Strain of bacillus for controlling plant diseases

Details Strain of bacillus for controlling plant diseases Strain of bacillus for controlling plant diseases

2000-03-21

2003-10-21

Marx, Irene (Department: 1651)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Bacteria or actinomycetales

C435S252400, C435S252500, C504S117000

Reexamination Certificate

active

06635245

ABSTRACT:

FIELD OF THE INVENTION
The present invention is in the field of biopesticides. More particularly, this invention relates to the finding that a novel strain of
Bacillus pumilus
, NRRL Accession Number B-30087, can inhibit a broad range of fungal plant diseases in vivo. The invention also relates to fungicidal compositions comprising this novel Bacillus strain, and the antibiotics and purified and non-purified fractions of this strain either alone, or in combination with other chemical and biological pesticides. The invention further relates to the synergistic fungicidal effect of using NRRL Accession No. B-30087 together with NRRL Accession No. B-21661, (CCRC 910106).
BACKGROUND
It is generally known that various microorganisms exhibit biological activity that are useful to control plant diseases. Although progress has been made in the field of identifying and developing biological pesticides for controlling various plant diseases of agronomic and horticultural importance, most of the pesticides in use are still synthetic compounds. Many of these chemical fungicides are classified as carcinogens by the Environmental Protection Agency (EPA), are toxic to wildlife and other non-target species. In addition, pathogens may develop resistance to chemical pesticides. See, e.g., Schwinn et al., in: Advances In Plant Pathology:
Phytopathora infestans
, The Cause of Late Blight of Potato, p. 244, Academic Press, San Diego, Calif. (1991).
Biological control offers an attractive alternative to synthetic chemical fungicides. Biopesticides (living organisms and the naturally produced compounds produced by these organisms) can be safer, more biodegradable, and less expensive to develop.
One commonly used biopesticide is the gram positive bacterium
Bacillus thuringiensis
. Pesticidal
B. thuringiensis
strains are known to produce crystal proteins during sporulation, which are specifically toxic to certain orders and species of insects and nematodes (See, e.g., U.S. Pat. No. 4,999,192 and U.S. Pat. No. 5,208,017). Proteinaceous endotoxins produced by
B. thuringiensis
also act as insecticidal agents against corn rootworm and other beetles (e.g., U.S. Pat. 5,187,09 and Johnson, T. J. et al. (1993),
J. Econ. Entomol
., 86:330-333).
B. thuringiensis
endotoxins have been shown to be effective as purified crystals, washed cell pellets, and expressed proteins. Warren et al. WO 96/10083, disclose non-endotoxin proteins produced during the vegetative stage of
Bacillus cereus
and
B. thuringiensis
. These vegetative proteins, called Vip1 and Vip2 have potent activity against corn rootworm (northern and western). See, Estruch et al. (1997),
Nature-Biotechnology
15:137-141.
One
B. thuringiensis
thermostable metabolite, termed beta-exotoxin has also been shown to have pesticidal properties. Burgjeron and Biache (1979),
Entomophaga
11:279-284, report a beta-exotoxin that is active against Colorado potato beetle (
Leptinotarsa decemlineata
). In addition, the known
B. thuringiensis
beta-exotoxins exhibit non-specific pesticidal activity, not only killing nematodes, but also flies, armyworm, mites, and corn rootworm. Sigma exotoxin has a structure similar to beta-exotoxin, and is active against Colorado potato beetle. See, Argauer et al. (1991),
J. Entomol. Sci
. 26: 206-213. Alpha-exotoxin is toxic to larvae of
Musca domestica
(Cluthy (1980),
FEMS Microbiol
.
Lett
. 8:1-7). Gamma-exotoxins are various proteolytic enzymes, chitinases and proteases. The toxic effects of gamma-exotoxins are only expressed in combination with beta-exotoxin or delta-endotoxin. See, Forsberg, C., “
Bacillus thuringiensis
: Its effects on Environmental Quality” National Research Council of Canada, Publication No. NRCC 15385, pp. 91-109 (1976). Stonard et al. (1994),
ACS Symposium Series
551:25, report a water-soluble secondary metabolite active against corn rootworm in the supernatant of a
Bacillus cereus
strain.
Zwittermicin A is a water soluble, acid stable linear arninopolyol molecule (see, He et al. (1994),
Tetrahedron Lett
. 35(16):2499-2502) with broad-spectrum activity against many fungal and bacterial plant pathogens. Zwittermicin A is also known to enhance the activity of
B. thuringiensis
. Manker et al. (WO 96/39037) were the first to determine the
B. thuringiensis
-enhancing abilities and properties of zwittermicin A. Subsequently, Schnepf et al. also reported that zwittermicin A enhanced
B. thuringiensis
(U.S. Pat. No. 5,702,703).
Bacilli are known to produce antifungal and antibacterial secondary metabolites. See, Korzybski et al. “Antibiotics isolated from the genus Bacillus (Bacillaceae)” in: Antibiotics—Origin, Nature and Properties, American Society for Microbiology, Washington, D.C. Vol. III (1978), and Berdy, CRC Handbook of Antibiotic Compounds, Vols. I-XIV, CRC Press, Inc., Boca Raton, Fla. (1980-87). Compounds produced by
B. pumilus
include micrococcin P, pumilin, and tetain.
Kawaguchi et al., in U.S. Pat. No. 4,250,170, isolated a novel water-soluble antibiotic from Bacillus with activity against a broad range of gram positive and gram negative bacteria. Stabb et al. (1990)
Applied Environ. Microbiol
. 60:4404-4412, have identified certain Bacillus spp. (Bacillus spp. include
B. subtilis, B. cereus, B. mycoides, B. thuringiensis
) strains that exhibit antifungal activity. These strains have been shown to produce zwittermicin A and/or kanosamine. See, Milner et al.,
Appl. Environ. Microb
. 62:3061-3066 (1996). These are antibiotic agents that are effective against the soil borne disease damping off, caused by
Phytopathora medicaginis, P. nicotianae, P. aphanidermatum
or
Sclerotinia minor
(See, Stabb et al., supra). Zwittermicin-A is a water soluble, acid stable linear aminopolyol molecule. See, He et al., (1994)
Tetrahedron Lett
. 35(16):2499-2502. It has broad spectrum activity against many fungal and bacterial plant pathogens. Kanosamine (Milner et al., 1996) also inhibits a broad range of fungal plant pathogens and a few bacterial species.
Handelsman et al., in U.S. Pat. No. 5,049,379, describe how Zwittermicin A-producing
B. cereus
controls damping off in alfalfa and soybeans. When the seed was coated with
B. cereus
ATCC 53522, the pathogenic activity of root rot fungus was inhibited. Similarly, application of spore-based formulations of certain
B cereus
strains to soybean seeds or the soil surrounding the seeds has been shown to improve soybean yield at field sites. See, Osburne et al. (1995)
Am. Phytopathol. Soc
. 79(6):551-556. Methods of applying biopesticides are well known in the art and include, for example, wettable powders, dry flowables, microencapsulation, and liquid formulations of the microbe, whole broth or antibiotic fractions from suitable cultures. See, e.g., U.S. Pat. No. 5,061,495 to Rossall and U.S. Pat. No. 5,049,379 to Handelsman et al.
Tsuno et al. (1986)
J. Antibiotics
XXXIX(7):1001-1003, report on a new amino sugar antibiotic from
B. pumilus
with activity against a broad range of bacteria in vitro.
Khmel, I. A. et al., (1995) in SU 1817875 disclose a novel strain of
Bacillus pumilus
VKM CR-333D, which is used to control fungal phytopathogens and bacteria.
Leifert et al.,
J. Appl. Bacteriol
. 78:97-108 (1995), report the production of anti-Botrytis and anti-Alternaria antibiotics by two Bacillus strains,
B. subtilis
CL27 and
B. pumilus
CL 45. The whole broth and cell-free filtrates are active against Botrytis and Alternaria in in vitro tests and are active against Botrytis in in vivo small plant tests on Astilbe. Leifert et al. (1997) U.S. Pat. No. 5,597,565 disclose
B. subtilis, B. pumilus
, and
B. polymyxa
that are particularly effective at inhibiting post harvest disease causing fungi,
Alternaria brassicicola
and
Botrytis cinerea
. They also disclose the presence of antibiotics produced in the cell-free culture filtrate and their activity at different pH values, but they do not identify these compounds. The compounds from
B. subtilis
lose activity at low pH, while the activity from the
B. pumilus
extracts occu

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