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| United States Patent |
6,239,103
|
|
Heins
, et al.
|
May 29, 2001
|
Strain of bacillus for controlling plant diseases and corn rootworm
Abstract
The present invention relates to a novel antibiotic-producing and
metabolite-producing Bacillus subtilis strain that exhibits insecticidal,
antifungal and antibacterial activity. The supernatant of this novel
strain contains effective insecticidal, antifungal and antibacterial
agents. Also included in the invention is a solvent extractable, small
molecular weight (<10,000 daltons) corn rootworm-active metabolite
produced in the supernatant. Also included in the invention are methods of
protecting or treating plants from fungal and bacterial infections and
corn rootworm infestations comprising the step of applying to the plant an
effective amount of the antibiotic/metabolite-producing novel Bacillus
subtilis strain, the antibiotic/metabolite produced by the novel Bacillus
subtilis strain or a combination thereof, optionally further comprising
another antibiotic-producing bacterial strain and/or a chemical pesticide.
The invention also includes methods of preventing or treating fungal and
bacterial infections using whole broth cultures or supernatants obtained
from cultures of the novel Bacillus subtilis strain alone or in
combination with chemical pesticides and/or other biocontrol agents. The
invention also includes novel antifungal and antibacterial compounds
designated agrastatins and a novel combination comprising an A-type
iturin, a plipastatin, a surfactin and an agrastatin. Methods of treating
or protecting plants from fungal and bacterial infections and corn
rootworm infestations comprising administering the novel agrastatins and
the novel combination comprising an A-type iturin, a plipastatin, a
surfactin and an agrastatin are provided.
| Inventors:
|
Heins; Sherry Darlene (Davis, CA);
Manker; Denise Carol (Davis, CA);
Jimenez; Desmond Rito (Woodland, CA);
McCoy; Randy Jay (Davis, CA);
Marrone; Pamela Gail (Davis, CA);
Orjala; Jimmy Ensio (Davis, CA)
|
| Assignee:
|
AgraQuest, Inc. (Davis, CA)
|
| Appl. No.:
|
312314 |
| Filed:
|
May 14, 1999 |
| Intern'l Class: |
A61K 038/00; A01N 063/00 |
| Field of Search: |
424/93.462,405
514/2,9,11
|
References Cited
U.S. Patent Documents
| 4904585 | Feb., 1990 | Shimada et al.
| |
| 4999192 | Mar., 1991 | Payne et al.
| |
| 5045314 | Sep., 1991 | Bone et al.
| |
| 5047239 | Sep., 1991 | Pusey.
| |
| 5049379 | Sep., 1991 | Handelsman et al.
| |
| 5055293 | Oct., 1991 | Aronson et al.
| |
| 5061495 | Oct., 1991 | Rossall.
| |
| 5187091 | Feb., 1993 | Donovan et al.
| |
| 5208017 | May., 1993 | Bradfisch et al.
| |
| 5344647 | Sep., 1994 | Rossall.
| |
| 5403583 | Apr., 1995 | Liu et al.
| |
| 5552138 | Sep., 1996 | Handelsman et al.
| |
| 5597565 | Jan., 1997 | Leifert et al.
| |
| 5645831 | Jul., 1997 | Chilcott et al.
| |
| 5733544 | Mar., 1998 | Marrone et al.
| |
| 5753222 | May., 1998 | Marrone et al.
| |
| Foreign Patent Documents |
| 62-210996 | Sep., 1987 | JP.
| |
| WO 96/10083 | Apr., 1996 | WO.
| |
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|
Primary Examiner: Tate; Christopher R.
Attorney, Agent or Firm: Konski; Antoinette F.
Baker & McKenzie
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. Ser. No. 09/074,870, filed May 8,
1998 now U.S. Pat. No. 6,060,051, which in turn is a continuation-in-part
of U.S. Ser. No. 08/853,753, filed May 9, 1997 now abandoned.
Claims
What is claimed is:
1. A compound having the formula (SEQ ID NO:6):
##STR9##
wherein R.sub.1 is a branched or straight aliphatic side chain of C8-C20;
R.sub.2 is an acetate or an ester derivative; and Glx is Gln or Glu.
2. A compound having the formula (SEQ ID NO:7):
##STR10##
3. A composition comprising the compound of claim 1 and a carrier.
4. A composition comprising the compound of claim 2 and a carrier.
5. The composition of claim 3, further comprising a biological or chemical
pesticide.
6. The composition of claim 4, further comprising a biological or chemical
pesticide.
7. The composition of claim 3 or 5, wherein the composition is formulated
as wettable powders, granules, flowables or microencapsulations.
8. The composition of claim 4 or 6, wherein the composition is formulated
as wettable powders, granules, flowables or microencapsulations.
9. A method for protecting or treating plants or fruit from corn rootworm
infestations comprising applying an effective amount of the compound of
claim 1 or 2, or to roots or soil around the roots of the plants.
10. A method for protecting or treating plants or fruit from fungal and
bacterial infections and corn rootworm infestations comprising applying an
effective amount of the composition of any of claims 3, 4, 5 or 6 to the
plant or fruit to the plants or fruit, or to roots or soil around the
roots of the plants.
11. A method for protecting or treating plants or fruit from corn rootworm
infestations comprising applying an effective amount of the composition of
any of claim 3, 4, 5, or 6 to the plants or fruit, or to roots or soil
around the roots of the plants.
12. A method for protecting or treating plants and fruit from fungal and
bacterial infections and corn rootworm infestations comprising applying an
effective amount of a compound of the formula (SEQ ID NO:9):
##STR11##
wherein R.sub.1 is a branched or straight aliphatic side chain of C.sub.8
-C.sub.20 ; R.sub.2 is an acetate or an ester derivative; X is Ala or Val;
and Glx is Gln or Glu to the plants or fruit, or to roots or soil around
the roots of the plants.
13. The method of claim 12 wherein the infections are caused by at least
one microorganism selected from the group consisting of Phytophthora
infestans, Rhizoctonia solani, Pythium ultimum, Botrytis cinerea,
Alternaria solani, Colletotrichum cocodes, Alternaria brassicicola,
Cladosporium cucumerinum, Monilinia fructicola, Venturia pyrina,
Acidovorax avenae, Pseudomonas syringae, Xanthomonas campestris, Erwinia
carotovora, Clavibacter michiganense, Plasmopara viticola, Sphaerotheca
fuliginea, Uncinula necator, and Peronospora parasitica.
Description
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
subtilis, AQ713, can inhibit a broad range of fungal and bacterial plant
diseases and also have activity against corn rootworm. The invention also
relates to fungicidal, bactericidal, and insecticidal compositions
comprising this novel Bacillus strain and the antibiotics and metabolites
produced by this strain either alone, or in combination with other
chemical and biological pesticides.
BACKGROUND OF THE INVENTION
For a number of years, it has been known that various microorganisms
exhibit biological activity so as to be 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 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., p. 244, ADVANCES IN PLANT
PATHOLOGY: PHYTOPHTHORA INFESTANS, THE CAUSE OF LATE BLIGHT OF POTATO
(Academic Press, San Diego 1991).
Every year 250-300 million dollars of chemical pesticides are used to
control corn rootworm infestations. Many of these chemical pesticides are
toxic to humans, wildlife and other nontarget species. Also some have been
found in the ground water. New chemical insecticides cost $100 million to
develop.
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.
Screening programs have identified certain Bacillus spp. (Bacillus spp.
includes B. subtilis, B. cereus, B. mycoides, B. thuringiensis) strains
that exhibit antifungal activity. (See e.g Stabb et al. (1990) Applied
Environ. Microbiol. 60: 4404-4412). These strains have been shown to
produce zwittermicin-A and or kanosamine (Milner et al. (1996) Appl.
Environ. Microb. 62: 3061-3066), two antibiotic agents that are effective
against the soil borne disease damping off, caused by Phytophthora
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) Tetra. Lett. 35 (16)
2499-2502.
U.S. Pat. No. 5,049,379 to Handelsman et al. describes how zwittermicin-A
produces damping off in alfalfa and soybeans. When the seed was coated
with B. cereus ATCC 53522, the pathogenic activity of root rot fungus is
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 of effective agents, liquid or solid
formulations of antibiotic fractions from suitable cultures. (See e.g.,
U.S. Pat. No. 5,061,495 to Rossall or U.S. Pat. No. 5,049,379 to
Handelsman).
Smith et al. (1993) Plant Disease 77(2) 139-142 report that the activity of
the soil-borne fungus, Pythium aphanidermatum, that causes cottony
cucumber leak can be suppressed using zwittermicin-producing B. cereus
strain UW85. Leifert et al. (1995) J. Appl. Bacteriol. 78: 97-108 report
that the production of anti-Botrytis and anti-Alternaria antibiotics by
two Bacillus strains, B. subtilis CL27 and B. pumilis CL 45. The whole
broth and cell-free filtrates were active against Botrytis and Alternaria
in in vitro tests and were 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. pumilis, and B. polymyxa that are particularly effective
at inhibiting post harvest disease causing fungi. 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.
Rossall (1994) U.S. Pat. No. 5,344,647 discloses Bacillus subtilis strains
with broad anti-fungal activity. Sholberg et al. (1995) Can. J. Microbiol.
41: 247-252, Swinburne et al. (1975) Trans. Brit. Mycol. Soc. 65: 211-217,
Singh and Deverall (1984) Trans. Br. Mycol. Soc. 83: 487-490, and
Ferreira, et al. (1991) Phytopathology 81: 283-287. Baker et al. (1983)
Phytopathology 73: 1148-1152 disclose the use of Bacillus spp. and
Bacillus subtilis as biocontrol agents of fungal plant pathogens. Baker et
al. (1983) Phytopathology 73: 1148-1152 also report on an antifungal
Bacillus subtilis for use on plant pathogens. Pusey et al. (1988) Plant
Dis. 72: 622-626, Pusey and Robins (U.S. Pat. No. 5,047,239), and McKeen
et al. (1986) Phytopathology 76: 136-139 disclose control of post harvest
fruit rot using B. subtilis. McKeen et al, supra, have shown that
antibiotics similar to the low molecular weight iturin cyclic polypeptides
contribute to this fungicidal activity of B. subtilis.
Liu et al. (1995) U.S. Pat. No. 5,403,583 disclose a Bacillus megaterium,
ATCC 55000 and a method to control the fungal plant pathogen, Rhizoctonia
solani. Islam and Nandi (1985) Journal of Plant Diseases and Protection
92(3): 241-246 disclose a Bacillus megaterium with antagonism to
Drechslera oryzae, the causal agent of rice brown spot. The same authors,
Islam and Nandi (1985) Journal of Plant Diseases and Protection 92(3)
233-240 also disclose in-vitro antagonism of B. megaterium against
Drechslera oryzae, Alternaria alternata and Fusarium roseum. They discuss
three components in the culture filtrate. The most active antibiotic was
highly soluble in water and methanol with a UV peak at 255 nm and a
shoulder at 260 nm, which proved to be a polyoxin-like lipopeptide. Cook
((1987) Proceedings Beltwide Cotton Production--Mechanization Research
Conference, Cotton Council, Memphis, p. 43-45) discloses the use of a
suspension of Bacillus megaterium to reduce the number of cotton plants
killed by Phymatotrichum omnivorum, a cause of cotton root rot.
Antibiotic production of B. megaterium has been recorded by Berdy (CRC
Handbook of Antibiotic Compounds, Vols. I-XIV, (CRC Press, Inc. Boca
Raton, Fla. 1980-87) who reports production of low-mammalian toxic peptide
antibiotics such as ansamitocin-PDM-O, bacimethrin, megacin, pentapeptide,
homopeptides.
Bacilli are known to produce antifungal and antibacterial secondary
metabolites (Korzybski et al. (1978)). University of Wisconsin and Cornell
researchers have identified a novel fungicidal compound, zwittermicin A,
produced by Bacillus sp. (He et al. (1994) Tetra. Lett. 35(16):2499-2502).
A second fungicidal metabolite produced by the same strain was recently
identified as the known amino-sugar, kanosamine (Milner et al. (1996)
Appl. Environ. Microb. 62:3061-3065).
Another group of previously described Bacillus metabolites are the cyclic
lipopeptides of the iturin class, some of which are potent fingicidal
agents. These agents consist of a cyclic octapeptide with seven
.alpha.-amino acids and one .beta.-amino acid with an aliphatic side
chain. There are several groups of iturins that differ in order and
content of the amino acid sequence. These are shown in Table 1 below.
Generally, a suite of related molecules is produced with differences in
the length and branching of the aliphatic amino acid residue. When tested
against Saccharomyces cerevesiae, mycosubtilin was found to be the most
active agent (LC50=10 .mu.g/mL) followed by iturin-A and bacillomycin L
(both haviing an LC50=30 .mu.g/mL) (Beeson et al. (1979) J. Antibiotics
32(8):828-833). The mode of action of these cyclic lipopeptides has been
reported to be due to interaction with fungal membranes creating
transmembrane channels that permit release of vital ions (Latoud et al.
(1986) Biochem. Biophys. Acta 856:526-535). Iturin-C is inactive against
fungi including Penicillium chrysogenum (Peypoux et al. (1978) Tetrahedron
34:1147-1152).
TABLE 1
Structures of the iturin family of antibiotics
Antibiotic L-Asz(X1) X4 X5 X6 X7
Iturin A L-Asn L-Gln L-Pro D-Asn L-Ser
Iturin C L-Asp L-Gln L-Pro D-Asn L-Ser
Bacillo- L-Asn L-Pro L-Glu D-Ser L-Thr
mycin D
Bacillo- L-Asp L-Ser L-Gln D-Ser L-Thr
mycin L
Bacillo- L-Asn L-Gln L-Pro D-Asn L-Thr
mycin F
Myco- L-Asn L-Gln L-Pro D-Ser L-Asn
subtilin
##STR1##
A research group at the USDA has investigated the structure/activity
relationship of the iturins by synthesizing a number of analogs differing
in the amino acid chain length. The researchers reported that the activity
of the iturins increased with the length of the fatty acid side chain and
the terminal branching in the order iso>normal>anteiso (Bland et al.
(1995) Proc. Plant Growth Regulation Soc. Am. 22nd: 105-107). They also
state that the "amounts of iturins obtained from natural production are
inadequate to be commercially viable" based on their work with a number of
iturin producing strains of Bacillus.
Another groups of cyclic lipopeptides isolated from B. cereus are the
plipastatins. These compounds are a family of acylated decapeptides, the
structures of which are shown in FIG. 1 (Nishikiori et al. (1986) J.
Antibiotics 39(6):755-761). These compounds were originally isolated as
inhibitors of porcine pancreatic phospholipase A.sub.2 (Umezawa et al.
(1986) J. Antibiotics 39(6):737-744), but were later found to inhibit some
plant pathogenic fungi including Botrytis, Pyricularia and Alternaria
(Yamada et al. (1990) Nippon Noyaku Gakkaishi 15(1):95-96). Yamada also
reported a synergistic effect observed between iturin A and the
plipastatins, both produced by the same B. subtilis strain.
Work has been carried out on fermentation improvements to increase
production of the iturins in both liquid (Phae and Shoda (1991) J.
Ferment. Bioeng. 71:118-121); Ohno et al. (1993) J. Ferment. Bioeng.
75:463-465) and solid state fermentations (Ohno et al. (1992) Biotech.
Lett. 14(9):817-822; Ohno et al. (1995) J. Ferment. Bioeng. 5:517-519).
There is a report of synergy between the closely related surfactins, that
are themselves inactive, and the iturins produced by the same B. subtilis
strain (Hiraoka et al. (1992) J. Gen. Appl. Microbiol. 38:635-640). The
nucleotide sequence for the gene that co-regulates biosynthesis of iturin
A and surfactin has been published (Huang et al. (1993) J. Ferment.
Bioeng. 76(6):445-450). Field work on iturin-producing strains has
concentrated on soil treatment for control of Rhizoctonia (Asaka and Shoda
(1996) Appl. Environ. Microbiol. 62:4081-4085) and foliar field
applications of iturins have not been reported.
Another cyclic lipopeptide compound produced by B. subtilis is surfactin,
which possesses an exceptional surfactant activity (Kaninuma et al. (1969)
Agric. Biol. Chem. 33:973-976). Surfactin contains a C14 or C15
.beta.-hydroxy fatty acid linked by a lactone ring to a heptapeptide
moiety with a LLDLLDL (SEQ ID NO:1) sequence (Arima et al. (1968) Biochem.
Biophys. Res. Commun. 31:488-494. Sandrin et al. ((1990) Biotechnol. Appl.
Biochem. 12:370-375) found B. subtilis strains that produced both
surfactin and iturin A, the bacillomycins F and L and mycosubtilin.
The novel microorganism AQ713 discovered by the inventors, previously
thought to be a strain of Bacillus megaterium and now identified as a
strain of Bacillus subtilis, produces A iturins, plipastatins and
surfactins. Production of this combination of lipopeptides by a
microorganism has not been previously reported. In addition, the inventors
have discovered that AQ713 also produces a newly described group of
compounds designated as "agrastatins." The combination of all three of the
above known compounds with the novel agrastatins is also novel.
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.
Nos. 4,999,192 and 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. No. 5,187,091; Johnson, T. J. et al. (1993),
J. Economic Entomology 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), discloses 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) (Estruch et al.
(1997), Nature Biotechnology 15:137-141 and Mullins et al. (1997), Appl
Environ. Microbiol. 63, (in press).
One B. thuringiensis thermostable metabolites, 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 exhibits non-specific pesticidal
activity, killing not only nematodes, but also flies, armyworms, mites,
and corn rootworms. Sigma exotoxin has a structure similar to
beta-exotoxin, and is active against Colorado potato beetle (Argauer et
al. (1991) J. Entomol. Sci. 26:206-213). Alpha-exotoxin is toxic against
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. Forsberg et al. (1976) "Bacillus
thuringiensis: Its effects in Environmental Quality," National Research
Council of Canada. 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.
There are no documented strains of Bacillus with bacteriacidal, fungicidal
and corn rootworm activity. There are no known metabolites produced by
Bacillus subtilis that are of less than 10,000 molecular weight and are
extractable in a non-polar solvent.
DISCLOSURE OF THE INVENTION
A novel antibiotic-producing and metabolite-producing strain of Bacillus
subtilis, previously identified as Bacillus megaterium, is provided that
exhibits broad fungicidal and bactericidal activity and also exhibits corn
rootworm activity. Also provided is a novel metabolite from the novel B.
subtilis with activity on corn rootworm. Also provided is a method of
treating or protecting plants from fungal and bacterial infections
comprising the step of applying an effective amount of the
antibiotic-producing Bacillus subtilis. The antibiotic-producing Bacillus
subtilis can be provided as a suspension in a whole broth culture or as an
antibiotic-containing supernatant obtained from a whole broth culture of
the antibiotic-producing strain of Bacillus. Also provided is a method of
treating or protecting plant roots from corn rootworm infestations
comprising the step of applying an effective amount of the novel
metabolite-producing Bacillus subtilis. The novel metabolite-producing
Bacillus subtilis can be provided as a suspension in a whole broth culture
or as a metabolite-containing supernatant or a purified metabolite
obtained from a whole broth culture of the microorganism. Also provided
are novel compounds, agrastatins, produced by the novel strain AQ713 and a
novel combination of compounds comprising inturin A, a plipastatin, a
surfactin and an agrastatin.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the structure of the plipastatin antibiotics where Plipastatin
A1 is SEQ ID NO:2, Plipastatin A2 is SEQ ID NO:3, Plipastatin B1 is SEQ ID
NO:4 and Plipastatin B2 is SEQ ID NO:5.
FIG. 2 shows the HPLC chromatogram of AQ713 metabolites.
MODES OF CARRYING OUT THE INVENTION
The present invention provides a novel strain, AQ713, of Bacillus subtilis,
previously identified as a Bacillus megaterium, or mutants thereof with
the broad antifungal and antibacterial activity and the novel combination
of antifungal and anti-corn rootworm activity. This novel strain is
designated AQ713 and was deposited with the NRRL on Mar. 7, 1997 under the
provisions of the Budapest Treaty on the International Recognition of the
Deposit of Microorganisms for the Purpose of Patent Procedure under
Accession No. B21661. The invention also includes methods of preventing
and treating fungal and bacterial diseases in plants using such bacterial
strains or antibiotic-containing supernatants or pure antibiotics obtained
from such bacterial strains. The invention also includes methods of
treating plant roots or soil to control corn rootworm larvae with a
bacterial suspension of AQ713 or a metabolite-containing supernatant of a
culture of AQ713 or purified metabolites from strain AQ713. The invention
also includes a solvent-extractable metabolite with activity on corn
rootworm with a molecular weight of less than 10,000 daltons. The
invention further includes novel compounds, agrastatins, produced by the
novel microorganism. Also included is a novel combination comprising an
A-type iturin, a plipastatin, a surfactin and an agrastatin.
Definitions
As used herein, "biological control" is defined as control of a pathogen or
insect by the use of a second organism. Known mechanisms of biological
control include enteric bacteria that control root rot by out-competing
fungi for space on the surface of the root. Bacterial toxins, such as
antibiotics, have been used to control pathogens. The toxin can be
isolated and applied directly to the plant or the bacterial species may
administered so it produces the toxin in situ.
The term "fungus" or "fungi" includes a wide variety of nucleated
spore-bearing organisms that are devoid of chlorophyll. Examples of fungi
include yeasts, molds, mildews, rusts, and mushrooms.
The term "bacteria" includes any prokaryotic organism that does not have a
distinct nucleus.
"Fungicidal" means the ability of a substance to increase mortality or
inhibit the growth rate of fungi.
"Antibiotic" includes any substance that is able to kill or inhibit a
microorganism. Antibiotics may be produced by a microorganism or by a
synthetic process or semisynthetic process. The term, therefore, includes
a substance that inhibits or kills fungi for example, zwittermicin-A or
kanosamine.
"Antifungal" includes any substance that is able to kill or inhibit the
growth of fungi.
The term "culturing" refers to the propagation of organisms on or in media
of various kinds. "Whole broth culture" refers to a liquid culture
containing both cells and media. "Supernatant" refers to the liquid broth
remaining when cells grown in broth are removed by centrifugation,
filtration, sedimentation, or other means well known in the art.
An "effective amount" is an amount sufficient to effect beneficial or
desired results. An effective amount can be administered in one or more
administrations. In terms of treatment and protection, an "effective
amount" is that amount sufficient to ameliorate, stabilize, reverse, slow
or delay progression of the fungal or bacterial disease states.
As used herein, the term "insects" includes all organisms in the class
"Insecta." "Pre-adult" insects refers to any form of an organism prior to
the adult stage, including, for example, eggs, larvae, and nymphs.
"Insecticidal" refers to the ability of a substance to increase mortality
or inhibit growth rate of insects. "Nematicidal" refers to the ability of
a substance to increase mortality or inhibit the growth rate of nematodes.
"Pesticidal" refers to the ability of a substance to increase mortality or
inhibit the growth rate of insects, nematodes and mites.
"Positive control" means a compound known to have pesticidal activity.
"Positive controls" include, but are not limited to commercially available
chemical pesticides. The term "negative control" means a compound known
not to have pesticidal activity. Examples of negative controls are water
or ethyl acetate.
The term "solvent" includes any liquid that holds another substance in
solution. "Solvent extractable" refers to any compound that dissolves in a
solvent and which then may be isolated from the solvent. Examples of
solvents include, but are not limited to, organic solvents like ethyl
acetate.
The term "metabolite" refers to any compound, substance or byproduct of a
fermentation of a microorganism that has pesticidal activity. Antibiotic
as defined above is a metabolite specifically active against a
microorganism.
The term "agrastatins" refers to a group of novel compounds having the
following general structures (SEQ ID NOS:6-9):
##STR2##
where R.sub.1 is a branched or straight aliphatic side chain, C8-C20; X is
either Ala or Val; R.sub.2 is an acetate or an ester derivative; and Glx
is Gln or Glu. These compounds have antibacterial and antifungal activity
as well as anti-corn rootworm activity.
SEQ ID NO:6 corresponds to the above general structure where Glx is either
Glu or Gln; and R.sub.1 is a branched or straight aliphatic chain of
C.sub.8 -C.sub.20 and R.sub.2 is an acetate or esters derivative; X is Ala
or Val; an example of which is:
##STR3##
SEQ ID NO:7 corresponds to the above general structure where Glx is either
Glu or Gln; and R.sub.1 is a branched or straight aliphatic chain of
C.sub.8 -C.sub.20 and R.sub.2 is an acetate or ester derivative or H; X is
Ala or Val: an example of which is:
##STR4##
SEQ ID NO:8 corresponds to the above general structure where Glx is either
Glu or Gln; and R.sub.1 is a branched or straight aliphatic chain of
C.sub.8 -C.sub.20 and R.sub.2 is an acetate or ester derivative or H; X is
Ala or Val: an example of which is:
##STR5##
SEQ ID NO:9 corresponds to the above general structure where Glx is either
Glu or Gln and where R.sub.1 is a branched or straight aliphatic chain of
C.sub.8 -C.sub.20 and R.sub.2 is an acetate or an ester derivative; X is
Ala or Val: an example of which is:
##STR6##
We describe a novel metabolite and antibiotic-producing strain of Bacillus
subtilis, previously identified as Bacillus megaterium, that has broad
antifungal and antibacterial activity and that also kills or stunts corn
rootworm larvae. In another aspect, the present invention provides a
method of treating or protecting plants from fungal and bacterial
infections comprising applying an effective amount of a supernatant
obtained from a whole broth culture of Bacillus subtilis AQ713 within the
present invention. The supernatant may be obtained well known in the art
including centrifugation, filtration, sedimentation and the like.
In another aspect, the invention encompasses a method of treating or
protecting plants from fungal and bacterial infections comprising applying
an effective amount of the whole broth of the novel strain Bacillus
subtilis.
In further aspect, the invention encompasses a method of treating or
protecting plants from fungal and bacterial diseases comprising applying
an effective amount of the antibiotic produced by the novel strain of
Bacillus subtilis.
In another aspect, the present invention provides a method of treating or
protecting plant roots from corn rootworm infestations comprising applying
an effective amount of a supernatant obtained from a whole broth culture
of Bacillus subtilis AQ713 within the present invention. The supernatant
may be obtained well known in the art including centrifugation,
filtration, sedimentation and the like.
In another aspect, the invention encompasses a method of treating or
protecting plants from corn rootworm infestations comprising applying an
effective amount of the whole broth of the novel strain Bacillus subtilis.
In further aspect, the invention encompasses a method of treating or
protecting plant roots from corn rootworm infestations comprising applying
an effective amount of the metabolite produced by the novel strain of
Bacillus subtilis.
In order to achieve good dispersion and adhesion of compositions within the
present invention, it may be advantageous to formulate the whole broth
culture, supernatant and/or metabolite/antibiotic with components that aid
dispersion and adhesion. Suitable formulations will be known to those
skilled in the art.
Compositions within the present invention can be formulated as wettable
powders, granules and the like, or can be microencapsulated in a suitable
medium and the like. Examples of other formulations include, but are not
limited to soluble powders, wettable granules, dry flowables, aqueous
flowables, wettable dispersible granules, emulsifiable concentrates and
aqueous suspensions. Other suitable formulations will be known to those
skilled in the art.
In yet a further aspect of the present invention, a novel group of
compounds designated "agrastatins" are provided. These compounds exhibit
antibacterial and antifungal activity in addition to anti-corn rootworm
activity.
In still a further aspect of the present invention, a novel combination
comprising an A-type iturin, a plipastatin, a surfactin and an agrastatin
is provided.
In another aspect of the present invention, methods of treating or
protecting plants from fungal and bacterial diseases comprising applying
an effective amount of a novel combination of compounds comprising an
A-type iturin, a plipastatin, a surfactin and an agrastatin are provided.
All patents and publications cited herein are hereby incorporated by
reference in their entirety. The following examples are provided to
illustrate the invention. These examples are not to be construed as
limiting.
EXAMPLES
Example 1
Characterization of Strain AQ713
The isolate was identified based on utilization of the Biolog microplate
panel (Biolog, Inc., Hayward, Calif.) as described in Bochner (1989)
Nature 339: 157-158. The Biolog microplate is comprised of prefilled and
dried panel wells with 95 different carbon substrates plates available for
gram positive and gram negative bacteria. The isolate was grown in liquid
medium at 28.degree. C. and after 24 hrs a washed cell suspension (0.85%
saline) was inoculated into each panel well of a GP Microplate (Biolog,
Inc.) After 24 hrs at 28.degree. C., carbon utilization reactions were
assessed. Substrate utilization profiles were then compared to the Biolog
Gram-Positive Data Base (release 3.50) and isolated to closest similar
species. Biolog results gave a similarity index of 0.883 to Bacillus
megaterium.
A more extensive characterization of AQ713 was conducted by the American
Type Culture Collection, Rockville, Md.
Isolate submitted as: Unknown; Strain AQ 713
Isolate identified as: Using the available physiological and biochemical
data, this strain most closely resembles Bacillus subtilis.
Cellular morphology: The motile cells are found in singly, with one
endospore formed in the central or subterminal region. The cells are
uniformly stained Gram positive.
Colonial morphology: The colonies are opaque and irregular with convex
elevation, a rough, dull surface and an erose margin.
Characterization Data of Strain AQ 713:
Rods + Colony opaque +
Rods straight + Colony entire -
Rods curved - Colony erose +
Cells single + Colony lobate -
Cells chained - Colony circular -
Ends tapered - Colony irregular +
Ends rounded + Colony rhizoid -
Ends squared - Colony low convex +
Endospore formed + Colony high convex -
Sporangium swollen - Colony flat -
One spore/cell + Colony raised -
Spore round - Colony glistening -
Spore cylindrical + Colony dull +
Spore oval + Colony dry -
Spore central + Colony smooth -
Spore terminal - Colony rough +
Spore subterminal + Soluble brown pigment -
Gram stained + Soluble black pigment -
Gram positive + Soluble yellow pigment -
Gram negative - Insoluble brown pigment -
Gram variable - Insoluble black pigment -
Vacuoles present - Insoluble yellow pigment -
Colony translucent - Insoluble orange pigment -
Colony transparent - Insoluble red pigment -
Cells motile + Acid from lactose -
Growth at 15.degree. C. + Gas from lactose -
Growth at 20.degree. C. + Acid from mannitol -
Growth at 26.degree. C. + Gas from mannitol -
Growth at 30.degree. C. + Acid from mannose -
Growth at 37.degree. C. + Gas from mannose -
Growth at 45.degree. C. + Acid from sucrose weak
Growth at 50.degree. C. weak Acid delayed >14 days weak
Growth at 55.degree. C. - Gas from sucrose -
Growth at 60.degree. C. - Acid from trehalose -
Growth at 65.degree. C. - Gas from trehalose -
Catalase + Acid from xylose -
Oxidase + Gas from xylose -
Casein hydrolysis + Aerobe -
Gelatin liquification + Facultative -
Hippurate hydrolysis - Microaerophile +
Lecithinase degradation - Anaerobe -
Starch hydrolysis + Gas from sealed nitrate -
Tween 80 hydrolysis + Gas from sealed glucose -
Tyrosine decomposition - Indole -
Growth in 2% NaCl + Nitrate to nitrite +
Growth in 5% NaCl + Nitrate to gas -
Growth in 7% NaCl + Methylene blue reduction +
Growth in 10% NaCl + Methylene blue reoxidation -
Growth in 0.2% Na azide V Litmus milk acid -
Growth at pH 4.5 + Litmus milk coagulated -
Growth at pH 6.0 + Litmus milk alkaline +
Acid from arabinose - Litmus milk reduced +
Gas from arabinose - Litmus Milk peptonized +
Acid from cellobiose weak VP (5198) positive +
Acid delayed >14 days weak VP (5331) positive +
Gas from cellobiose - pH VP 5198 6.0 or less -
Acid from fructose + pH VP 5198 6.5-7.5 +
Acid delayed >14 days - pH VP 5198 8.0 or more -
Gas from fructose - Citrate utilization +
Acid from glucose + Propionate utilization -
Acid delayed >14 days - Phenylalanine deamination -
Gas from glucose -
Comments: Using the available physiological and biochemical data, this
strain most closely resembles Bacillus subtilis.
Key Characterization Results
Characterization Tests Strain AQ 713 Bacillus subtilis
Swollen sporangium - -
Anaerobic growth microaerophilic microaerophilic
VP reaction + +
pH of VP 7.0 5.0-8.0
Maximum temperature growth 55.degree. C. 45-55.degree. C.
7% NaCl growth + +
Acid from glucose + +
Acid from arabinose - +
Acid from xylose - +
Acid from mannitol - +
Casein decomposition + +
Tyrosine decomposition - -
Citrate utilization + +
Propionate utilization - -
Reference:
Gordon, R. E., W. C. Haynes and C. H. N. Pang. 1973. The Genus Bacillus.
Handbook No. 427. U.S. Department of Agriculture, Washington, D.C.
Example 2
Activity of AQ713 Against Corn Rootworm
Bacillus samples were grown in a Bacillus culture media. Medium 2 contained
5% peptone, 5% dextrose, 3% yeast extract, 3% malt extract, 1.5% proflo
cotton seed extract (59% protein, 4.26% fat, 6.73% ash, 3.19% fiber and
trace amounts of gossypol; the balance is water), 10% soy flour, and 0.5%
MgSO.sub.4.times.7H.sub.2 O. Medium 3 contained the same ingredients,
except with 20% peptone and 3.4% KH.sub.2 PO.sub.4 and 4.3% K.sub.2
HPO.sub.4. One day old streaked cultures were used to inoculate 250 mL
baffled shake flasks. Flasks were shaken at 200 rpm at 29.degree. C. for 5
days. To assay insecticidal activity, 35 mL of culture broth were
centrifuged at 5,200 rpm for 20 minutes and the supernatant used in
microassay described below.
Assays were performed in 96-well microplates. Each well contained a solid
agar substrate, a test organism and either a positive control, a negative
control or supernatant obtained as described in Example 1 from the novel
Bacillus strain.
To assay insecticidal activity, an agar substrate was prepared for the
wells of the microplate according to Marrone et al. (1985), J. Econ.
Entomol. 78: 290-293. To assay nematicidal activity, plain agar (1.5%) was
used in the wells instead.
A 1 ppm solution of Avid.RTM. (avermectin) was used as a positive control.
Deionized water was used as a negative control. Two replicates of test
sample or control were used for each assay. 40 uL of supernatant sample or
whole broth grown in medium 1, 2 or 3 were dispensed into each sample
well. Plates were then placed in a fume hood to dry for approximately 2-3
hours until the agar solution was dried.
Test organisms were either pre-adult corn rootworms (Diabrotica
undecimpunctata), pre-adult German cockroaches (Blatella germanica),
pre-adult beet armyworms (Spodoptera exigua), pre-adult flies (Drosophila
melanogaster), or the N2 strain of the nematode Caenorhabditis elegans.
Test organisms were diluted in 0.1% agar to a concentration of
approximately 5 organisms per 25 uL of agar dispensed into each well. The
microplate was sealed with an airtight substance such as Mylar.RTM., and
each well ventilated with a pin press. The plates were incubated at
27.degree. C. for up to 7 days.
After incubation, wells were scored by noting neonate mortality or the
degree of larval development. Sample wells containing all dead or stunted
larvae were given a score of 1, wells containing some dead and other
severely stunted larvae were given a score of 2, live but stunted larvae
were scored as 3 and sample wells containing no dead larvae were given a
score of 4. Scores were averaged to among replicates within each sample.
Results are summarized in Tables 2 and 3.
TABLE 2
Score Rating of AQ7l3 Against Insect Pests Whole broth
C. Corn Beet Fruit Positive Negative
elegans rootworm armyworm Fly Control Control
Medium 2 NT 1.0 4.0 4.0 1.0 4.0
Medium 3 NT 2.0 4.0 4.0 1.0 4.0
NT = not tested
TABLE 2
Score Rating of AQ7l3 Against Insect Pests Whole broth
C. Corn Beet Fruit Positive Negative
elegans rootworm armyworm Fly Control Control
Medium 2 NT 1.0 4.0 4.0 1.0 4.0
Medium 3 NT 2.0 4.0 4.0 1.0 4.0
NT = not tested
These tests show that AQ713 was active in both media as a whole broth
culture, with the best activity in medium 2. The supernatant was only
active when AQ713 was grown in medium 2.
Example 3
Chemical Properties of the AQ713 Metabolite
Active Against Corn Rootworm
50 mL of AQ713 was grown in media 2. To each culture was added 50 mL ethyl
acetate and the mixture was shaken in a separatory funnel for 2 minutes.
The aqueous layer was removed and the organic layer was collected in a
bottle containing magnesium sulfate. The organic filtrate was then
filtered into a round bottom flask and the solvent removed on the rotovap.
For the bioassay, the dried organic extract was redissolved in 2.5 mL
acetone. A 40 uL aliquot was removed and diluted to 800 uL with 70%
acetone/water. This is a 10.times. concentration of the organic extract.
Serial dilutions were carried out to obtain samples on neonate corn
rootworm with percent mortality recorded of neonate larvae (1 per well in
a microtiter plate as prepared above) after 7 days. The results are
recorded in Table 4.
TABLE 4
Activity of Ethyl Acetate Extracts of AQ7l3 Against Corn Rootworm
Sample Percent Mortality
AQ713: Organic extract 10X 89
Organic extract 5X 93
Organic extract 1X 65
Whole broth 100
70% acetone/water 27
Water 59
The results show that AQ713 produces a solvent-extractable metabolite that
kills corn rootworms.
To determine the molecular weight range of the active metabolite, a 50-mL
culture of AQ713 was grown in media 2. One mL was placed into a microfuge
tube and spun at 12,000 rpm for 15 minutes. The supernatant was removed.
500 microliters of supernatant was placed on top of a 10,000 dalton
molecular weight centricon filter. These were centrifuged according to the
manufacturer's instructions (12,000 rpm for 35 minutes). The filtrate was
collected and the retentate recovered by centrifugation and washing of the
filter. Samples of the supernatant, filtrate and retentate were tested
against neonate corn rootworm larvae (96 well-plate with insect diet,
Marrone et al., supra as above; 40 uL of sample per well and 8 wells for
each sample, 1 larva/well). The results of the test are shown in Table 5.
TABLE 5
Molecular Weight Cutoff of AQ713
Percent Mortality
Against Corn Rootworm
AQ713: supernatant 43
filtrate 63
retentate 17
The results show that the supernatant and filtrate were active, thus the
molecular weight of the metabolite is less than 10,000 daltons.
Example 4
Chemical Properties of the AQ713 Metabolite Active Against Plant Pathogens
50 mL of AQ713 was grown in media 2. To each culture was added 50 mL ethyl
acetate and the mixture was shaken in a separatory funnel for 2 minutes.
The aqueous layer was removed and the organic layer was collected in a
bottle containing magnesium sulfate. The organic filtrate was then
filtered into a round bottom flask and the solvent removed on the rotovap.
For the bioassay, the dried organic extract was redissolved in 2.5 mL
acetone. A 40 uL aliquot was removed and diluted to 800 uL with 70%
acetone/water. This is a 10.times. concentration of the organic extract. A
96-well plate assay (described below) plant pathogen assay with Pythium
ultimum and Botrytis cinerea was conducted to determine activity of the
organic extract. The whole broth gave 100% control (score of 1), but the
10.times. organic extract gave no control of the two plant pathogens
(score of 4). This indicates that the active antibiotics, unlike the corn
rootworm active metabolites produced by AQ713 are not extractable in an
organic solvent such as ethyl acetate.
Further testing provided for the isolation of a novel compound, agrastatin
A. A butanol extract was made of the fermentation broth by first
extracting the broth two times with an equal volume of ethyl acetate and
separating the layers. The aqueous fraction was then extracted two times
with an equal volume of butanol. The butanol extracts were combined and
solvent was removed with a rotary evaporator. A powder was obtained by
freeze drying the resulting extract.
The powder was dissolved in 80% acetonitrile/water and sonicated. The
solution was applied to a C-18 solid phase extraction (SPE) cartridge that
had been activated with methanol and equilibrated with 80%
acetonitrile/water. The SPE cartridge was eluted with 80% ACN/water and
this eluent was collected and the solvents removed. The eluent was further
purified by HPLC. A C-18 HPLC column (1 cm.times.25 cm) was used (UV
detection at 210 nm) with an acetonitrile+0.05% TFA/water+0.05% TFA
solvent gradient as follows: 0-20 minutes, 33% ACN; 20-30 minutes, 40%
ACN; 30-45 minutes, 45-55% ACN; and 45-63 minutes, 55% ACN.
An HPLC chromatogram of AQ713 shows the presence of the iturins,
iturin-like compounds (plipastatins and agrastatins) and surfactins, see
FIG. 2. Iturins A2, A3, A4, A7 and A6 were identified by a combination of
NMR data and LC mass spectrometry data and comparison to literature
values. Surfactins were identified by comparison to purchased surfactin
standards by HPLC and by LC mass spectrometry.
The iturin-like compounds were determined to be a mixture of plipastatins
and the novel agrastatins by a combination of amino acid analysis and LC
mass spectrometry. Extensive NMR data was also collected for one of the
novel compounds (HPLC peak 20), designated agrastatin A. Agrastatin A was
found to contain the following amino acids: Thr; 3 Glu; Pro; Ala; Val; 2
Tyr; and Orn. This make up differs from plipastatin A by the presence of
Val and the loss of Ile. The molecular weight of agrastatin A was
determined to be 1448 which corresponds to the following structure (SEQ ID
NO:2):
##STR7##
The straight chain nature of the fatty acid portion was confirmed by .sup.1
H NMR. The position of the amino acids in the cyclic peptide was
determined by detailed analysis of the TOCSY and ROESY datasets.
Mass spectrometry and amino acid analysis of agrastatin B (HPLC peak 26)
suggest that its structure is similar to plipastatin B2 with the
substitution of the Ala residue with Val. The structure (SEQ ID NO:5) is
shown below:
##STR8##
Example 5
Activity of AQ713 Against Plant Pathogens in in-vitro Culture (96-well
plate)
To determine if AQ713 is effective against the fungi, Phytophthora
infestans, Pythium ultimum, Botrytis cinerea, Rhizoctonia solani,
Alternaria solani, the following experiments were performed. 96-well
plates (flat-bottomed, 400 microliters per well, Nunc brand) were filled
with an agar medium (potato dextrose agar) (PDA, Difco). Phytophthora
infestans cultures were grown for three days in liquid YPG-1 medium (0.4 g
yeast, 0.1% KH.sub.2 PO, 0.5% MgSO.sub.4.times.7 H.sub.2 O, 1.5% glucose).
For the other fungi, spores were scraped from the surface of petri plates
and 0.1-0.2 mL aliquots of deionized water and spore suspension
(concentration approximately 2.times.10.sup.6 spores/mL) of pathogen were
spread onto the agar.
AQ713 was grown for 72 hours in the medium 2 or 3 as described in Example
2. To obtain supernatants, the whole broth culture was centrifuged at
5,200 rpm for 20 minutes. The fungal plant pathogens were pipetted onto
the 96-well plates (8 wells/pathogen). The presence or absence of fungal
growth was recorded for each of 8 wells. Approximately 40 uL of AQ713
supernatant or 20 uL of whole broth was added to each well. A score of "1"
means complete inhibition of fungal growth. A score of "4" means no
inhibition of fungal growth. Results are shown in Table 6.
TABLE 6
In-Vitro Inhibition of Fungal Growth (96-well plate)
Media 2 Media 3
Score Score
AQ713 Supernatant
Phytophthora infestans 1
Pythium ultimum 1 1
Botrytis cinerea 1 1
Rhizoctonia solani 4 1
Alternaria solani 1 1
AQ713 whole broth
Colletotrichum cocodes 1 NT
Alternaria brassicicola 1 NT
Botrytis cinerea 1 NT
Cladosporium cucumerinum 1 NT
Monilinia fructicola 1 NT
Venturia pyrina 1 NT
Rhizoctonia solani 1 NT
Alternaria solani 1 NT
NT Not tested
The results show that AQ713 has broad fungicidal spectrum in-vitro and that
both the whole broth and supernatant are highly active. The supernatant
was active on Rhizoctonia solani in medium 3 but not medium 2.
Example 6
Activity of AQ713 Against Plant Pathogens in in-vitro Culture (zone assay)
To determine the activity of AQ713 in an agar diffusion (zone) assay, plant
pathogen spores were spread over the surface of potato dextrose agar in 10
cm petri dishes. 7.0 mm wells were removed from the agar and a 100 uL
sample of the supernatant of AQ713 grown in medium 2 was placed in the
well. Supernatant was prepared by centrifuging at 4200 rpm for 40 minutes.
The supernatant was then spun again at 4200 rpm for another 40 minutes.
Typical results consisted of a zone of no growth and/or reduced growth of
the pathogen around the well. The zone size in millimeters was measured
and recorded. The results are shown in Table 7.
TABLE 7
In-Vitro Inhibition of Fungal Plant Pathogen Growth (Zone Test)
Alternaria Botrytis Monilinia
brassicicola cinerea fructicola
AQ713 supernatant 16 23 14
Zone size (mm)
AQ713 Whole broth 22 15 18
Example 7
Activity of AQ713 Against Bacterial Plant Pathogens
A standard agar diffusion assay was set up as in example 6. A lawn of each
bacterial pathogen was spread over the surface of a petri plate. 100 uL of
AQ713 whole broth grown in medium 2 was placed in each well. The size of
the zone was measured in millimeters.
TABLE 8
In-Vitro Inhibition of Bacterial Plant Pathogens (Zone Test)
AQ713 Whole broth: Inhibition Zone (mm)
Acidovorax avenae subsp. citrulli 18
Pseudomonas syringae pv. tomato 11
Xanthomonas campestris pv. campestris 18
Erwinia carotovora subsp. carotovora 11
Clavibacter michiganense subsp. michiganense 22
AQ713 was active against all species of bacterial plant pathogens tested
in-vitro.
Example 8
Activity of AQ713 Against Plant Pathogens in Plant Tests
The activity of AQ713 was tested against gray mold, Botrytis cinerea, on
beans and geranium leaves, Alternaria solani on tomato seedlings, and
downy mildew of lettuce, Bremia lactucae.
For A. solani, tomato seedlings at the 2-3 leaf stage planted in 6-packs
were sprayed to runoff with AQ713 whole broth (media 2). After spraying,
the seedlings were allowed to dry (about 1.5 hours). The seedlings were
then sprayed with 5.0.times.10.sup.4 spores/mL. Seedlings were covered
with a plastic dome and kept at 28.degree. C. in a Percival incubator.
Water with no AQ713, with and without spores of the pathogen was used as a
negative control and a positive pathogen control. Four days later the test
was read. On the water A. solani control, there were uniform lesions over
all the leaves and the cotyledons were detached and severely infected
(rating of 5=complete infection, no control). AQ713 treated plants had a
few light lesions scattered on the true leaves. The cotyledons were
attached but with some small lesions (rating of 1). The negative control
was not infected.
A second test was set up using detached tomato seedlings (stems broken off
at the ground level) placed in mason jars filled with water put under
domes and stored as above. The plants were sprayed as above and the
symptoms of A. solani were recorded four days later. There were no
symptoms on the negative control. On the positive control, there were
uniform lesions over the seedlings. The AQ713 treatment was rated 1 (few
or no lesions). Two days later, the plants in the positive control were
destroyed, but the AQ713 treated seedlings were virtually clean and looked
the same as the negative controls (water sprayed plants).
For the test on Botrytis cinerea, the first true leaves of a bean plant
were wounded by pressing the mouth of a 13.times.100 culture tube onto
each leaf. Each leaf received two wounds/leaf. The leaves were sprayed
with AQ713 whole broth (media 2) or water alone or the pathogen alone.
When dry, they were again sprayed with B. cinerea spores
(0.8.times.10.sup.6 spores/mL). The leaves were placed in flats covered
with plastic domes and stored at 18-20.degree. C. in a Percival incubator.
Five days later, the positive control (pathogen alone) was rotted in an
area about 25 mm in diameter. The negative control (water alone) had no
rotting. AQ713 showed no infections on 7 of 8 circles where the leaves
were wounded. The one that was infected had light infection at two
locations around the circle.
For the Bremia test, lettuce seeds were planted in a layer of sterilized
potting mix containing peat, perlite and vermiculite in small clear
plastic plant condominiums about 8 centimeters high and wide. After the
lettuce germinated (one week), the lettuce seedlings were sprayed with the
AQ713 broth or supernatant sample. The plants were allowed to dry and then
downy mildew spore suspension from infected lettuce seedlings was sprayed
onto the seedlings. The plastic covers were placed over the plants and
incubated at 18-20 20.degree. C. in a Percival incubator. One week later,
the test was evaluated. AQ713 did not prevent downy mildew from Bremia on
lettuce seedlings.
Example 9
Efficacy of AQ713 Against Plant Diseases (Greenhouse Test)
Grape Downy Mildew
AQ713 was grown in a soy-based medium in a 400 liter fermenter for 48
hours. Grape plants (cultivar Chardonnay) were sprayed with a hand-held
sprayer to run-off with whole broth from the 400 liter fermentation run
diluted with sterile water to 0.5.times. and 0.25.times. concentrations.
When the foliage dried, the plants were sprayed a second time. After
drying, the plants were inoculated with the pathogen causing grape downy
mildew, Plasmopara viticola. Three plants were treated for each dose. Each
plant was evaluated by estimating the percent disease control based on a
scale from 0 to 100% control. 100% control is a plant with no visible
lesions. A chemical fungicide, metalaxyl, was used for comparison. The
results were as follows:
AQ713 0.5X whole broth 97.7% control
AQ713 0.25X whole broth 100% control
Metalaxyl 30 ppm 100% control
Metalaxyl 10 ppm 98.3% control
Metalaxyl 1 ppm 80% control
The results demonstrate that AQ713 effected control of grape downy mildew
as well as the chemical fungicide.
Example 10
Efficacy of AQ713 Against Squash Powdery Mildew
AQ713 was grown in a soy-based medium in a 400 liter fermenter for 48
hours. Squash plants (Crookneck and Acorn) were sprayed with a hand-held
sprayer to run-off with whole broth from the 400 liter fermentation run
and a sample diluted with sterile water to 0.5.times. concentration. After
drying, the plants were inoculated with the squash powdery mildew
pathogen, Sphaerotheca fuliginea. Two plants were treated for each dose.
Spray dried powder of the whole broth was also tested. The 400 liter
fermentation broth was spray dried to remove the water. 10% and 2.5% spray
dried powder solutions were sprayed on the plants to run-off as above. The
incidence of powdery mildew disease was rated on a score from 0 to 5. The
5 rating is 100% disease whereas the 0 rating is no disease. The results
are shown below in Table 9.
TABLE 9
Crookneck Crookneck
Test Acorn Squash Acorn Squash Squash Squash
Suspension Plant 1 Plant 2 Plant 1 Plant 2
AQ713 1X 0 0 0 0
whole broth
AQ713 0.5X 0 0 0 0
whole broth
AQ713 10% 0 0 0 0
spray dried
powder
AQ713 2.5% 0 0 0.5 1
spray dried
powder
AQ713 whole broth and spray dried powder provided nearly complete control
of squash powdery mildew.
Example 11
Efficacy of AQ713 on Late Blight, Gray Mold, Grape Powdery Mildew, Cereal
Powdery Mildew, Sheath Blight and Rice Blast in the Greenhouse
AQ713 was grown in a soy-based medium for 72 hours in a 250 mL shake flask.
The disease, causative pathogen and host are listed in Table 10 below.
This whole broth culture was tested on the plants as shown in Table 11
below.
TABLE 10
Disease Plant Pathogen Host
Late Blight Phytophthora infestans Tomato
Gray Mold Botrytis cinerea Pepper
Sheath Blight Rhizoctonia solani Rice
Rice Blast Pyricularia oryzae Rice
Powdery Mildew Uncinula necator Grape
Powdery Mildew Drysiphe graminis f. sp. Wheat
graminis
Each broth was sprayed to run-off at 1.times. concentration on the test
plants with a hand held sprayer, allowed to dry and then sprayed a second
time. Three plants were treated for each disease and treatment. After
drying, the plants were inoculated with the pathogens. Each plant was
evaluated by estimating the percent disease control based on a scale from
0 to 100% control 100% control refers to a plant with no visible lesions.
Chemical fungicides were used for comparison. Disease index is the
severity of the disease on the untreated control.
TABLE 11
P. B. E. U. P. R.
infestans cinerea graminis necator oryzae solani
AQ713 70 100 84 100 100 100
Metalaxy 100
30 ppm
Metalaxyl 77
10 ppm
Propico- 87
nazole
10 ppm
Propico- 57
nazole
5 ppm
Propico- 100
nazole
0.5 ppm
Propico- 54
nazole
0.2 ppm
Myclo- 100
butanil
30 ppm
Myclo- 100
butanil
10 ppm
Pency- 100
curon
50 ppm
Pency- 100
curon
10 ppm
Benomyl 100
100 ppm
Benomyl 77
40 ppm
Disease 80 95 70 50 60 80
Index (%)
AQ713 showed activity that was equivalent to chemical fungicides on all the
pathogens tested.
Example 12
Efficacy of AQ713 Against Brassica Downy Mildew
Bacillus strain AQ713 was grown in a ten liter fermenter in a soy-based
medium for 48 hours. The whole broth culture at 1.times. strength was
sprayed onto three week-old cauliflower and brussel sprouts plants at the
full cotyledon stage with an artist's air brush powered by compressed air.
Three replicates of 15-25 seedlings/pot were sprayed per treatment.
Quadris.TM., an azoxystrobin fingicide from Zeneca, was also sprayed on
plants (three per treatment) at rates of 250 ppm and 125 ppm. A spore
suspension of downy mildew, Peronospora parasitica, at 1-5.times.10.sup.4
spores/mL was sprayed onto the Brassica plants after the AQ713 and Quadris
sprays dried. The plants were held at 15-17.degree. C. for 24 hours for
infection, then the seedlings were incubated at 20-24.degree. C. for six
days. The pots were returned to 15-17.degree. C. overnight to allow
sporulation of the pathogen until the test was rated. Each plant was
evaluated by estimating the percent disease control based on a scale from
0 to 100% control. 100% control is a plant with no sporulating lesions.
The results averaged across replicate pots are shown below in Table 12.
TABLE 12
Reading taken Reading taken Reading taken
December 23 December 30 January 6
AQ713 whole broth 100 90 75
Quadris 250 ppm 100 NT NT
Quadris 125 ppm NT 100 100
Water Control 0 0 0
NT = Not Tested
AQ713 controlled downy mildew effectively for three weeks in duration.
Example 13
Synergism of AQ713 and a Commercial Fungicide
AQ713 was grown in a ten liter fermenter in a soy-based medium for 72
hours. The bacterial culture was diluted with sterile water to 0.5.times.
and 0.25.times. concentrations. The culture at 1.times., 0.5.times. and
0.25.times. concentrations was sprayed onto three week-old pepper plants
with an artist's air brush powered by compressed air. Three plants were
sprayed per treatment. Quadris.TM., an azoxystrobin fungicide from Zeneca,
was also sprayed on plants (three per treatment) at concentrations of 500
ppm, 250 ppm and 125 ppm. In addition, combinations of Quadris plus the
whole broth culture of AQ713 in a 1:1 ratio were sprayed onto pepper
plants (three per treatment). The treatments with and without Quadris are
outlined in Table 13 below. A spore suspension of Botrytis cinerea, gray
mold, at 1.times.10.sup.6 spores/mL was sprayed onto the pepper plants
after the AQ713 and Quadris sprays dried. The plants were held at
20-22.degree. C. for 3 days until the test was rated. The incidence of
gray mold disease was rated on a score from 0 to 5. The 5 rating indicates
100% disease whereas the 0 rating indicates no disease. The results are
shown in Table 13 below.
TABLE 13
Rating Rating Rating Rating
Treatment Replicate 1 Replicate 2 Replicate 3 Average
AQ713 1X 0.5 0.5 1.5 0.8
AQ713 0.5X 2.0 2.5 2.0 2.2
AQ713 0.25X 3.0 3.0 2.0 2.7
Quadris 4.0 3.5 4.0 3.8
500 ppm
Quadris 2.5 3.5 3.0 3.0
250 ppm
AQ713 1X + 0.5 1.0 1.0 0.8
Quadris 500 ppm
AQ713 1X + 1.0 1.0 0.5 0.8
Quadris 250 ppm
AQ713 0.5X + 0.5 1.0 1.0 0.8
Quadris 250 ppm
AQ713 0.25X + 0.5 1.0 2.5 1.3
Quadris 250 ppm
Water control 4.0 5.0 5.0 4.7
Water control 2 5.0 5.0 5.0 5.0
The results clearly show that combinations of Quadris and AQ713 control
gray mold disease significantly better than either Quadris or AQ713 alone.
SEQUENCE LISTING
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<200> SEQUENCE CHARACTERISTICS:
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<213> ORGANISM: Bacillus subtilis
<400> SEQUENCE: 1
Leu Leu Asp Leu Leu Asp Leu
1 5
<200> SEQUENCE CHARACTERISTICS:
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<211> LENGTH: 10
<212> TYPE: PRT
<213> ORGANISM: Bacillus subtilis
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<221> NAME/KEY: MOD_RES
<222> LOCATION: (1)...(1)
<223> OTHER INFORMATION: Glu has CH3(CH2)12COH2 attached.
<400> SEQUENCE: 2
Glu Xaa Tyr Thr Glu Ala Pro Gln Tyr Ile
1 5 10
<200> SEQUENCE CHARACTERISTICS:
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<211> LENGTH: 10
<212> TYPE: PRT
<213> ORGANISM: Bacillus subtilis
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<222> LOCATION: (1)...(1)
<223> OTHER INFORMATION: Glu has a CH3CH2CHCH3(CH2)10CH(OH)CH2CO
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<400> SEQUENCE: 3
Glu Xaa Tyr Thr Glu Ala Pro Gln Tyr Ile
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 4
<211> LENGTH: 10
<212> TYPE: PRT
<213> ORGANISM: Bacillus subtilis
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<221> NAME/KEY: MOD_RES
<222> LOCATION: (1)...(1)
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<400> SEQUENCE: 4
Glu Xaa Tyr Thr Glu Val Pro Gln Tyr Ile
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 5
<211> LENGTH: 10
<212> TYPE: PRT
<213> ORGANISM: Bacillus subtilis
<220> FEATURE:
<221> NAME/KEY: MOD_RES
<222> LOCATION: (1)...(1)
<223> OTHER INFORMATION: Glu has a CH3CH2CHCH3(CH2)10CH(OH)CH2CO
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<400> SEQUENCE: 5
Glu Xaa Tyr Thr Glu Val Pro Gln Tyr Ile
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 6
<211> LENGTH: 10
<212> TYPE: PRT
<213> ORGANISM: Bacillus subtilis
<220> FEATURE:
<221> NAME/KEY: MOD_RES
<222> LOCATION: (1)...(1)
<223> OTHER INFORMATION: Glx is Gln or Glu and has an R1-CH(OR2)CH2(CO)
attached where R1 is a branched or straight aliphatic chain of
C8-C20 and R2 is an acetate or ester derivative.
<400> SEQUENCE: 6
Xaa Xaa Tyr Thr Xaa Ala Pro Xaa Tyr Val
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 7
<211> LENGTH: 10
<212> TYPE: PRT
<213> ORGANISM: Bacillus subtilis
<220> FEATURE:
<221> NAME/KEY: MOD_RES
<222> LOCATION: (1)...(1)
<223> OTHER INFORMATION: Glu has a CH3(CH2)12-CH(OH)CH2CO attached.
<400> SEQUENCE: 7
Glu Xaa Tyr Thr Glu Ala Pro Gln Tyr Val
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 8
<211> LENGTH: 10
<212> TYPE: PRT
<213> ORGANISM: Bacillus subtilis
<220> FEATURE:
<221> NAME/KEY: MOD_RES
<222> LOCATION: (1)...(1)
<223> OTHER INFORMATION: Glu has a CH3CH2CH(CH2)10-CH(OH)CH2CO attached.
<400> SEQUENCE: 8
Glu Xaa Tyr Thr Glu Val Pro Gln Tyr Val
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 9
<211> LENGTH: 10
<212> TYPE: PRT
<213> ORGANISM: Bacillus subtilis
<220> FEATURE:
<221> NAME/KEY: MOD_RES
<222> LOCATION: (1)...(1)
<223> OTHER INFORMATION: Xaa is either Glu or Gln; and Xaa has a
R1CH(OR2)CH2CO attached, where R1 is a branched or straight chain
aliphatic side chain of C8-C20 and R2 is an acetate or an ester
derivative.
<400> SEQUENCE: 9
Xaa Xaa Tyr Thr Glx Xaa Pro Glx Tyr Val
1 5 10
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