Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Reexamination Certificate
1999-06-15
2001-08-28
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
C424S093200, C435S252310, C435S252500
Reexamination Certificate
active
06280722
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the identification of a mutant strain of
Bacillus thuringiensis
(Bt) which has improved antifungal activity.
BACKGROUND OF THE INVENTION
Plant diseases reduce both the quantity and quality of plant products. Annual crop losses caused by disease amount to about 15% of expected harvest even in industrialized countries. The causes of plant diseases are many and diverse.
Infectious agents of plant diseases include such different groups as viroids, viruses, mollicutes, rickettsias, bacteria, actinomycetes, fungi, algae, protozoans, nematodes, insects and mites. A large percentage of all plant diseases is caused by fungi. These diseases are often spread by insects.
The soil microbe
Bacillus thuringiensis
(Bt) is a gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions can be observed microscopically as distinctly shaped crystals. The proteins are generally toxic to certain insects and contain specific endotoxic activity. Numerous Bt toxin genes have been isolated, sequenced, and recombinant DNA-based Bt products have been produced and approved for use.
Since the 1950's, formulations of Bt have been used as biological insecticides to control agricultural insect pests and more recently, insect vectors of a variety of human and animal diseases. Preparations of spores and crystals of
B. thuringiensis
var. kurstaki have been used for many years as commercial insecticides for lepidopteran pests.
Aflatoxins are highly carcinogenic compounds that are metabolic products of the ubiquitous fungi,
Aspergillus flavus
and
A. parasiticus.
Aflatoxins can contaminate peanuts and other crops such as corn whenever this commodity is invaded by the fungi. Reduction of aflatoxin contamination is a priority need in the production of peanuts (
Arachis hypogaea
). Aspergillus-like fungi invade peanut tissue when hot, dry weather prevails, especially during the last 4-6 weeks of the growing season. Fungal invasion of the developing peanut seed is facilitated when the pods are damaged in any way, such as feeding damage caused by the soilborne larvae of the lesser cornstalk borer (LCB),
Elasmopalpus lignosellus,
(Lepidoptera: Pyralidae).
Peanut pods and seed develop underground which makes preharvest control of aflatoxin contamination extremely difficult. Fungi that produce aflatoxins survive well in soil and invade developing peanut pods throughout growing season. Because no chemicals are effective against aflatoxin contamination of peanuts and culture control tactics are impractical, alternatives such as biological control are needed.
Organisms exhibiting biological control activity and natural products are among the control alternatives being sought for aflatoxin control. Organisms with biological control activity have an advantage of colonizing and, presumably, increasing in population as control is manifested. Natural products and biological control do not have the same negative impacts on the environment that synthetic pesticides may have. In addition, as synthetic pesticides become more difficult and expensive to discover and register, natural products are becoming increasingly important as alternatives.
One class of “biological control” compounds which have been investigated are the chitinases. Chitinases have been implicated in helping to control both insects and fungi, and also to help degrade crustacean exoskeltons, as they collect after the meat has been harvested from shellfish such as shrimp and crab. As a result, numerous chitinase genes have been cloned and sequenced from bacteria, plants, and actinomycetes. Interestingly, many of these chitinase genes display little sequence homology with other chitinase genes.
Therefore, there is needed biocontrol strains which are effective against insects and fungi.
SUMMARY OF THE INVENTION
A novel
Bacillus thuringiensis
(Bt) strain is disclosed having increased activity against plant pathogenic fungi. The strain retains insecticidal activity against certain lepidopteran plant pests. Further, DNA sequences which encode the chitinase activity of the strain are also encompassed. These chitinase genes encode proteins with potential antifungal and/or insecticidal activity. The invention further discloses the use of the strain and DNA sequences for protection of plants from fungal and insect damage.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an improved biocontrol strain of Bt which can be used to control pathogenic attack on crop plants. The strain is able to aggressively control fungal and insect attack. The strain is an enhanced chitinolytic isolate of
Bacillus thuringiensis
subsp. dendrolimus, (HD-548).
The strain produced by mutagenesis and selection shows enhanced fungicidal activity against certain plant pathogenic fungi. Such fungi include: Botrytis, particularly
B. cineria;
Alternaria, particularly
A. solani;
Aspergillus, etc. Additionally the strain retains the insecticidal activity normally attributed with the wild-type, or parent Bt. That is, the Bt strain of the invention protects plants against damage by lesser cornstalk borer and other lepidopteran pests typically susceptible to Cry1Ab and Cry1Ac toxins. The Bt strain is designated AU634 and has been deposited with the USDA NRRL in Peoria, Ill. as NRRL B-21620.
Bt strain AU634 inhibits growth and reduces aflatoxin production by Aspergillus, particularly
A. flavus
and
A. parasiticus
in peanuts. The strain additionally has substantial activity against lesser cornstalk borer. Accordingly, the novel strain finds use as a pesticidal spray or alternatively as a seed treatment.
General methods for employing the strains of the invention in pesticide control or engineering other organisms as pesticidal agents are known in the art. See, for example, U.S. Pat. No. 5,039,523 and EP 0480762A2. Preferred methods of applying active strains of the invention or an agrochemical composition of the present invention are leaf application, seed coating and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pests and other agronomic factors.
Bt strain AU634 is a result of mutagenesis and selection of Bt strain HD-548 obtained from the USDA NRRL collection in Peoria, Ill., NRRL HD-548. Strain HD-548 was incubated with N-methyl-N′-nitro-N-nitrosoguanidine. Colonies were selected for enhanced chitinase activity with subsequent demonstrated fungicidal activity against
Aspergillus flavus
and
Alternaria solani.
The strain was additionally tested to ensure that lepidopteran activity was not affected.
Fungal invasion of developing peanut seed is facilitated when the pods are damaged in any way. Feeding damage by the soilborne larvae of the lesser cornstalk borer insect, in particular, has been shown to be closely related to peanut seed invasion by
A. Flavus
-type fungi. Peanut seed develop in the soil over an extended period of time. Throughout peanut pod development, seed may become invaded by aflatoxigenic fungi. Thus, strain AU634 is an ideal biocontrol agent, as it prevents seed pod damage by the insect and has antifungal activity.
A general characterization of strain AU634 is as follows: Parent strain is HD-548, serotype 4a:4b, colony morphology: colony edge is rounded and mostly smooth. The isolate can degrade sufficient chitin on a Nutrient agar plant supplemented with 0.4% colloidal chitin plate so that a visible clearing zone can be detected after 36 hours. Exhibits putative antibiosis against
Botrytis cinerea
and
Alternaria solani.
The present invention is also drawn to a novel chitinase and the DNA sequence encoding the novel chitinase in strain AU634. The chitinase (SEQ. ID NO: 1) comprises an N-terminal sequence as follows:
Ala-Asn-Asn-Leu-Gly-Ser-Glu-Leu-Ileu-Val-Gly-Tyr-Phe-Pro-Asn-Phe-Asp-Asn-Gly-Thr
This N-terminal sequence exhibits little homology with any of the published chitinases. For the N-terminal sequence see also SEQ ID NO: 1.
To obtain
Alston & Bird LLP
Auburn University
Mosher Mary E.
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