 and the results in Table 1 
were obtained the following summer. At each sampling period of four weeks, 
the plots were rototilled and soil samples were taken for assay. The 
numbers of sclerotia per 100 g of soil were determined and expressed as 
the percentage of the numbers that were present at the beginning of the 
experiment. 
The rate of application and the results of the experiment are shown in 
Table 1. The results indicate that the two highest treatments, 2,000 and 
20,000 lbs/acre of the Sporidesmium-sand mixture were responsible for a 
significant reduction in the numbers of sclerotia after 20 weeks. The 
results of further sampling at 24 weeks are also shown in Table 1. The 
population of sclerotia per 100 g of soil prior to addition of 
Sporidesmium is shown at zero weeks in Table 1. 
TABLE 1. 
______________________________________ 
Treatments and results of biological control 
field test using Sporidesmium sclerotivorum to reduce 
the population of Sclerotinia minor. 
Rate of Population of Percent 
Sporidesmium 
S. minor Reduction of 
mixture added 
(Sclerotia/100 g) S. minor 
to field plots 
zero 20 24 20 24 
(lbs/acre) 
weeks weeks weeks weeks weeks 
______________________________________ 
0 22.2 13.8 14.6 37.8 34.2 
20 20.0 12.2 14.8 39.0 26.0 
200 16.2 9.8 12.2 39.5 24.7 
2,000 24.0 6.4 5.2 73.3 78.3 
20,000 24.0 1.8 1.4 92.5 94.2 
______________________________________