Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Reexamination Certificate
2001-02-05
2003-08-05
Naff, David M. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
C424S405000, C435S252500, C435S838000
Reexamination Certificate
active
06602500
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed toward a biocontrol agent and pesticide and, in particular, biocontrol agent and pesticide for inhibition of disease-causing fungi.
BACKGROUND OF THE INVENTION
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.
SUMMARY OF THE INVENTION
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 “isolated” or “purified” bacterial strain is substantially free of materials from its natural environment including soil and biological matter including other bacterium or plant matter. The language “substantially free of materials from its natural environment” 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 “substantially free of materials from its natural environment” 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 “bioactive mutants or modified forms of strain PKB1” 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
Paenibacil
Beatty Perrin Hudson
Coleman Richard Nigel
Jensen Susan Elaine
Kharbanda Prem Dutt
Tewari Jalpa P.
Bennett Jones LLP
Naff David M.
The Governors of the University of Alberta
Ware Deborah K.
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