Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Fungus
Reexamination Certificate
1999-06-02
2002-07-23
Marx, Irene (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Fungus
C435S255100, C435S255400, C435S255500
Reexamination Certificate
active
06423310
ABSTRACT:
Novel methods and compositions are provided for the biocontrol of plant diseases by use of a biological coating that has both a protective and a curative effect for the control of postharvest decay. The coating includes chitosan salts, at least one antagonistic microorganism, and a softener.
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 fungicides 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 fungicides 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 fruits 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.
Present coatings (mostly waxes) for postharvest commodities are somewhat effective in delaying ripening, but in general, do not prevent decay. Moreover, these coatings are under scrutiny as possible health hazards. Synthetic fungicides which have been added to the coatings to alleviate the problems of decay have recently been withdrawn from the market, and there is also public pressure to remove the petroleum-based coatings because of health and environmental concerns. A critical need therefore exists for alternatives to presently available coatings for agricultural commodities. The coatings need to be fungicidal as well as safe for the consumer and the environment.
Antagonistic yeasts have been reported to be effective agents for the biological control of postharvest diseases (Wilson and El Ghaouth, 1993). However, antagonistic microorganisms currently available have not been demonstrated to provide control of fruit and vegetable postharvest decay comparable to that obtained with synthetic fungicides. Limitations include the microorganisms' inability to cure previously-established infections in the crops and to prevent the resumption of quiescent infections.
Recently, chitosan, an animal-derived polymer, has been shown to have some potential as an antifungal preservative. Chitosan, a &bgr;-1,4-glucosamine polymer, is commercially produced from chitin of arthropod exoskeletons that have been deacetylated to provide sufficient free amino groups to render the polymer readily soluble in diluted organic acids. Chitosan and its derivatives are known to form a semi-permeable film (Averbach, 1978), to be inhibitory to a number of pathogenic fungi (Allan and Hadwiger, 1979), and to activate a number of biological processes in plant tissues, including the stimulation of chitinases, the accumulation of phytoalexins, the synthesis of proteinase inhibitors, and increased lignification (El Ghaouth et al., 1992, a, b). The polycationic nature of chitosan is believed to provide the basis for its physico-chemical and biological functionality. Chitosan is regarded safe as indicated by feeding trials with domestic animals. When applied as a coating, chitosan controlled decay and delayed ripening of strawberry, bell pepper, tomato, and cucumber by acting as a selective barrier to gas diffusion (El Ghaouth et al., 1991). The control of decay by chitosan is believed to originate, in part, from its antifungal property. Indeed, in vitro studies showed that chitosan not only inhibited the radical growth of major postharvest pathogens, but also induced severe morphological alterations in
Rhizopus stolonifer
and
Botrytis cinerea,
as well as increased cellular leakage in both fungi, presumably by interfering with fungal plasma membranes (El Ghaouth et al., 1992 b).
Because infection of commodities such as fruit can occur either prior to harvest or during harvesting, an ideal biological control agent is expected to display both a protective and curative activity comparable to that observed with synthetic fungicides. Currently available antagonistic microorganisms do not appear to be able to control previously-established infections and are most effective when applied prior to infection by the pathogen.
New safe and effective means of controlling postharvest diseases are needed. The present invention provides such means.
SUMMARY OF THE INVENTION
The present invention is directed to unique compositions that are a combination of antifungal agents with antagonistic microorganisms and a softener. The invention also relates to methods of preventing and curing postharvest decay of plants caused by various pathogenic fungi, by applying the compositions of the invention to plants.
The combinations of the present invention form a “biological coating.” In an embodiment of a composition of the present invention, the antagonistic microorganism is a yeast in particular
Candida saitoana
and the antifungal agents include chitosan salts amended with CaCl
2
. Other suitable microorganisms include bacteria, for example
Pseudomonas syringae
and
Bacillus subtilis.
In a preferred embodiment, the biochemical additive are chitosan salts amended with CaCl
2
or chitosan propionate amended with CaCl
2
, and a yeast, in particular
Candida saitoana.
Other suitable genera of yeast include Candida spp; Cryptococcus spp; Pichia spp; Debaryomyces spp; Bulleromyces spp; Sporobolomyces spp; and Rhodotorula spp.
Other suitable chitosan salts include chitosan acetate; chitosan sorbate; chitosan propionate; chitosan lactate; chitosan glutamate; chitosan benzoate.
A softener is an additive that renders the yeast resistant to the adverse effects of chitosan and organic acids. Monovalent and divalent cations of Ca, Mg, Zn, or K are suitable. The softener is not considered to affect the pathogen.
The combination of the antifungal property of chitosan salts e.g. chitosan acetate and the biocontrol activity of antagonistic microorganisms such as the yeast
C. saitoana,
wherein the yeast functions against the pathogen in the presence of chitosan salts amended with CaCl
2
, provides improved consistency and efficacy in controlling postharvest decay. In addition, the combination of antagonistic yeast (
C. saitoana
) with chitosan salts amended CaCl
2
offers control of postharvest decay of fruit and vegetables superior to that obtained with antagonistic yeast alone or chitosan salts amended with CaCl
2
alone. This improvement is unexpected and synergism was demonstrated.
Native chitosan and organic acids (acetic; sorbic; propionic; and lactic) are known to inhibit the growth of yeasts such as Candida spp and filamentous fungi such as
Botrytis cinerea, Penicillium expansum
Link, and
Penicillium digitatum.
Thus it is expected that the combination of chitosan-organic acid with
C. saitoana
will adversely affect the biocontrol activity of the selected yeast. However, unexpectedly, the combination produced improved control of pathogenic fungi.
The combination of antagonistic yeast (
C. saitoana
) with chitosan salts (chitosan acetate) amended with CaCl
2
may be applied to harvested crops either before or after infection
El Ghaouth Ahmed
Wilson Charles
Barnes & Thornburg
Biotechnology Research and Development Corporation
Martin Alice O.
Marx Irene
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