Antifungal biocontrol agents, a process for preparing and...

Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales

Reexamination Certificate

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C424S405000, C435S252400, C435S253500, C435S267000, C435S886000

Reexamination Certificate

active

06280719

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to antifungal biocontrol agents, and to a process for preparing and treating the same. In particular, the present invention relates to two antifungal biocontrol strains of Streptomyces sp., WYE 20 and WYE 324, capable of enhancing plant growth and reducing plant diseases by suppressing fungal pathogens colonized in rhizosphere soils of host plants, and the preparation method and application method thereof.
BACKGROUND OF THE INVENTION
It is well known that soilborne fungal phytopathogens cause enormous economic losses in the agricultural and horticultural industries. In particular,
Rhizoctonia solani
is one of the major fungal phytopathogens exhibiting strong pathogenicity; it is associated with seedling diseases as well as foliar diseases such as seed rot, root rot, damping-off, leaf and stem rot of many plant species and varieties, resulting in enormous economic losses. For example,
Rhizoctonia solani
AG 1 (IB) causes plant diseases in crops such as cucumber (
Cucmumis sativus
L.) and pepper (
Capsicum annuum
L.) as well as brown patches of golf green creeping bentgrass,
Rhizoctonia solani
AG 2—2 causes large patches in fairway turfgrass on large areas of golf courses resulting in enormous economic losses. In addition,
Phytophthora capsici
is a widespread and highly destructive soilborne fungal phytopathogen that causes root and crown rot as well as the aerial blight of leaves, fruit, and the stems of peppers (
Capsicum annuum
L.). It is very difficult to suppress Phytophthora blight in pepper plants if they are infected by a
Phytophthora capsici. Phytophthora capsici
is a particularly destructive phytopathogen responsible for blight of pepper plants during hot and humid rainy seasons resulting in the killing of the pepper plants and consequently enormous economic losses. As mentioned above, both
Rhizoctonia solani
and
Phytophthora capsici
are major fungal phytopathogens which exhibit strong pathogenicity and produce spores which survive for a long time in harsh conditions. Thus, they repeatedly cause plant diseases in large areas when conditions for incidence of the disease are optimal.
Therefore, growers generally apply a mixture of fungicides to plants on a regular basis to control plant diseases caused by both
Rhizoctonia solani
and
Phytophthora capsici
. However, it is becoming increasingly difficult to control Phytophthora blight of pepper plants and Rhizoctonia brown patches of creeping bentgrass because of the emergence of strains of fungal phytopathogens resistant to fungicides. It is also becoming increasingly difficult to control these diseases due to the widespread incidence of
Rhizoctonia solani
resistant to fungicides in golf courses and the proliferation of
Phytophthora capsici
resulting from the successive cultivation of pepper plants.
Intensive use of agrochemicals has also provoked residual toxicity and environmental problems. Furthermore, agrochemicals are considered ineffective in controlling soilborne plant diseases due to wash out, and the lack of an efficient application which allows effective penetration into rhizosphere soils. It is also very difficult to expect long term protection of plants with an application of chemical fungicides. Therefore, using certain rhizosphere bacteria as a biocontrol agent can provide not only more effective and economical practices for the control of plant diseases such as Phytophthora blight caused by
Phytophthora capsici
and golf course turfgrass diseases caused by
Rhizoctonia solani
, but also increased environmental conservation.
It has been shown that use of antagonistic microorganisms is an attractive way to control fungal pathogens (Suh, Ph.D. Dissertation. University of Idaho, Idaho USA. 1992; Crawford et al, Appl. Environ. Microbiol. 59:3899-3905, 1993; U.S. Pat. No. 5,403,584). The inventors disclosed in the prior art that certain Streptomyces species strains can be used to control plant pathogens by using a delivery medium containing peat moss, sand, and cornmeal, in the potting mixture/soil or by coating plant seeds with sodium alginate containing the biocontrol agent, resulting in the colonizing of the roots (U.S. Pat. No. 5,403,584). It has also been disclosed in the prior art that peat containing a beneficial biocontrol agent, can be used to control plant pathogens (U.S. Pat. No. 4,595,589). The delivery medium methods in the prior arts are suitable for the application of seedlings/potting mixtures, but are not quite suitable for direct application to plant seeds or plant roots to achieve efficient protection against fungal phytopathogens.
The object of the present invention is to provide novel strains of Streptomyces species which are capable of controlling fungal phytopathogens by direct application to plant seeds or plant roots utilizing specially designed formulations comprising delivery media containing a biologically pure culture selected fiom the newly isolated strains of Streptomyces species.
Further, it is an object of the present invention to provide antifungal biocontrol agents and describe their use in protecting plants from infections caused by soilborne fungal phytopathogens.
SUMMARY OF THE INVENTION
The present invention has been achieved through the isolation of Streptomyces sp. WYE 20 and WYE 324 which are capable of protecting plants against
Rhizoctonia solani
and
Phytophthora capsici
. The strains, Steptomyces sp. WYE 20 and WYE 324, are effective in preventing the incidence of fungal diseases in plants and in enhancing plant growth in greenhouse and agricultural field trials. Therefore, one aspect of the present invention is microbially pure cultures of Streptomyces sp. WYE 20 and WYE 324.
The present invention also encompasses the various delivery media suitable for the treatment of plant seeds, seedling beds/pots, or potting mixtures/soil with Streptomyces sp. WYE 20 and WYE 324. The delivery medium is very useful for carrying Streptomyces sp. WYE 20 and WYE 324 to inhibit the plant diseases caused by fungal phytopathogens.
In a particular embodiment according to the present invention, the delivery medium consists of wood sawdust, wheat bran, chitin, chitosan, and PHARMAMEDIA (trade name; made by The Budkeye Oilseed Products Company, Texas, USA; aka cottonseed flour having the following components: 59.2% protein, 24.13% carbohydrate, 4.02% fat, 2.55% fiber, and 6.71% ash, as described in the
Manual of Industrial Microbiology and Biotechnology
(1985), by A. L. Demain and N. A. Solomon, editors, p. 130-131.) together with Streptomyces sp. WYE 20 or WYE 324. In another particular embodiment, the delivery medium consists of pectin and colloidal chitin and water together with Streptomyces sp. WYE 20 or WYE 324. In the preferred embodiment, the delivery medium comprises at least 10 colony forming units of Streptomyces sp .WYE 20 or WYE 324 per gram of delivery medium.
The present invention also encompasses methods for reducing fungal infection in seeds, seedlings and/or growing plants prior to or during sowing and growing seasons through the treatment of plant seeds, seedling beds/pots, potting mixture/soils, spraying or in-furrow application.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, Streptomyces sp. WYE 20 and WYE 324 are provided that are effective in inhibiting fungal diseases and in enhancing the growth of plants such as pepper, cucumber, and turfgrasses, although their scope is not limited to these examples.
The present invention encompasses the various delivery media that are useful for carrying Streptomyces sp. WYE 20 and WYE 324 to inhibit the plant diseases caused by fungal phytopathogens.
In a particular embodiment of the present invention, the delivery medium consists of 40 to 65 w/w % of wheat bran, 1 to 5 w/w % of chitosan, 30 to 55 w/w % of wood sawdust, 1 to 3 w/w % chitin, and 1 to 3 w/w % of PHARMAMEDIA on the basis of the total weight of the delivery medium. In the preferred embodiment, the delivery medium further consists of 0.2 to 3.5 w/w % of sporulatio

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