Note: Descriptions are shown in the official language in which they were submitted.
2183275
BIOLOGICAL CONTROL OF
PLANT DISEASE ON ROOTS OF CONIFER SEEDLINGS
The U.S. Government has a paid-up license in
this invention and the right in limited circumstances
to require the patent owner to license others on
reasonable terms as provided for by the terms of Grant
Nos.: USDA-FS 24-42-TN-031114; USDA-FS 152103; and
USDA-FAS-RSED SF08 awarded by the United States
Department of Agriculture.
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, WI. 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 soilfumigation 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.
s
. 2183275
-2-
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 app.
a Corynebacterium sp., and two Bacillus app., 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~
biofungicide, contains an isolate of S. griaeoviridis
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 Fuaarium spp. and other fungi. M-L.
Lahdenper~, et al. (1991), "Mycostop - A novel
biofungicide based on Streptomyces bacteria", pp.258-
263 j,~ Biotic Interactions and Soil-Borne DiRPaseR,
A.B.R. Beemster, et al., eds., Elsevier, Amsterdam.
The Mycostop~ Biofungicide Directions for Use (Kemira
Biotech, Helsinki, Finland) recommends Mycostop~ for
use on pine and other conifers.
Another Streptomyces ap. 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
S
2183275
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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 (Paeudomonas solanacearum), described in
El-Abyad, et al. (1993), "Inhibitory effects of UV
mutants of Streptomycea corchorusii and Streptomyces
apiroverticillatus on bean and banana wilt pathogens",
Can.J.Bot. 71:1080-1086, and S. hygroscopicua 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 yeasta, 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. No. 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 mot cells, and
ectomycorrhizae, which infect many forest tress such as
pines and other conifers. Compositions and methods
have been developed to help efforts to artificially
2183275
-4-
inoculate plants with mycorrhizae. See, for example,
U.S. Pat. No. 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
i~ Mycorrhizae and Plant Health, F.L. Pfleger and R.G.
Linderman, eds., APS Press, St. Paul.
Ectcmycorrhizal fungi are generally capable
of infecting many species of plants. The
ectomycorrhizal fungus which has been the most
extensively investigated, Pi.tholithua sp., has been
used to infect seUeral species of the following woody
plants: pine iPinus), oak (guercus), acacia (Acacia),
and eucalyptus (Eucalyptus). Ice. 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 ,~lr., 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
2183275
-5-
Fusarium damping-off in Pinus banksiana aeedlings~~,
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 Paxillua involutus and Pinus resinosa induces
resistance to Fusarium oxysporum~~, Can. ~T. 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
CA 02183275 1998-02-12
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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
F~sarium 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 (HEl) deposited September 29, 1997 with the National
Center for Agricultural Utilization Research in Peoria,
Illinois (NRRL 21841, and preferred bacterial control agents
are Methylobacterium mesophilicum (BC19) deposited September
CA 02183275 1998-02-12
-6A-
29, 1997 and as a viable deposit on October 10, 1997 (NRRL
B-21842), the actinomycetes Streptomyces Iavendulae (BCB176)
deposited September 29, 1997 and as a viable deposit on
December 12, 1997 (NRRL 21838), S. rochei subsp. rochei (BCT
19b) deposited September 29, 1997 and as a viable deposit on
October 10, 1997 (NRRL 21839), and S. violaceusniger subsp.
violaceusniger (BCTSa) deposited September 29, 1997 and as a
CA 02183275 1998-02-12
viable deposit on October 10, 1997 (NRRL 21840), and a
mixture of biologically pure cultures of the bacteria
Rhodococcus erythropolis, Kocuria varians, and Pseudomonas
diminuta (BC 20) having the identifying characteristics of
NRRL B-21843 deposited on September 29, 1997 and as a viable
deposit on October 10, 1997.
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
F~sarium 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.
CA 02183275 1998-02-12
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rochei NRRL 21839, 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 ectomcycorrhizal 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 tc 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, WI. Another
ectomycorrhizal fungus, Laccaria bicolor
was isolated from a mushroom growing in General Andrews
Nursery, willow River, I~1. Laccaria bicolor is often
treated as the same species as Laccaria laccata. See,
for example N.E. Strobel and W.A. Sinclair (1991)
2183275
-9-
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
mycorrhizai 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 Cylindrocladiuin) 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 Fuaarium will cause root
rot and can generally be treated interchangeably.
Preferred for this invention are pathogenic isolates of
four Fuaarium species, F. oxysporum, F. oxyaporum var.
redolena, 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
21 X32.75
-1~-
diseased roots of eastern white pine seedlings and
associated nursery soil", Can. J. Plant Pathol. 17:325-
330. Fuaarium can be stored and grown in a variety of
ways. See pp. 25-27 and 61-75 ~ O.D. Dhingra and J.B.
Sinclair (1995), Dasic Plant Pat oloc~y 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° C. Fusarium isolates
are preferably grown from silica gel crystals on
carnation leafagar (CLA) (see p. 347 in O.D. Dhingra
and J.B. Sinclair, sugra).
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 Fusari.um 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 Fuearium 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, suora)._ 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
' 2183275
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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 Nonmyaorrhizal BCAs
The nonmycorrhizal BCAa (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 oroatmeal broth. See for example R.A.
Lelliott and D.E. Stead (1987), Methods for the
Diagnosis of Bacter~a~ D~aeases of P~anta, 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
rhizoaphere than from non-rhizosphere soil. Therefore,
CA 02183275 1998-02-12
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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
2183275
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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 Fuaarium, preferably a mixture of isolates
of four Fusarium identified as root rot-causing
isolates of F. oxyaporum, F. oxysporum var. redolens,
F. proliferatum, & F. solani. In a continuation of the
study referred toabove, 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 fox 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
' 2183275
i
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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' to 105 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
BCAs 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 aeries of the soil, by
standard methods. See, for example p. 86 in O.D.
Dhingra and J.B.,Sinclair, su8ra.
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-tainera (Stuewe & Sons,
Corvallis, OR), 17 cm long and 24 mm in diameter, are
plugged with--5 cc of non-infested plant-growing medium
CA 02183275 1998-02-12
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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. Numbers 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 (Pinua resinosa)",
Can. J. For. Res. 21:1459-1465. In the preferred
embodiment, Hebeloma arenoas 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
CA 02183275 1998-08-10
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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
l0 will contain about 250 to 550 colony forming units. In
the preferred embodiment, the ratio of bacterial agent
to Hebelonra,'in colony forming units, is between about
19 and 37.
Since all ectomycorrhizal fungi will infect a
number of plant species, Xebeloena arenoaa NRRL 21841
will be beneficial for any conifer. Xebeloma has been
utilized as an ectomycorrhizae on pine, Sitka spruce,
(E. N. Loopstra, C.G. Shaw, III, and R.C. Sidle,
"Ectomycorrhizal Inoculation Fails to Improve
Performance of Sitka Spruce Seedlings on Clear Cuts in
Southeastern Alaska," Western Journal of Applied
Forestry Vol. 3, 4/88, pp. 110-112), black spruce,
(M.H.R. Browning and R.D. Whitney, "Field Performance
of Black Spruce and Jack Pine Inoculated with Selected
Species of Ectomycorrhizal Fungi," Canadian Journal of
Forestry Research Vol. 22, 1992, pp. 1974-1982), and
angiosperm species, (G. Gay, R. Marmeisse, P.
Fouillet, M. Bouletreau and J.C. Debaud,
"Genotype/Nutrition Interactions in the
Ectomycorrhizal Fungus Hebeloma cylindrosporun
Romagnesi," New Phytologist Vol. 123, 1993, pp. 335-
343 ) .
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.
CA 02183275 1999-03-29
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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
CA 02183275 1999-03-29
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healthy; 2= over 500 length of one lateral root is
exhibiting rot; 3= lower 1/3 of tap root is symptomatic or
greater than 500 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 0.5o 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-
tamers and a field soil. A loamy sand field soil with an
organic content of 1-2% and average bulk density of 1.20
g/cm3, was collected from a white pine field at F.G. Wilson
State Forest Nursery in Boscobel, WI. 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-tamers 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-tamer. 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
CA 02183275 1998-02-12
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evaluated after six months. The commercial
Streptomyces griseoviridis preparation Mycostopm was
included in these evaluations. In these studies, the
commercial product Mycostopm 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~ 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: HCT 19b =
Streptomyces rochei subsp. rochei NRRL 21839; BCT SA =
Streptomyces violaceusniger subsp. violaceusniger NRRL
21840; BCB 176 = Streptomyces lavendulae NRRL 21838; BC 19 _
Methylobacterium mesophilicum NRRL B-21842; and BC 20 = a
mixture of biologically pure cultures of the bacteria
Rhodococcus erythropolis, Kocuria varians, and Pseudomonas
diminuta, NRRL B-21843.
CA 02183275 1999-03-29
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Table 1. Greenhouse evaluation of rhizosphere BCAs for
Fusarium root rot using 4 pathogenic Fusarium isolates. The
claimed BCAs are in bold type.
BCA candidate Mean Root Rot Severity % Healthy Seedlings
B . y
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 llb 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
Streptl5 1.2 87
Strept32 1.8 39
Strept93 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
CA 02183275 1999-03-29
-20
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-tamers. 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 and Laccaria bicolor. The
cone-tamers 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 40o 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
3850 greater number of healthy seedlings and a 68% lower
mean root rot rating than Laccaria, Fusarium combination
treatments. In general,
CA 02183275 1998-02-12
-21-
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.
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CA 02183275 1999-03-29
-23
Example 3
Combinations of the non-mycorrhizal BCAs 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 70o Canadian peat,
20o perlite, l00 #3 vermiculite, and 5 lb dolomite/cubic
yard. The methods of preparing cone-tamers, 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~ 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° was not effective
when used as a single-application seed coat. Effective
results were only achieved by repeated Mycostop~ 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
2183275
-24-
controlling Fusarium root rot. However, the .
combination of the Hebeloma and the BCAs were more
effective than the BCAs alone or in combination with
Laccarfa 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.
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CA 02183275 1998-02-12
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All of the strains utilized in this invention have
been deposited with the National Center for Agricultural
Utilization Research in Peoria, Illinois, U.S.A. and are
identified herein by their NRRL number. Thesestrains may
also be obtained from Cynthia M. Ocamb, U.S. Department of
Agriculture, Forest Service, North Central Forest Experiment
Station, 1992 Folwell Avenue, St. Paul; Minnesota 55108.
