The present invention is directed toward a biocontrol agent and pesticide and, in particular, biocontrol agent and pesticide for inhibition of disease-causing fungi.
Blackleg is a fungal disease of canola that is responsible for losses in crop yield and seed quality. The disease has spread throughout most of the Canadian prairies despite the use of fungicides and blackleg resistant canola cultivars. No successful inhibitor to the spread of blackleg exists, therefore another method of disease control is sought.
Canola is an economically important crop in Canada and considerable losses in seed quality and yield are seen every year due to fungal diseases such as blackleg. The canola cultivars presently grown show varying degrees of susceptibility to blackleg, and to date, there are also no varieties resistant to Sclerotinia white stem rot.
Canola is an important agricultural product in Canada, with a cash value of over $300 million per year in Alberta alone. In 1994 agronomists reported that the global consumption of vegetable oils is increasing by about 4% every year. To meet this global demand for canola oil Canada would need to grow 15% more than the 1994 acreage, and since that time there has been no abatement in the demand for canola oil. One of the major blocks to increasing the production of canola is the loss of crops to fungal diseases such as blackleg, Sclerotinia, Alternaria and Rhizoctonia. Much work has been done on developing blackleg disease tolerant canola cultivars. These new varieties help to improve the crop yield, however the canola cultivars are still not resistant to the fungal diseases. Some measure of control is achieved using chemical fungicides as a seed treatment, but newly emerged seedlings are still susceptible to disease. As a result, crop rotation on a four year cycle is an essential element in the control of blackleg disease, which further restricts the amount of canola which can be produced.
Fungal diseases also adversely affect other crops. As an example, fairy ring, caused by Marasmius oreades, is a common disease of turf grass.
Fungicidal seed treatments are used for chemical control of disease-causing fungus. However, single applications at the time of seeding do not provide sustained protection for the plants, and considerable losses can still occur before the crop reaches a stage of growth where some natural resistance has developed. Multiple fungicide applications are undesirable from both an economic and environmental perspective, and fungicides are not effective against the fungal spores that can persist in infected canola stubble from year to year. Constant use of fungicides can select for fungicide-resistant fungi, so improved methods of controlling fungal diseases are needed to protect plants in the vulnerable seedling stage and throughout the growing season. Biological control of blackleg and other fungal diseases may offer an environmentally sound method for plant disease control.
After considerable research and effort, a soil bacterium has been isolated from canola roots in a canola production plot near Sedgewick, Alberta Canada. The soil bacterium is a new strain of Paenibacillus polymyxa (formerly defined as Bacillus polymyxa) and has been called PKB1. A sample of the bacterium was deposited on May 18, 1998 with the American Type Culture Collection (ATCC), bearing ATCC Accession Number 202127. The soil bacterium produces an antibiotic after sporulation of the vegetative cells. The antibiotic is primarily spore associated. The antibiotic exhibits pesticidal activity against some bacteria and fungi. In particular, the P. polymyxa strain, PKB1 and the peptide antibiotic from PKB1 offer antifungal activity against Leptosphaeria maculans, the fungus that causes blackleg disease in canola, as well as other economically important disease-causing fungi including Sclerotinia sclerotiorum, Marasmius oreades, Pythium pythioides, Rhizoctonia solani, Fusarium avenaceum and Alternaria brassicae. When freeze dried or living cells of this P. polymyxa strain are applied to canola seeds they provide the plants germinating from the seed with protection against L. maculans in the stubble. Strain PKB1 of P. polymyxa can be used as a biocontrol agent against blackleg and other fungal diseases of canola.
The present invention relates to a novel strain of bacteria, referred to herein as PKB1, which has an inhibitory affect on fungi such as L. maculans and S. sclerotiorum. The present invention also relates to the antibiotic isolated from the bacterial strain PKB1, referred to herein as the PKB1 antibiotic, and the peptides of the antibiotic, referred to herein as the PKB1 peptides, which provide the inhibitory affect against fungi. The bacteria, antibiotic and peptides of the present invention can be used as pesticides and biocontrol agents against disease-causing fungi, for example, in crop plants.
One aspect of the invention pertains to an isolated Paenibacillus polymyxa strain PKB1 that acts as an inhibitory agent against Leptosphaeria maculans and other disease-causing fungi such as for example. Sclerotinia sclerotiorum, Marasmius oreades, Pythium pythioides, Rhizoctonia solani, Fusarium avenaceum and Alternaria brassicae. An xe2x80x9cisolatedxe2x80x9d or xe2x80x9cpurifiedxe2x80x9d bacterial strain is substantially free of materials from its natural environment including soil and biological matter including other bacterium or plant matter. The language xe2x80x9csubstantially free of materials from its natural environmentxe2x80x9d includes preparations or cultures of the bacterium in which the bacterium is separated from components of the environment in which it is naturally found. In one embodiment, the language xe2x80x9csubstantially free of materials from its natural environmentxe2x80x9d includes cultures having less than about 20% (by count) of non-PKB1 bacteria (also referred to herein as contaminating bacteria, contaminating bacteria does not include bioactive mutants or modified forms of strain PKB1), more preferably less than 10% (by count) of non-PKB1 bacteria and most preferably less than about 5% non-PKB1 bacteria.
The invention also pertains to bioactive mutants or modified forms of strain PKB1 which retain their inhibitory affect against L. maculans. As used herein, the term xe2x80x9cbioactive mutants or modified forms of strain PKB1xe2x80x9d is intended to include bacterium which have naturally mutated or by manipulations such as, for example, chemical or UV mutation or genetic modification or transformation been modified to have other characteristics such as, for example, antibiotic resistance.
As used herein, inhibition is a reduction in the growth or development of the fungi, for example, against control systems. Standard assays, such as those described herein, can be used to determine the ability of the strain or bioactive mutants or modified forms thereof to act against the fungi of interest. The standard assays can be conducted in vitro or in the field.
The strain or bioactive mutants or modified forms thereof can be in vegetative or spore state. They can be in culture, cell suspension, dried, dead or viable or in any other form such that they are capable of inhibiting L. maculans and preferably other disease-causing fungi.
Another aspect of the invention pertains to methods for detecting the presence of the bacterial strain of the present invention in a biological sample. In a preferred embodiment, the methods involve contacting a biological sample (e.g. a soil sample) with a compound or an agent capable of detecting PKB1 cells or nucleic acids such that the presence of PKB1 is detected in the biological sample. The compound or agent can be, for example, a labeled or labelable nucleic acid probe capable of hybridizing to PKB1 bacterium nucleic acids.
Another aspect of the invention pertains to an isolated antibiotic which is capable of inhibiting the development of L. maculans. In a preferred embodiment, the isolated antibiotic contains at least one of the spore-associated peptides of Paenibacillus polymyxa strain PKB1 or contains at least one peptide which is sufficiently homologous to the amino acid sequence of one of the spore-associated peptides of Paenibacillus polymyxa strain PKB1 and maintains the ability to inhibit the development of L. maculans. 
In one embodiment, the antibiotic of the present invention comprises at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain and that have inhibitory affect against L. maculans, or at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain deposited with ATCC as Accession Number 202127 or, preferably, at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain PKB1 having molecular weights of between 883 to 884 or between 896 to 897.
The invention also provides an isolated preparation of the PKB1 antibiotic. In preferred embodiments, the antibiotic preparation comprises at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain, or at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain deposited with ATCC as Accession Number 202127 or, preferably, at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain PKB1 having molecular weights of between 883 to 884 or between 896 to 897 and has a inhibitory affect against L. maculans. 
In one embodiment, the antibiotic includes two primary peptides. The peptides are each comprised of eight amino acids in a branched cyclic sequence and are nearly identical in form. One peptide has a molecular weight (MW) of about 897 to 898 and the second form has a molecular weight of about 883 to 884.
Another aspect of the invention pertains to an isolated peptide of the present invention or a fragment, or portion, e.g. a bioactive fragment or portion, thereof. In a preferred embodiment, the isolated peptide or bioactive fragment thereof can inhibit the development of L. maculans. In another preferred embodiment, the isolated peptide or bioactive fragment thereof is sufficiently homologous to the amino acid sequence of one of the spore-associated peptides of Paenibacillus polymyxa strain PKB1 and maintains the ability to inhibit the development of L. maculans. 
In one embodiment, the peptide or bioactive fragment of the present invention comprise the amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain, or the amino acid sequence of a spore-associated peptides of Paenibacillus polymyxa strain deposited with ATCC as Accession Number 202127 and preferably those spore-associated peptides of Paenibacillus polymyxa strain PKB1 having molecular weights of between 883 to 884 and/or between 896 to 897 and have inhibitory affect against L. maculans. In one preferred embodiment, the peptide or bioactive fragment of the present invention comprises one valine, one alanine, one threonine, two aspartic acids, one proline, one acetyl-modified leucine and one glutamic acid and being capable of inhibiting L. maculans. In another preferred embodiment, the peptide or bioactive fragment of the present invention comprises the amino acid sequence according to FIG. 6B.
The invention also provides an isolated preparation of the peptide according to the present invention. In preferred embodiments, the peptide of the preparation comprise the amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain, or the amino acid sequence of a spore-associated peptides of Paenibacillus polymyxa strain deposited with ATCC as Accession Number 202127 and preferably those spore-associated peptides of Paenibacillus polymyxa strain PKB1 having molecular weights of between 883 to 884 and/or between 896 to 897 and have inhibitory affect against L. maculans. In one preferred embodiment, the peptide or bioactive fragment of the present invention comprises one valine, one alanine, one threonine, two aspartic acids, one proline, one acetyl-modified leucine and one glutamic acid and being capable of inhibiting L. maculans. In another preferred embodiment, the peptide or bioactive fragment of the present invention comprises the amino acid sequence according to FIG. 6B. In yet another embodiment, the peptide is at least about 50-60%, preferably at least about 65-70%, more preferably at least about 75-80%, and even more preferably at least 85, 90, 95% or more homologous to the entire amino acid sequence of FIG. 6B. In other embodiments, the isolated peptide comprises an amino acid sequence which is at least about 60-70% or more homologous to the amino acid sequence of FIG. 6B and has an one or more of the following activities: 1) it can inhibit development of L. maculans and 2) it can inhibit development of S. sclerotiorum. 
The peptide or a bioactive fragment thereof, as defined above, can be operatively linked to another peptide to form a fusion protein. In addition, the PKB1 peptide or a bioactive fragment thereof can be incorporated into a pesticide composition comprising the peptide and an acceptable carrier or enhancer.
The P. polymyxa antibiotic producing strain PKB1 according to the present invention offers promise for use as a biocontrol agent because it is active against many fungal diseases. Biocontrol agents are relatively inexpensive to mass produce because they are grown in large volume fermenters and require little or no downstream processing other than, in some embodiments, drying. Unlike the situation with chemical fungicides, once a crop has been treated with a biocontrol agent, it may not need to be treated again for several years because the biocontrol agent is a live organism and can sustain itself in the soil environment. In this regard, the bacterium of the present invention is a preferred organism for use as a biocontrol agent because it forms dormant spores which are extremely resistant to heat, drying, chemical treatment, radiation and ultraviolet light exposure. This makes it possible to store dried cells of Paenibacillus polymyxa. PKB1 for long periods with little loss of viability. The same-spore forming ability occurs in the soil, making it possible for the introduced P. polymyxa strain to survive long periods of dryness or harsh conditions.