Biologically pure culture of bacteria which suppresses...

Details Biologically pure culture of bacteria which suppresses... Biologically pure culture of bacteria which suppresses...

1997-07-10

2002-12-17

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S876000, C424S093470

Reexamination Certificate

active

06495362

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a novel bacteria strain of
Pseudomonas fluorescens
designated as ATCC 55939 and a method for large scale screening of native rhizosphere microflora, to identify and characterize naturally occurring rhizosphere-competent biocontrol bacteria from non-sterilized soil, which could effectively colonize plant roots. The present invention relates more specifically to the use of a novel strain of Pseudomonas fluorescens as a biocontrol agent for controlling plant fungal disease particularly those diseases caused by fungus of the genus Fusarium sp., Rhizoctonia sp. and Pythium sp.
BACKGROUND OF THE INVENTION
The fungal pathogens play a major role in the development of diseases on many important field and horiculture crops which often results in the poor plant yields. Considering the cost of chemical pesticides and hazard involved, biological control of plant diseases is now increasingly capturing the imagination of plant microbiologists. Frequent failure of the added microorganisms to become established is not surprising because the biological associations and antagonisms within the ecosystem determine the composition of the microflora, the climax population being a reflection of the physical and chemical characteristics of the habitat.
A major factor in the unsuccessful commercialization of rhizosphere bacteria has been the inconsistency of field test results. Reasons for the reported variability include nonpersistence on seed before it is planted and poor bacterial establishment on seed and roots, please refer Burr, T, J., and A. Caesar, Crit. Rev. Plant Sci. 2: 1-20 (1984); Gaskins, M. H. et al. Ecosystems Environ. 12: 99-116 (1985); Liang, L. et al. Appl. Environ. Microbiol. 44: 708-714 (1982); O'Sullivan, D. J., and F. O'Gara, Micrbiol. Rev. 56: 662-676 (1992); Schrotk, M. N., and J. G. Hancock. Disease suppressive soil and root colonizing bacteria. Science 216: 1376-1381 (1981); Weller, D. M., Ann. Rev. Plant Pathol. 26: 379-407 (1988). The introduced microorganism must colonize plant roots and demonstrate rhizosphere competence before its further utilization as biological control and/or, plant growth promoting agent. When the proper bacterial strain is used, plant roots are extensively colonized by the introduced strain, which suggests a close bacteria-plant association that allows for beneficial plant growth or disease protection, see for example, Schmidt, E. L., Ann. Rev. Microbiol. 33: 355-376 (1979).
The isolation and development of plant beneficial bacteria applicable to a varietv of crops. soils. and locations will depend on the development of improved detection and screening procedures that more rapidly identify beneficial bacteria. While prior methods have been somewhat effective, such methods have had inherent shortcomings. For example, the current methods which are required to ascertain bacterial root colonization capacity are laborious and often produce highly variable results. Bennett and Lynch J. Gen. Microbiol. 125: 95-102 (1981), developed a closed test tube assay for measuring root colonization capacity of bacteria under gnotobiotic conditions which proved useful for studying specific microbial interactions in the rhizosphere. However, it is not possible to extrapolate results obtained in sterilized soils to those expected under field conditions, see for example, Klopper, J. W. Plant growth-promoting rhizobacteria and plant growth under gnotibiotic conditions. Phytopathology. 71: 642-644 (1981). Scher, F. M. et al. Can. J. Microbiol. 30: 151-157 (1984) measured the root colonization capacity of bacteria on maize in raw soil-sand closed test tube assay and demonstrated that root population densities determined in the soil-sand assay were comparable with those determined with plants grown in soils under greenhouse conditions. However, Scher et al. did not compare the competitive fitness of Rif mutants with the wild type strain. Compeau, G. et al. Appl. Environ. Microbiol. 54: 2432-2438 (1988) have demonstrated that colonization of soil by a species which is isogenic to a challenging organism may preempt the colonization of the soil by the second organism. This is true even when organisms display identical fitness. This interaction may be important in the failure of introduced strains to increase in number when introduced into their own environment. Thus, it is desirable to obtain novel strains of biocontrol agents which effectively control the growth of plant pathogens, particularly fungi, and are able to aggressively compete with indigenous bacteria and other microflora that exist in the rhizosphere of the plant.
SUMMARY OF THE INVENTION
This invention relates to a simple sand-live soil assay method, for large scale screening of the rhizosphere-competent bacteria that are effective in suppressing plant pathogens, that has been developed. Screening for chickpea rhizosphere competitive bacteria having biological control property was conducted at three different stages: development of screening method for large scale initial selection of bacteria isolates from chickpea rhizosphere, testing of biocontrol activity under in vitro conditions and screening of antibiotic resistant mutants for rhizosphere competence in nonsterile field soil, which assay has disclosed one
Pseudomonas fluorenscens
NBRI 1303 (ATCC 55939) that is effective in suppressing plant pathogens, including
Fusarium oxysporum f. sp. ciceri, Rhizoctonia bataticola
and Pythium sp. in chickpeas. The purified bacterial strain can be used as an active agent for biocontrol compositions and can also be used for enhancement of chickpea plant growth and yield, as well as for the production of antibiotics directed against phytopathogenic fungal diseases.
Accordingly, it is an object of the present invention to provide a method of raw (non-sterile) soil assay for large scale screening of native rhizosphere microflora of chickpeas that identifies and characterizes naturally occurring rhizosphere bacteria that effectively colonize chickpea roots.
Another object of the present invention is to provide a biocontrol agent that is useful in methods for suppression of fungal infection in chickpeas and thereby enhances plant yields.
Still another object of the present invention is the use of a biological control agent(s) that produces one or more metabolites capable of inhibiting fungal pathogens of chickpeas or other plants. The use of such a biological control agent instead of seed treatment and soil treatment chemical fungicides allows reduction of environmental contamination.
Yet another object of the present invention is to provide a method for biocontrol of pathogenic fungi
F. oxysporum f. sp. ciceri, R. bataticola
and Pythium sp. in plants by application of an isolated
P. fluorenscens
strain.
Other objects and advantages of the invention will become apparent from the ensuing description.
Accordingly, the present invention provides a method for large scale screening of native rhizospheric competent bacteria for strains that will control plant pathogens thereby promoting plant growth in field grown chickpea crops, said method comprising:
a. harvesting chickpea crops from a disease-suppressive field after five months of plant growth;
b. subjecting the said harvested crop roots to drying in the field for a post harvest period of four weeks;
c. isolating bacterial strains from the said chickpea roots by standard methods;
d. selecting rifampin resistant bacterial strains showing growth comparable to wild type on agar plates containing 100 (&mgr;g rifampin/ml);
e. evaluating the root colonization capability of the bacterial strain obtained in step (d) in the greenhouse on the basis of average root colonization values of rifampin resistance strains after four weeks of post planting period;
f. selecting bacterial strains from the strains evaluated in step (e) with 10
7
-10
8
CFU/g root; and
g. screening a novel strain from the bacterial strains obtained in step (f) for its invitro inhibition against
F. oxysporum f. sp. ciceri, R. bataticola
and Pythium s

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