Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Reexamination Certificate
1999-09-02
2002-02-19
Marx, Irene (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
C435S253300
Reexamination Certificate
active
06348193
ABSTRACT:
GOVERNMENT FUNDING
None
CROSS-REFERENCE TO RELATED APPLICATIONS
None
BACKGROUND OF THE INVENTION
(1). Field of the Invention
The present invention relates to a method for controlling bacterial and fungal diseases in plants using
Pseudomonas aureofaciens
in admixture with its metabolites from growth of the
Pseudomonas aureofaciens
. In particular, the present invention relates to a novel strain of
Pseudomonas aureofaciens
which is useful in the method.
(2). Description of Related Art
Development of new chemical bacteriocides, and fungicides generally occurs through the mass screening of novel synthetic compounds. Utilization of antifungal compounds produced by microbial organisms, such as antibiotics, have been highly exploited in the development of medicinal compounds. Application of medicinal antibiotics for the management of plant diseases has been restricted due to concerns of the development of resistance to these compounds by potential human pathogens.
Several bacteria have been identified as producing a variety of classes of compounds that are antifungal in nature, including enzymes, siderophores, hydrogen cyanide, ethylene, and antibiotics. Although all of these compounds have been implicated in biological control activity by bacteria, the commercial application of enzymes for plant disease management is not likely due to their sensitivity to environmental conditions. Another class of compounds that would not be feasible for study are volatile compounds such as hydrogen cyanide and ethylene.
The use of bacteria as biological control agents is one of the fastest growing fields of research in disease management. The concept of the management of disease through the application of soilborne bacteria is attractive due to its sensitivity to environmental concerns. However, significant breakthroughs yielding biological controls that provide consistent disease management have not yet been realized.
Pseudomonas aureofaciens
is a gram negative, rod-shaped bacterium possessing one or more flagella, is strictly aerobic, and chemoorganotrophic.
P. aureofaciens
is included in the class of fluorescent pseudomonads and was included taxonomically as a biovar of
Pseudomonas fluorescens
by Stanier et al (Journal of General Microbiology 43:159-271 (1966)). Inclusion of
P. aureofaciens
in the group of fluorescent pseudomonads is based on the ability of most strains to produce the fluorescent pigment pyoveridin. The name “
aureofaciens
” literally means to “make golden” which refers to its ability to turn artificial media to an orange-gold color. This color is caused by production of non-fluorescent phenazine pigments. Phenazine pigments reported to be produced by
P. aureofaciens
are phenazine-1-carboxylic acid (PCA), phenazine 2-oxophenazine and 2-oxophenazine-1-carboxylic acid (Trutko, S. M., et al., Biokhimima 54:1329-1336 (1990)). Evidence has been presented that the role of phenazine compounds produced by
P. aureofaciens
allows for the removal of excess reducing equivalents from NADH and NADPH under substrate and/or oxygen limitations.
The role of microflora in relation to the reduction of disease severity was brought to light with the identification of “suppressive soils”. “Suppressive soils” refers to soils which reduce the level of disease intensity to a particular pathogen (Rovira, A. D., et al., The nature and mechanism of suppression. Pages 385-415 in: Biology and Control of Take-All, M. J. C. Asher and P. J. Shipton, eds. Academic Press, New York, N.Y., 538 pp. (1981)). Suppressive soils may be divided into two classes; general antagonism and specific antagonism (Gerlagh, M., Netherlands Journal of Plant Pathology 74:1-97 (1968)).
General antagonism may be found to some degree in all soils and can be directly related to high soil bacteria populations (Rovira, A. D., et al., The nature and mechanism of suppression. Pages 385-415 in: Biology and Control of Take-All, M. J. C. Asher and P. J. Shipton, eds. Academic Press, New York, N.Y., 538 pp. (1981)). Characteristics common to this type of antagonism include the maintenance of soil suppressiveness after heating to 70° C. for 30 minutes, inability for transfer to other soils, and the exhibition of greater suppression in undisturbed soils. It is fostered by the addition of organic amendments, increased suppression in soil at temperatures above 25° C., and is promoted by the use of ammonium-nitrogen (NH
4
+
—N) rather than nitrate-nitrogen (NO
3−
—N) (Cook, R. J., et al., Biological and cultural tests for control of plant diseases. 3:53 (1988)). Smith (Smith, A. M., Soil Biology and Biochemistry 8:293-298 (1976)) suggested that ethylene (C
2
H
4
) biosynthesis by soil microflora may be a major factor involved in general antagonism. Factors supporting the role of ethylene in general antagonism are that ethylene production in soil increases as soil temperatures increase up to 35° C., is promoted by ammonium-nitrogen but inhibited by nitrate-nitrogen, is fostered by the addition of organic amendments, and is greater in undisturbed bulk soils. Ethylene has also been shown to be inhibitory to
G. graminis
var. tritici at concentrations less than 5 parts-per-million in the soil atmosphere (Rovira, A. D., et al., The nature and mechanism of suppression. Pages 385-415 in: Biology and Control of Take-All, M. J. C. Asher and P. J. Shipton, eds. Academic Press, New York, NY, 538 pp. (1981)).
Specific antagonism occurs through continuous monoculture of a crop in the presence of a pathogen (Gerlagh, M., Netherlands Journal of Plant Pathology 74:1-97 (1968)). This results from the buildup of specific antagonistic microbial populations that are antagonistic to the pathogen. This type of antagonism occurs in soils of lower temperature than general antagonism (15-25° C.), is eliminated by 60° C. moist heat, can be transferred to other soils by mixing, and is related to the build-up of specific bacteria in the rhizosphere (Cook, R. J., et al., Biological and cultural tests for control of plant diseases. 3:53 (1988)).
One of the most studied models of suppressive soils involves take-all disease of wheat and other grasses as caused by the fungus
G. graminis
. General antagonism to this disease involves all of the factors previously listed. Research interest has been focused on the phenomenon known as “take-all decline” which is a form of specific antagonism in which “suppression (of take-all) develops with 2 or 3 years of wheat monoculture and severe take-all; the soil becomes “immune” to subsequent outbreaks of take-all if cropped exclusively thereafter to wheat and barley” (Cook, R. J., et al., Biological and cultural tests for control of plant diseases. 3:53 (1988)). This occurrence was first reported by Glynne (Glynne, M. D., Annals of Applied Biology 22:225-235 (1935)) in 1935, who noted a reduction in take-all severity after 4 consecutive wheat crops.
Several studies in the mid 1970's correlated fluorescent pseudomonads with the occurrence of take-all decline. Evaluation of 100 bacterial strains for specific antagonism to
G. graminis
var. tritici in greenhouse conditions by Cook and Rovira (Cook, R. J., et al., Biological and cultural tests for control of plant diseases. 3:53 (1988)), identified eight (8) strains which yielded suppression greater than or equal to those of natural suppressive soils. All eight strains were Pseudomonas spp., seven of which were fluorescent. Further evaluation of bacterial populations by Cook and Rovira (Cook, R. J., et al., Biological and cultural tests for control of plant diseases. 3:53 (1988)) indicated suppressive soils contained 1000 times more fluorescent pseudomonads than non-suppressive soils. Simon and Ridge (Simon, A., et al., Journal of Applied Bacteriology 37:459-460 (1974)) similarly found 100 to 1000 fold increases of fluorescent pseudomonads on infected root tissues than on healthy roots. Agar plate tests demonstrated that over 70% of the fluorescent pseudomonads isolated from suppressive soils were antagonistic to
G. graminis
. Increases in fluorescent pseudomonad
Detwiler Alvin Ronald
Dykema Nancy M.
Nair Muraleedharan G.
Vargas, Jr. Joseph M.
Board of Trustees of Michigan State University
Marx Irene
McLeod Ian C.
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