Biological control of plant disease on roots of conifer seedlings

A novel method and composition is provided to control Fusarium root rot and damping off on conifer seedlings. When certain bacteria and an ectomycorrhizal fungus are placed in contact with the conifer seed or seedling, the combination reduces or eliminates disease symptoms caused by several Fusarium species.

BACKGROUND OF THE INVENTION 
The production of conifers from seed is greatly affected by Fusarium root 
rot, in which seedling roots are destroyed by infection with the 
soil-inhabiting fungus Fusarium. That disease, which can be caused by 
Fusarium oxysporum, F. oxysporum var. redolens, F. proliferatum, or F. 
solani, kills 20-90% of conifer seedlings grown in the Lake States region 
each year, and reduces the vigor and growth of infected seedlings which 
are not killed, as discussed in the 1983, 1985, and 1986 editions of Prey, 
et al., Forest Pest Conditions in Wisconsin, Annual report. Department of 
Natural resources, Div. of Resource Management, Bureau of Forestry, 
Madison, Wis. Fusarium can also cause damping-off disease, in which the 
stem of the seedling near the soil line is destroyed. 
Current measures for controlling soil Fusarium spp. include soil fumigation 
with methyl bromide-chloropicrin in bareroot nurseries, and frequent 
fungicide applications to greenhouse-grown plants. These measures are 
often ineffective in controlling seedling Fusarium diseases because 
Fusarium spp. is often present in the seed. Also, methyl 
bromide-chloropicrin soil fumigation may not be allowed after the year 
2000 because its use is viewed as an environmental hazard. R. S. Smith and 
S. W. Fraedrich (1993), "Back to the future--pest management without 
methyl bromide", Tree Planters' Notes 44:87-90. Thus, alternatives are 
needed for controlling Fusarium diseases in tree nurseries. 
The use of biological control agents (living microorganisms used to control 
pests) is gaining recognition as an alternative disease control. The 
effective use of bacteria, actinomycetes, and fungi as agents for 
biological control of soil-borne plant disease has been demonstrated in 
several instances. Among the useful biological control bacteria are 
Bacillus megetarium, which controls Rhizoctonia solani on soybean, as 
disclosed in U.S. Pat. No. 5,403,583, and a mixture of combinations of 
three Pseudomonas spp. a Corynebacterium sp., and two Bacillus spp., which 
controls Aphanomyces root rot of peas, as disclosed in U.S. Pat. No. 
5,244,658. 
Actinomycetes are bacteria with fungus-like growth characteristics. Several 
isolates of the actinomycete Streptomyces have proved effective as 
biological control agents against soil-borne plant pathogens. A commercial 
product, Mycostop.RTM. biofungicide, contains an isolate of S. 
griseoviridis as its active ingredient. That product is effective as a 
seed and soil treatment against seed rots, root and stem rots, and wilt 
diseases of various ornamental plants, caused by Fusarium spp. and other 
fungi. M-L. Lahdenpera, et al. (1991), "Mycostop--A novel biofungicide 
based on Streptomyces bacteria", pp.258-263 in Biotic Interactions and 
Soil-Borne Diseases, A. B. R. Beemster, et al., eds., Elsevier, Amsterdam. 
The Mycostop.RTM. Biofungicide Directions for Use (Kemira Biotech, 
Helsinki, Finland) recommends Mycostop.RTM. for use on pine and other 
conifers. 
Another Streptomyces sp. isolate, designated WYEC 108, disclosed in U.S. 
Pat. No. 5,403,584, is effective as a seed treatment against damping-off 
of chickpea caused by Pythium spp. That patent also described some 
inhibitory activity against Fusarium growing in agar-solidified growth 
media in petri plates. 
Other Streptomycetes used for biological control include mutants of S. 
corchorusii and S. spirovirticillatus, effective against Fusarium wilt of 
French bean and the organism which causes bacterial wilt of banana 
(Pseudomonas solanacearum), described in El-Abyad, et al. (1993), 
"Inhibitory effects of UV mutants of Streptomyces corchorusii and 
Streptomyces spiroverticillatus on bean and banana wilt pathogens", Can. 
J. Bot. 71:1080-1086, and S. hygroscopicus var. geldanus, which controlled 
Rhizoctonia root rot of pea, caused by R. solani, described in C. S. 
Rothrock and D. Gottlieb (1984), "Role of antibiosis in antagonism of 
Streptomyces hygroscopicus var. geldanus to Rhizoctonia solani in soil", 
Can. J. Microbiol. 30:1440-1447. 
Various fungi have been utilized as biological control agents to control 
fungal plant pathogens. Two yeasts, Pichia guilliermondii and 
Hanseniasporum uvarum, are effective in controlling preharvest and 
postharvest development of several pathogens on numerous commodities, as 
disclosed in U.S. Pat. No. 5,413,783. Several isolates of Trichoderma spp. 
have also been employed to control soil-borne diseases, as disclosed in 
U.S. Pat. Nos. 4,996,157 and 5,192,686, including Fusarium spp. on cotton, 
disclosed in U.S. Pat. No. 4,713,342. 
Mycorrhizae are fungi which infect and form mutualistic relationships with 
plant roots. These fungi can improve plant growth by increasing the 
plant's assimilation of nutrients, especially phosphorus, which are 
sparingly soluble in the soil. Mycorrhizal infection will often make the 
plant roots more resistant to various soil-borne fungal pathogens. There 
are two major types of mycorrhizae: vesicular-arbuscular (VA) mycorrhizae, 
which infect most cultivated plants and produce specialized structures 
(vesicles or arbuscules) in the root cells, and ectomycorrhizae, which 
infect many forest trees such as pines and other conifers. Compositions 
and methods have been developed to help efforts to artificially inoculate 
plants with mycorrhizae. See, for example, U.S. Pat. Nos. 4,551,165 and 
5,178,642. Also see a review of these efforts in M. A. Castellano (1994), 
"Current status of outplanting studies using ectomycorrhizae-inoculated 
forest trees", pp. 261-281 in Mycorrhizae and Plant Health, F. L. Pfleger 
and R. G. Linderman, eds., APS Press, St. Paul. 
Ectomycorrhizal fungi are generally capable of infecting many species of 
plants. The ectomycorrhizal fungus which has been the most extensively 
investigated, Pitholithus sp., has been used to infect several species of 
the following woody plants: pine (Pinus), oak (Quercus), acacia (Acacia), 
and eucalyptus (Eucalyptus). Id. Additionally, many genera of 
ectomycorrhizal fungi, including Hebeloma and Laccaria, have been shown to 
be capable of infecting herbacious plants such as corn and wheat, as 
disclosed in U.S. Pat. No. 5,178,642. Thus, ectomycorrhizal fungi can be 
generally considered to be somewhat nonspecific in the plants they infect. 
Both VA mycorrhizae and ectomycorrhizae have been utilized as biological 
control agents, with limited success. That work is reviewed in R. G. 
Linderman (1994), "Role of VAM fungi in biocontrol", pp. 1-25 Id., and L. 
C. Duchesne, "Role of ectomycorrhizal fungi in biocontrol", pp. 27-45 Id. 
Ectomycorrhizae have shown some promise in controlling soil-borne diseases 
on conifer seedlings, but the protection to date has been unreliable due 
to the extreme variability of results. For example, Laccaria spp. 
exhibited limited control against Fusarium root rot and damping off on 
Douglas fir (described in N. E. Strobel and W. A. Sinclair (1991), 
"Influence of temperature and pathogen aggressiveness on biological 
control of fusarium root rot by Laccaria bicolor in douglas-fir", 
Phytopathol. 81:415-420) and pine (in P. Chakravarty and S. F. Hwang 
(1991), "Effect of an ectomycorrhizal fungus, Laccaria laccata, on 
Fusarium damping-off in Pinus banksiana seedlings", Eur. J. For. Path. 
21:97-106, and Paxillus involutus increased resistance of pine seedlings 
by 47% to Fusarium root diseases, as described in L. C. Duchesne, et al. 
(1988), "Interaction between the ectomycorrhizal fungus Paxillus involutus 
and Pinus resinosa induces resistance to Fusarium oxysporum", Can. J. Bot. 
66:558-562. Because of the limited and conditional control exhibited in 
these studies, the authors have expressed pessimism that they could be 
used effectively without further extensive research. See N. E. Strobel and 
W. A. Sinclair, supra, and L. C. Duchesne (1994), supra. 
The present invention addresses a long felt need to provide an alternative 
to chemical control methods by utilizing a strategy employing novel 
ectomycorrhizae and Streptomyces isolates alone and in combination to 
effectively control conifer seedling diseases caused by Fusarium. 
FIELD OF THE INVENTION 
The present invention relates to the use of ectomycorrhizal fungi in 
combination with soil bacteria to inhibit disease caused by Fusarium spp. 
and to establish ectomycorrhizal infections. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, conifer seeds or nascent 
seedlings are contacted with a composition comprising a mixture of two 
genera of microorganisms, namely, a biologically pure culture of an 
ectomycorrhizal fungus capable of colonizing the roots of a conifer, and a 
biologically pure culture of a bacterial control agent inhibitory to the 
growth of Fusarium spp. This composition may be applied to seeds prior to 
planting, or to young seedlings undergoing transplantation. The invention 
thus provides a method for reducing the incidence of Fusarium infection in 
conifer seedlings grown from conifer seeds. This is an important advance 
in the art since Fusarium infestations in nurseries can obliterate conifer 
stocks, and reduce the survival of more mature seedlings which must be 
thinned and transplanted. 
In an alternative method, conifer seeds are first coated with a culture of 
the bacterial biological control agent. The residue is allowed to dry to 
form a protective coating, and upon planting, the region of planting 
medium surrounding the seed is impregnated with a culture of the 
ectomycorrhizal fungus. A further embodiment involves first coating the 
seed with the biological control agent, and then later, after the seed has 
germinated seedling has emerged, further treating the nascent root with a 
culture of ectomycorrhizae upon transplantation, or adding it to the 
plant-growth medium in sufficient quantity to saturate the region 
surrounding the rhizosphere. Since the principal manifestations of 
Fusarium infection are the formation of root rot and damping off of plant 
stems, the methods of the invention result in reduction in the incidence 
of root rot and damping off. 
The present invention can also be adapted to providing a preformed 
plant-growth media, which comprises conventional soil and processed 
support material such as vermiculite, perlite, sand, and the like. 
Preferably the final mix is sterilized or pasteurized by heat or steam. 
Cultures of the combination of an ectomycorrhizal fungus capable of 
colonizing conifer roots, and a biologically pure culture of a bacterial 
control agent inhibitory to Fusarium spp. are then blended with the 
conventional medium to obtain a media ready for conifer seed planting. 
This preformed medium is especially efficacious for large nurseries where 
large numbers of seedlings are managed, and labor factors are critical. 
In the methods and compositions of the present invention, a preferred 
ectomycorrhizal fungus is Hebeloma arenosa NRRL 21841, and preferred 
bacterial control agents are Methylobacterium mesophilicum, NRRL B-21842, 
the actinomycetes Streptomyces lavendulae NRRL 21838, S. rochei subsp. 
rochei NRRL 21839, and S. violaceusniger subsp. violaceusniger NRRL 28140, 
and a mixture of biologically pure cultures of the bacteria Rhodococcus 
erythropolis, Kocuria varians, and Pseudomonas diminuta, NRRL B-21843. All 
cultures or mixtures identified by "NRRL" number herein identify cultures 
or mixtures of cultures prepared or isolated as described below, which 
were deposited with the National Center for Agricultural Utilization 
Research, 1815 North University Street, Peoria, Ill. 61604, in accordance 
with the terms of the Budapest Treaty. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to the utilization of a newly isolated 
ectomycorrhizal fungus and combinations of that fungus with newly isolated 
bacteria on conifer seedlings to reduce the incidence and severity of root 
rot and damping off caused by Fusarium species, and efficiently establish 
a beneficial ectomycorrhizal relationship on those seedlings. The newly 
isolated fungus, a Hebeloma, is unusual in its superior ability to reduce 
the effects of Fusarium root rot and establish an ectomycorrhizal 
relationship on conifer seedlings alone in soil and in combination with 
the newly isolated bacteria. These bacteria are also superior to other 
bacteria in the prior art in their ability to reduce the effects of 
Fusarium root rot when applied alone or in combination with Hebeloma. 
These bacteria also help the Hebeloma to establish an ectomycorrhizal 
relationship with conifer seedlings. 
A "biological control agent" or BCA is herein defined as a microorganism 
which can reduce the effects of plant disease when applied in the environs 
of the plant disease--causing organism. 
The utilization of a combination of two different genera of microorganisms 
for biological control is novel, and the combination disclosed in this 
invention satisfies a need for alternatives to chemical plant disease 
control, as by methyl bromide-chloropicrin. 
The nonmycorrhizal bacteria, which are also referred herein as "bacterial 
control agents" or nonmycorrhizal BCAs, are Streptomyces rochei subsp. 
rochei NRRL 21841, Streptomyces violaceusniger subsp. violaceusniger NRRL 
21840, Streptomyces lavendulae NRRL 21838, Methylobacterium mesophilicum 
NRRL B-21842, and mixtures of biologically pure cultures of Rhodococcus 
erythropolis, Kocuria varians, and Pseudomonas diminuta, NRRL B-21843. 
These are the first bacterial control agents known for these species. They 
are highly effective bacterial control agents when used alone and in 
combination with Hebeloma arenosa NRRL 21841. 
The term "biologically pure culture" is used herein to refer to cultures of 
organisms which have subcultured to species homogeneity by mass transfer. 
Isolation of the ectomycorrhizal fungi 
Although ectomycorrhizal fungi have not been shown to be effective BCAs in 
the past, isolates of Hebeloma and Laccaria were obtained from conifer 
nurseries having an unusually high incidence of Fusarium root rot. Species 
derived from healthy plants in such an environment may be more robust in 
colonizing conifer roots then in less infested environments. The Hebeloma 
isolated is unusually effective in its ability to colonize roots in the 
presence of Fusarium and in its ability to protect conifer seedlings from 
Fusarium root rot. 
Ectomycorrhizal fungi may be isolated from the roots of trees or, 
preferably, from the fruiting body of the fungus, such as a mushroom, 
which arise from the soil adjacent to the trees infected with the fungus. 
The ectomycorrhizal fungus Hebeloma arenosa NRRL 21841 was isolated from a 
mushroom found in a conifer nursery, specifically the F. G. Wilson State 
Forest Nursery in Boscobel, Wis. Another ectomycorrhizal fungus, Laccaria 
bicolor, was isolated from a mushroom growing in General Andrews Nursery, 
Willow River, Minn. Laccaria bicolor is often treated as the same species 
as Laccaria laccata. See, for example N. E. Strobel and W. A. Sinclair 
(1991) supra. Laccaria, but not Hebeloma has been previously studied as a 
potential BCA, exhibiting limited control of Fusarium root rot. These two 
fungi were isolated from single-spore colonies by the method described in 
R. L. Doudrick and N. A. Anderson (1989), "Incompatibility factors and 
mating competence of two Laccaria spp. (Agaricales) associated with black 
spruce in northern Minnesota", Phytopathology 79:694-700. 
These ectomycorrhizal fungi may be cultured in a culture medium under 
conditions suitable for rapid growth and retention of the ability to form 
an ectomycorrhizal relationship with the conifer root and ability to 
inhibit plant disease. The preferred medium is Melin-Norkrans' nutrient 
solution, which is described in D. H. Marx (1969), "The influence of 
ectotrophic mycorrhizal fungi on the resistance of pine roots to 
pathogenic infections. I. Antagonism of mycorrhizal fungi to root 
pathogenic fungi and soil bacteria", Phytopathology 59:153-163. Isolates 
may be stored on agar slants of Modified Melin-Norkrans' medium as 
described in D. H. Marx, supra. 
Preparation and use of root rotting fungi 
Root rotting isolates of Fusarium are the preferred pathogens for 
evaluation of the BCAs. Although other organisms causing root rot on 
conifers (such as Cylindrocladium) could be used, Fusarium is preferred 
because it causes the most incidences of root rot in conifer nurseries, it 
is present essentially everywhere conifers are grown, and it is easy to 
grow, store, and manipulate. 
Several species of Fusarium will cause root rot and can generally be 
treated interchangeably. Preferred for this invention are pathogenic 
isolates of four Fusarium species, F. oxysporum, F. oxysporum var. 
redolens, F. proliferatum, & F. solani, which are the most frequent 
species causing Fusarium root rot of conifers. See C. M. Ocamb and J. 
Juzwik (1995), "Fusarium species associated with rhizosphere soil and 
diseased roots of eastern white pine seedlings and associated nursery 
soil", Can. J. Plant Pathol. 17:325-330. Fusarium can be stored and grown 
in a variety of ways. See pp. 25-27 and 61-75 in O. D. Dhingra and J. B. 
Sinclair (1995), Basic Plant Pathology Methods, CRC Press, Boca Raton. To 
assure the maintenance of pathogenicity and morphology, the following 
methods are preferred. Each isolate is grown from a single spore and 
stored on silica gel at 5.degree. C. Fusarium isolates are preferably 
grown from silica gel crystals on carnation leaf agar (CLA) (see p. 347 in 
O. D. Dhingra and J. B. Sinclair, supra). 
For petri dish overlay studies, the Fusarium may be overlaid onto growing 
cultures of BCA candidates, then incubated so that BCA candidates which 
inhibit the growth of the Fusarium in the overlay will cause a zone of 
inhibition around the candidate organism. More effective BCA candidates 
will show a larger zone of inhibition than less effective candidates. The 
following method, which is similar to that summarized in C. M. Ocamb 
(1994), "Microbes isolated from white pine nursery soil to suppress 
pathogenic Fusarium species", Phytopathology 84:1137-1138, is the 
preferred overlay method because it is simple to carry out and each BCA 
candidate which inhibits Fusarium growth causes a reproducible zone of 
inhibition. 
The BCA candidates, either in a biologically pure culture or in a diluted 
matrix (such as soil) which contains many organisms, may be grown for 
about 72 hours in agar-solidified medium containing various mineral salts 
(see p. 390 in O. D. Dhingra and J. B. Sinclair, supra). A carbon source 
consisting of cellulose, pectin, or chitin is added. The entire Fusarium 
culture may be homogenized, diluted into molten, cooled Czepak-Dox agar 
(see p. 349-350 in O. D. Dhingra and J. B. Sinclair, supra), and overlaid 
onto the growing culture of the BCA candidate. BCAs will be selected 
having the greatest inhibition of Fusarium growth or largest inhibition 
zone. 
For testing BCAs, the Fusarium is first inoculated into growing medium and 
grown into a dense biologically pure culture. This culture may be 
conveniently mixed with the plant-growing medium and reliably cause root 
rot on a large proportion of conifer seedlings sown therein. In a 
preferred method, a 5-mm agar plug is taken from 10- to 14-day old CLA 
cultures and transferred to sterile cornmeal-sand medium (97 g sand, 3 g 
cornmeal, 40 ml distilled water) in 150 ml glass jars. The cornmeal-sand 
cultures are incubated 4-6 wk at 25 C. The cultures are then dried 
thoroughly in a laminar flow hood. Dried inoculum is then thoroughly mixed 
into the plant-growing medium to give a final concentration of 
approximately 15,000-25,000 colony-forming units per gram of oven-dried 
soil. 
Isolation and use of Nonmycorrhizal BCAs 
The nonmycorrhizal BCAs (bacterial agents)are grown using known culture 
methods. A suitable medium will allow the bacteria to achieve rapid growth 
and retain the ability to inhibit plant disease. BCA candidates are 
screened for the ability to enhance the fungus' ability to form an 
ectomycorrhizal relationship. There are several commercially available 
media which are suitable, with the preferred media being King's B or 
oatmeal broth. See for example R. A. Lelliott and D. E. Stead (1987), 
Methods for the Diagnosis of Bacterial Diseases of Plants, Blackwell 
Scientific Publications, Oxford. 
Although BCAs which are effective against root rot might be found in any 
soil, they may be more abundant in the rhizosphere (the area of soil 
around plant roots) than in areas of soil which do not contain plant 
roots. U.S. Pat. No. 5,403,584 discloses greater numbers of potential BCA 
actinomycetes isolated from a rhizosphere than from non-rhizosphere soil. 
Therefore, the BCAs of the present invention are isolated from rhizosphere 
soil. 
U.S. Pat. No. 5,360,606 discloses the isolation of BCAs from the 
rhizosphere of peas which are effective in controlling root rot of peas. 
In the present invention, however, BCAs effective against conifer root rot 
are derived from white pine (since it is desirable to obtain a BCA 
effective against conifer diseases). BCAs isolated from corn rhizosphere, 
Methylobacterium mesophilicum NRRL B-21842, and a mixture of biologically 
pure bacteria Rhodococcus erythropolis, Kocuria varians, and Pseudomonas 
diminuta, NRRL B-21843 were surprisingly effective. Another aspect of the 
identification of these BCAs is that they were not isolated from corn 
rhizospheres using an overlay method. Rather, they were found in 
association with a Fusarium isolated from corn roots. In overlay tests, 
however, these BCAs were found to strongly inhibit Fusarium. 
When isolating BCAs from rhizosphere soil using the plate dilution method 
described supra, the soil may be diluted in any of a number of media which 
will allow the BCAs to grow. However, cellulose, pectin, and chitin media 
is preferred because they select for specifically adapted microorganisms, 
ones believed to compete well in the rhizosphere. Collected soil may be 
diluted 2000-fold in molten but cooled cellulose, pectin, or chitin media, 
poured into petri dishes and incubated approximately 72 hours. A Fusarium 
overlay may be added, and the petri dishes incubated. The microorganisms 
which are selected for further study as BCA candidates will be those at 
the center of zones of inhibition greater than 1 mm in diameter. In one 
study, 586 BCA candidates were isolated by this method. 
These BCA candidates may be tested directly in soil with target conifer 
seedlings, but preferably, candidates are retested by a petri 
dish--Fusarium overlay method similar to that used with the soil 
dilutions. The candidates are transferred onto a petri dish containing 
Czepak-Dox agar, where they grow out in biologically pure culture. The 
overlay comprises a mixture of Fusarium, preferably a mixture of isolates 
of four Fusarium identified as root rot-causing isolates of F. oxysporum, 
F. oxysporum var. redolens, F. proliferatum, & F. solani. In a 
continuation of the study referred to above, 61 BCA candidates, out of the 
originally selected 586, showed strong ability to prevent the combined 
four species of Fusarium from growing on petri dishes. 
BCA candidates which have survived at least one round of selection using 
Fusarium mixtures overlaid are further tested for their ability to control 
Fusarium root rot in conifer seedlings in a second step selection. It is 
believed the BCA contacts the seed or seedling parts which are below the 
soil surface, such as roots of seedling transplants, or the seed itself. 
In a preferred embodiment, the BCA is inoculated into sterile medium, 
preferably oatmeal broth, in containers suitable for culturing 
microorganisms, which are maintained in aerobic growth phase. Conifer 
seeds are stratified (a cold or chemical treatment required for many 
conifer seed varieties to germinate). They are then contacted with the 
liquid culture of the appropriate BCA for time sufficient to bind the BCA 
to the seed, as by immersion or spraying. "Immerse" is herein defined as, 
to thoroughly wet. A very brief immersion, long enough to allow the seed 
surface to become completely wet, is sufficient; however the seed may be 
kept covered with the liquid culture for as long as six hours at room 
temperature and 24 hours at 4 C without deleterious effect on the seed. 
The preferred time is 60 min. 
The seeds may then be dried by any method which would retain the viability 
of the seed and a sufficient amount of the BCA to be effective. The seeds 
may be air-dried by any convenient method, for example by spreading the 
wet seeds on a screen and blowing air through the screen. The preferred 
embodiments of the seed soaking method have yielded approximately 10.sup.3 
to 10.sup.5 colony-forming units per seed. 
The BCAs may be used in soil or an artificial planting mix such as those 
used by professional growers of conifers. These planting mixes may contain 
peat, bark, perlite, vermiculite, sand, compost, or other ingredients well 
known in the art. The BCAs may also be used with container-grown seedlings 
or seedlings grown in the ground, and seedlings grown outdoors, in 
greenhouses, in shadehouses, or in growth chambers. 
When testing candidate BCAs, the plant growing medium can be naturally 
infested with a plant disease, or the causal agent of the plant disease 
can be added artificially. Artificial addition of a mixture of the 4 
pathogenic Fusarium species previously mentioned is preferred because one 
can then discern ECAs which are effective against several species of 
Fusarium. The pathogen is added to the plant-growing medium in a way to 
get consistent, reliable root rot symptoms in seedlings grown in the 
medium from seed or added by transplanting growing seedlings into the 
medium. Each of the four Fusarium isolates, prepared in sterile 
cornmeal-sand medium as disclosed above, is added to the plant-growing 
medium at a rate of about 0.0025 to 0.010 g/cc plant growing medium. The 
medium may be evaluated for propagule numbers of each Fusarium species by 
conducting a dilution series of the soil, by standard methods. See, for 
example p. 86 in O. D. Dhingra and J. B. Sinclair, supra. 
When growing seedlings in containers, any container and plant-growing 
medium where an added BCA will provide control of root rot may be used. 
For example, pine cell cone-tainers (Stuewe & Sons, Corvallis, Oreg.), 17 
cm long and 24 mm in diameter, are plugged with 5 cc of non-infested 
plant-growing medium then, if a pathogen is desired, a quantity of 
Fusarium-infested growing medium is added via a sterile PVC tube (18 mm 
OD, 12 mm ID) attached to a sterile 65 mm polypropylene funnel. The pine 
cells are then almost filled with non-infested soil. The conifer is sown, 
in duplicate, either as seedling transplants, or preferably as stratified 
seed. If the seed or seedlings have not been previously been treated with 
a BCA, for example by using the seed coating method described above, the 
BCA is added in a manner which will provide sufficient contact with the 
seed or seedling to provide control of Fusarium root rot. Examples of 
alternative methods of applying effective amounts of BCAs in soil include 
application as a culture or on a carrier to the soil at planting near the 
seed or seedling, or adding the BCA as a liquid culture or in a solid or 
liquid carrier to the soil after planting. U.S. Pat. Nos. 5,415,672; 
5,403,58; 5,403,583; 4,996,157; 4,534,965; and 4,713,432 disclose suitable 
methods. 
An ectomycorrhizal fungus may be contacted with the seed or seedling by any 
means known in the art, including using a plant-growing mix which contains 
the fungus prepared before planting, as was described as a "Ball mix" in 
J. S. MacFall and S. A. Slack (1991), "Effects of Hebeloma arenosa on 
growth and survival of container-grown red pine seedlings (Pinus 
resinosa)", Can. J. For. Res. 21:1459-1465. In the preferred embodiment, 
Hebeloma arenosa NRRL 21841 is grown in modified Melin-Norkrans' nutrient 
solution, as described in Marx, supra. Prior to application to the 
plant-growing medium, the cultures are leached with sterile distilled 
water to wash away nutrients. Sterile glass beads may be added to break up 
the mycelial pieces with manual agitation. The fungus is added to the soil 
by adding some of this preparation to the plant-growing medium at the time 
of planting. 
It is important to note that these organisms are controlled participants in 
an environment in which microecological balance in the numbers of 
organisms is effected naturally as the seedlings develop. Therefore, the 
ratios and numbers of the respective ectomycorrhizae and BCA do not 
conform to set ranges and may be arbitrarily selected so long as a 
sufficient inoculum is used to ensure a generally uniform distribution of 
organisms in the rhizosphere. Generally, a one ml aliquot of this fungal 
preparation will contain about 250 to 550 colony forming units. In the 
preferred embodiment, the ratio of bacterial agent to Hebeloma, in colony 
forming units, is between about 19 and 37. 
Since all ectomycorrhizal fungi will infect a number of plant species, 
Hebeloma arenosa NRRL 21841 will be beneficial for any conifer. Hebeloma 
has been utilized as an ectomycorrhizae on pine, Sitka spruce, (E. M. 
Loopstra, et al., 1988), black spruce, (M. H. R. Browning and R. D. 
Whitney, 1992), and angiosperm species, (G. Gay, et al., 1993). 
Containers in with the treated seed or seedling is planted may be placed 
outdoors, in a greenhouse, in a shadehouse, or in a growth chamber as 
desired, where they may be maintained following good horticultural 
practice. 
The BCA and ectomycorrhizal fungus may be added together or separately, in 
either order. It is important that they be in proximity to the rhizosphere 
of the emerging root. 
Fusarium root rot symptoms can develop within 4 months after sowing, and 
any time thereafter seedlings may be evaluated for the presence of 
disease. The preferred method involves removal of seedlings from the 
containers. Excess soil is then washed off the roots. Severity of root rot 
is then assessed for each seedling. Root rot is manifested as brown root 
tissue which is soft or macerated. Root rot is assessed by rating the 
roots on a 1 to 5 scale: 1=apparently healthy; 2=over 50% length of one 
lateral root is exhibiting rot; 3=lower 1/3 of tap root is symptomatic or 
greater than 50% of two or more lateral roots is necrotic; 4=lower 2/3 of 
tap root is rotted (with or without lateral root injury); 5=upper 1/3 of 
tap root is rotted or entire root system is affected. If roots exhibit 
rot, then small segments of tissues are excised from the edge of the 
necrotic areas, disinfested in 0.5% NaOCl for 1 minute, then embedded into 
solidified pentachloronitrobenzene-peptone agar supplemented with 
aureomycin (Nash medium). Cultures on Nash medium are incubated up to 21 
days at 24 C with indirect lighting. Confirmation of Fusarium species may 
be done by transferring colonies to potato dextrose agar and CLA for 
classification according to methods known to the art. See for example O. 
D. Dhingra and J. B. Sinclair, supra, at p. 25.

EXAMPLES 
Example 1 
Evaluation of BCA candidates for effectiveness in controlling Fusarium root 
rot 
The effectiveness of 61 BCA candidates, isolated by the Fusarium overlay 
method, was determined using cone-tainers and a field soil. A loamy sand 
field soil with an organic content of 1-2% and average bulk density of 
1.20 g/cm.sup.3, was collected from a white pine field at F. G. Wilson 
State Forest Nursery in Boscobel, Wis. The soil was pasteurized by 
enclosing it in plastic shoe boxes, and steaming at 12 psi for 60 min on 
each of two consecutive days. The cone-tainers were placed in a greenhouse 
where the seeds germinated and the seedlings were grown up. Starting at 
four weeks, the seedlings were fertilized weekly with a 20-7-19 liquid 
fertilizer, at 150 ppm. About six weeks after sowing, the seedlings were 
thinned to a single seedling per cone-tainer. The study was conducted 
twice. In the first test, the seedlings were evaluated for root rot after 
four months; in the second test, seedlings were evaluated after six 
months. The commercial Streptomyces griseoviridis preparation 
Mycostop.RTM. was included in these evaluations. In these studies, the 
commercial product Mycostop.RTM. was added as a soil drench every four 
weeks, unlike the other BCAs tested, which were only applied as a seed 
coat at planting. Results of studies using a one-time seed coating at 
planting showed Mycostop.RTM. to be ineffective in reducing Fusarium root 
rot on conifer seedlings. Table 1 summarizes the averaged results of both 
evaluations for the bacterial agents claimed herein (in bold) along with 
other, representative, BCA candidates. The isolates which were the most 
effective in reducing the incidence and/or severity of root rot were: BCT 
19b =Streptomyces rochei subsp. rochei NRRL 21839, deposited Oct. 10, 
1997; BCT 5a =Streptomyces violaceusniger subsp. violaceusniger NRRL 
21840, deposited Oct. 10, 1997; BCB 176=Streptomyces lavendulae NRRL 
21838, deposited Dec. 12, 1997; and BC 19=Methyobacterium mesophilicum, 
deposited Oct. 10, 1997; and BC 20=a mixture of biologically pure cultures 
of the bacteria Rhodococcus erythropolis, Kocuria varians, and Pseudomonas 
diminuta, NRRL B-21843, deposited Oct. 10, 1997. 
TABLE 1 
______________________________________ 
Greenhouse evaluation of rhizosphere BCAs for 
Fusarium root rot using 4 pathogenic Fusarium isolates. 
The alarmed BCAs are in bold type. 
BCA candidate 
Mean Root Rot Severity 
% Healthy Seedlings 
______________________________________ 
BCT 3b 1.4 59 
BCT 5a 1.5 67 
BCT 5b 1.9 39 
BCT 6b 2.3 29 
BCT 8a 2.3 39 
BCT 8b 2.3 21 
BCT 11a 1.4 71 
BCT 11b 1.6 58 
BCT 12a 2.0 44 
BCT 12b 2.4 20 
BCT 19b 1.3 83 
BCT 48 2.4 35 
BCT 62 2.1 29 
BCT 124 1.9 40 
BCT 205 2.2 34 
BCB 41 2.1 38 
BCB 70 1.6 60 
BCB 151 1.9 48 
BCB 152 1.6 60 
BCB 172 1.4 64 
BCB 175 1.4 72 
BCB 176 1.1 90 
BCB 191a 2.6 24 
BCB 226 1.8 49 
BCB 229 1.6 55 
BCB 282 1.7 58 
BCB 284a 1.9 48 
BCB 284b 1.7 45 
BCB 285 1.9 33 
BCB 311 1.4 76 
BCB 314 2.0 34 
Strept 15 1.2 87 
Strept 32 1.8 39 
Strept 93 1.6 71 
BC 18 2.0 25 
BC 19 1.5 57 
BC 20 1.0 97 
BC 23 2.6 15 
Mycostop 1.5 65 
infested control 2.2 24 
______________________________________ 
Example 2 
Example 2 was conducted to evaluate two ectomycorrhizal fungi for 
effectiveness as BCAs and in establishing mycorrhizal infection on eastern 
white pine growing in the field soil used in Example 1. The pasteurized 
soil was used in the preferred method where BCA-coated seeds are planted 
in cone-tainers. In some cases the four isolates of Fusarium were added. 
To infect the planted seed with an ectomycorrhizal fungus, one ml of the 
fungus, which had been grown in modified Melin-Norkrans' nutrient 
solution, was added to the soil. The seeds were then covered with soil, 
then further covered with a layer of perlite. 
The two isolates of ectomycorrhizal fungi used here were Hebeloma arenosa 
NRRL 21841, deposited Sep. 29, 1997, and Laccaria bicolor. The 
cone-tainers were placed in a growth chamber for 16 weeks where the seeds 
germinated and grew. The soil was removed and the roots evaluated. The 
percentage of the root system with mycorrhizal roots was also determined 
by visually determining the number of lateral roots and the number of 
those roots colonized by the fungus. The results are in Table 2. 
These results show that Hebeloma is much more effective than Laccaria in 
establishing a mycorrhizal relationship. The Hebeloma-treated seedlings 
had a 40% greater proportion of ectomycorrhizal infection in the absence 
of Fusarium than the Laccaria-treated seedlings. In the presence of 
Fusarium, the Hebeloma was also able to maintain a mycorrhizal 
relationship when subjected to pathogenic Fusarium spp., whereas Laccaria 
was not. 
Most importantly, Table 2 shows that Hebeloma was more successful than 
Laccaria in controlling Fusarium root rot, since the Hebeloma, Fusarium 
combination treatments had a 385% greater number of healthy seedlings and 
a 68% lower mean root rot rating than Laccaria, Fusarium combination 
treatments. In general, any ectomycorrhizae having 30- root colonizing 
incidence greater than Laccaria and a 50% lower root rot rating than 
Laccaria will have efficacy as a separate, independent seedling root 
treatment. Thus, Laccaria, the only ectomycorrhizae heretofore described 
as a BCA for controlling Fusarium root rot, was inferior to Hebeloma 
arenosa NRRL 21841 and not acceptable for that purpose. 
TABLE 2 
__________________________________________________________________________ 
Growth chamber evaluation of ectomycorrhizal fungi for biocontrol of 
Fusarium root rot..sup.1 
Fusarium Added 
Mycorrhizal Treatment 
Mean Root Rot Severity 
% Healthy Seedlings 
% Roots Ectomycorrhizal 
__________________________________________________________________________ 
- Hebeloma 1.1 c 89 a 65 a 
- Laccaria 1.1 c 94 a 46 b 
+ Hebeloma 1.6 b 68 a 63 a 
+ Laccaria 2.7 a 14 b 21 c 
+ none 2.5 a 7 b 1 d 
__________________________________________________________________________ 
.sup.1 Data followed by the same letters are not significantly different 
at P = 0.05. 
Example 3 
Combinations of the non-mycorrhizal ECAs and the ectomycorrhizal fungi 
disclosed herein were evaluated for effectiveness in: a) establishing an 
ectomycorrhizal infection and b) controlling Fusarium root rot on eastern 
white pine. 
The plant-growing medium used was Fafard #2, which is a medium commonly 
used by commercial growers to grow conifer seedlings. Fafard #2 mix is 
composed of 70% Canadian peat, 20% perlite, 10% #3 vermiculite, and 5 lb 
dolomite/cubic yard. The methods of preparing cone-tainers, adding 
Fusarium (isolates of all four species) to the growing mix, treating seed 
with BCAs and greenhouse cultivation were the same as in Example 1. The 
method of inoculating the cone-tainers with ectomycorrhizal fungi were the 
same as in Example 2. Each of the biological control agents which were 
effective against Fusarium root rot in the studies described in Example 1 
were evaluated eleven months after sowing. Determination of root volume 
was by a determination of the weight of the water displaced by the root 
mass. The results of studies using the claimed BCAs and Mycostop.RTM. with 
the two newly isolated ectomycorrhizal fungi are shown in Table 3. The 
non-mycorrhizal biological control agents were quite effective by 
themselves as biological control agents. As disclosed in Example 1 supra, 
Mycostop.RTM. was not effective when used as a single-application seed 
coat. Effective results were only achieved by repeated Mycostop.RTM. 
treatments. The claimed BCAs were effective as shown with a single 
treatment, however. Thus, the claimed BCAs are superior to the prior art 
because they are more effective than a successful commercial product. In 
combination with Hebeloma, these agents were even more effective than when 
used alone in controlling root rot. The Hebeloma by itself was ineffective 
under these conditions in becoming established in a mycorrhizal 
relationship and in controlling Fusarium root rot. However, the 
combination of the Hebeloma and the BCAs were more effective than the BCAs 
alone or in combination with Laccaria in preventing root rot. Indeed, 
since, under these conditions, Hebeloma alone was ineffective as an 
ectomycorrhizae and in reducing Fusarium root rot, one would expect the 
combination of Hebeloma with the bacterial agents to be no better than the 
bacterial agents alone. However, the Hebeloma/BCA combinations were much 
more effective than the Laccaria/BCA combinations, even though the 
Laccaria alone was somewhat effective. Therefore, the bacterial agents 
were unexpectedly effective in combination with Hebeloma. The 
Hebeloma-bacterial agent combination is thus the most effective of any 
known treatments in establishing mycorrhizal infections. This superiority 
is reflected in some cases by the significant increases in root volume of 
the combinations. 
TABLE 3 
__________________________________________________________________________ 
Greenhouse evaluation of ectomycorrhizal and bacterial agent combinations. 
.sup.1 
Mycorrhizal 
Bacterial % Healthy 
Treatment Agent Mean Root Rot Severity Seedlings % Roots Ectomycorrhizal 
Root Volume (g) 
__________________________________________________________________________ 
Hebeloma 
5a 1 f 100 43 bc 1.5 bcde 
arenosa 19b 1.4 cdef 79 46 bc 2.1 abc 
176 1 f 100 65 a 2.5 a 
Mycostop 1 f 100 65 a 2.4 ab 
None 2.2 ab 18 6 fgh 0.9 e 
Laccaria 5a 1.4 cdef 69 16 efgh 1.7 abcde 
19b 2.4 a 23 14 efgh 1.4 cde 
176 1.8 bcdef 57 24 de 1.6 bcde 
Mycostop 1.8 bcdef 41 33 cd 1.1 de 
None 1.4 cdef 63 21 def 1.2 cde 
None 5a 1.8 bcde 42 4 gh 1.2 cde 
19b 1.9 bcde 24 3 gh 1.4 cde 
176 2.1 bc 13 2 gh 1.1 de 
19 2.5 a 62 2 gh 1.5 bcde 
20 1.3 def 67 1 h 1.1 de 
Mycostop 2.1 bcd 17 6 fgh 1.2 cde 
None 2.5 a 0 1 gh 0.8 e 
__________________________________________________________________________ 
.sup.1 Data followed by the same letters are not significantly different 
at P = 0.05.