Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Fungus
Reexamination Certificate
1999-06-02
2002-07-16
Marx, Irene (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Fungus
C424S094600
Reexamination Certificate
active
06419922
ABSTRACT:
Novel methods and compositions are provided for the biocontrol of plant diseases, in particular diseases causing postharvest decay. The methods and compositions are both protective and curative. Combinations of antagonistic microorganisms and antifungal agents are used.
BACKGROUND OF THE INVENTION
The U.S. market for biocontrol of tree fruit postharvest diseases could exceed $100 million by the year 2000 (
Industrial Bioprocessing,
September 1992). In
Postharvest News and Information
(1991) it was estimated that approximately 25% of harvested fruit and vegetables are lost because of postharvest diseases. Synthetic fingicides have been the primary means for controlling postharvest diseases of fruit and vegetables. However, increased concern of the public over the carcinogenicity of synthetic fungicides, has led to the withdrawal of some fungicides from the market. The development of fungicide-resistance in pathogens has limited chemical fingicides as a means of controlling them.
Control of plant diseases is not a problem confined to the U.S. The European Parliament has voted in favor of a total ban on postharvest treatment of fruit and vegetables with pesticides as soon as this ban becomes feasible. The withdrawal of current fungicides from use in the United States and other parts of the world is creating a large, new market for biological control agents (“biocontrol”). Baker (1987) has defined biological control as “the decrease of inoculum or the disease-producing activity of a pathogen accomplished through one or more organisms, including the host plant but excluding man.” The cost of commercializing a biological control agent is much less expensive than the cost of commercializing a synthetic pesticide because only Tier 1 toxicology tests (Hofstein et al 1994.) are required. Also, if a biological control agent is properly selected, fewer new environmental impact studies are required.
A type of biological control agent is a microorganism that is antagonistic to postharvest pathogens. For example, antagonistic yeasts have been disclosed as effective biocontrol agents for the biological control of postharvest disease (Wilson and El Ghaouth, 1993, 1997). However, microorganisms currently available have not been accepted as providing control comparable to control obtained by the use of synthetic fungicides. Some limitations are due to the microorganisms'inability to cure previously-established infections in the crops and to prevent the resumption of quiescent infections. Improved and broader biocontrol is desirable.
Antifungal hydrolases such as chitinase, &bgr;-1,3-glucanase, lysozyme, and lyticase are low molecular proteins that hydrolyze the main components of fungal and yeast cell walls, &bgr;-glucan and chitin (Bowles, 1990; Mauch et al., 1988; Schlumbaum et al, 1986; Kendra et al., 1989; Sahai and Manocha, 1993). These enzymes are reported to play a major role in disease resistance of plants against invading pathogens and may be responsible for the biocontrol activity of some microbial antagonists. The action of glucanohydrolases was reported to inhibit fungal growth (Schlumbaum et al., 1986; Sela-Buurlage et al., 1993).
New means of controlling postharvest diseases are needed that are safe, effective, and economically feasible. The present invention provides such means.
SUMMARY OF THE INVENTION
The present invention is directed to novel compositions that are combinations of antagonistic microorganisms with antifungal agents, and to methods of preventing or curing plant diseases caused by various postharvest pathogens that cause decay of plants by applying the compositions of the present invention to plants. “Prevention or curing” is included under the general term “biocontrol” of plants. Percent of plants infected is a measure of control. Postharvest decay is one of the detrimental phenomena that is controlled by methods and compositions of the present invention.
The combinations of the present invention form a “biocontrol cocktail.” Suitable antagonistic yeasts include those from the following genera: Candida spp; Cryptococcus spp; Pichia spp; Debaryomyces spp; Bulleromyces spp; Sporobolomyces spp; Rhodotorula spp; Aureobasidium spp; Issatchenkia spp; Zygosaccharomyces spp; Dekkera spp; and Hansenula spp. Other suitable microorganisms include bacteria, for example
Pseudomonas syringae
and
Bacillus subtilus.
Suitable enzymes and biochemicals include the enzymes chitinase, laminarase, chitosanase, &bgr;-1,3-glucanase, lectins; and the biochemicals: &bgr;-1,3-glucan, &bgr;-1,4-glucan, a polysaccharide of fungal or yeast origin, and polycations such as glycoprotein.
In a preferred embodiment of a composition of the present invention, the antagonistic microorganism is a yeast and the antifungal agent is an enzyme. For example, the combination of the antifungal property of an enzyme, e.g., lysozyme and/or lyticase and the biocontrol activity of an antagonistic yeast, e.g.,
C. saitoana,
wherein the yeast can function against the pathogen in the presence of the enzymes, provides improved consistency and efficacy in controlling postharvest rot (decay). In addition, the combination of antagonistic yeast (
C. saitoana
) with lysozyme or lyticase offers control of postharvest decay of fruit and vegetables superior to that obtained with an antagonist yeast alone or with an enzyme alone such as lysozyme or lyticase. This improvement and synergism is unexpected.
The combination of antagonistic microorganisms such as a yeast with antifungal enzymes (lysozyme or lyticase) was not expected to successfully work to effect biocontrol of plant pathogens or to produce a synergistic effect because of the known actions of each agent individually. Antifungal hydrolases such as chitinase, &bgr;-1,3-glucanase, lysozyme, and lyticase are known to hydrolyze the main components of fungal and yeast cell walls, &bgr;-glucan and chitin, and thereby negatively affect the growth of the yeasts such as Candida spp. and of filamentous fungi. Therefore, a combination of an antagonist yeast with antifungal enzymes was expected to impair the biocontrol activity of the selected yeast. However, unexpectedly, the combination produced improved control of pathogens.
Compositions including antagonistic microorganisms such as a yeast (
C. saitoana
) with antifungal agents such as the enzymes lysozyme or lyticase may be applied to plants either before or after infection because the compositions have both a protective and a curative effect against major postharvest pathogens and consequently offer a level of control of decay better than that of synthetic chemical fingicides.
It is contemplated that a plurality of antagonistic microorganisms may be combined, as long as the microorganisms do not adversely affect the antagonism, viability, or other parameters that are needed for antagonistic microorganism activity according to the present invention. In addition, not only may a plurality of microorganisms be effective in combination with one antifungal agent, but a plurality of antifungal agents may also be effective, provided the plurality of antifungal agents do not deleteriously affect the biocontrol action either of other agents or microorganisms combined with them.
The compositions of the present invention are applied to plants by means such as spraying, drenching, or dipping, which are known in the art. Effective amounts of antifungal agents, e.g. enzymes, have been found to range from 20 &mgr;g/ml to about 1000 &mgr;g/ml, with about 100 &mgr;g/ml being preferred. Effective amounts of yeasts have been found to range from about 10
6
CFU to about 10
8
CFU with 10
8
CFU preferred. It is understood, however, that optimal concentrations will vary with particular situations, and it is well within the level of skill in the art to arrive at optimal formulations by following conventional testing procedures such as those described by the Examples herein.
The complexity of the mode of action displayed by the combined agents of the present invention makes the development of pathogen resistance in the target plant
El Ghaouth Ahmed
Wilson Charles
Barnes & Thornburg
Biotechnology Research and Development Corporation
Martin Alice O.
Marx Irene
LandOfFree
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