The invention relates to plant protection compositions including pentacyclic triterpenes and methods for use thereof.
Outer layers of plants such as leave cuticle, fruit peels, as well as bark protect the plant against abrasion, prevent water loss, and also protect against pathogenic microorganisms. The breaking through the plant cuticle is a prerequisite for a pathogen to be able to enter the plant""s internal tissue.
The mechanism, by which plants naturally defend themselves against this early stage of pathogenesis has not been fully understood. The initial process of fungal propagules attaching to a host plant is essential to the successful establishment of pathogenesis. The established facts that aerial fungal pathogens bind strongly to very hydrophobic surfaces suggests that hydrophobic forces are involved in the attachment processes.
The attachment of aerial fungal pathogens involves an active process of secretion of extracellular mucilages or adhesives, which may start within minutes after contact with the host. Other reported components in the adhesive secretions include enzymes, among them esterases and cutinases. The erosion of cuticular waxes adjacent to and underlying the conidium may became observable within 20 min of liquid release. The growth of the appressorial germ tube appears to be limited to the zone of deposition of the liquid film.
Some studies have suggested that preparation of the infection court involves active dissolution of the host cuticle by foliar pathogen. It is also believed that this dissolution is the purpose of these enzymes. Nicholson, R. L., and L. In: The Fungal Spore and Disease Initiation in Plants and Animals., Eds. Cole, G. T., and Hoch, H. C.,1991 Plenum Press, New York, 3-23. Thus, the cuticle has to be penetrated by the attacking pathogen before the sequential steps of disease development occur. Some fungal spores help themselves with mechanical force exerted by the infection structure in addition to the enzymatic degradation. (Kxc3x6ller, W. in: The Fungal Spore and Disease Initiation in Plants and Animals., Eds. Cole, G. T., and Hoch, H. C., 1991 Plenum Press, New York, 219-246.
Pentacyclic triterpenes (PT) are among the most common plant secondary metabolites, but their function in plants have not been understood. They are usually concentrated in the outermost layers such as plant cuticle, fruit peel and bark. In some cases, these layers contain very high concentration of pentacyclic triterpenes. For example, an apple peel contains about 0.1 grams of ursolic acid per fruit, and the outer bark of white birch species contains up to 40% w/w of betulin. The amount of betulin obtainable from the birch bark waste in the wood-working industry in Finland is estimated at about 150,000 tons per annum. At present, waste bark is used as a low value fuel for energy production. Jxc3xa4xc3xa4skelxc3xa4ien, P. (1981) Pap. Puu 63, 599-603.
Literature supplies numerous examples of enzymes that can be inhibited by PT, indicating the ability of PT to act broadly in a non-specific mode on multiple targets. See for example, (a.) Bxc3xcchler et al. (1991) Biochem. Biophys. Acta 1075, 206-212, Inhibition of rat renal 11xcex2-hydroxysteroid dehydrogenase by steroidal compounds and triterpenoids; structure/function relationship; (b.) Koch et al. (1994) Phytother. Res. 8, 109-111, In vitro inhibition of adenosine deaminase by a group of steroid and triterpenoid compounds.; (c.) Najid et al. (1992) FEBS 299, 213-217, Characterization of ursolic acid as a lipoxygenase and cyclooxygenase inhibitor using macrophages, platelets and differentiated HL60 leukemic cells.; (d.) Pengsuparp et al. (1994) J. Nat. Prod. 57, 415-418, Pentacyclic triterpenes derived from Maprounea africana are potent inhibitors of HIV-1 reverse transcriptase.; (e.) Simon et al. (1992) Biochem. Biophys. Acta 1125, 68-72, Inhibition of lipoxygenase activity and HL60 leukemic cell proliferation by ursolic acid isolated from heather flowers (Calluna vulgaris).; (f.) Ying et al. (1991) Biochem. J. 277, 521-526 Inhibition of human leucocyte elastase by ursolic acid. Evidence for a binding site for pentacyclic triterpenes. The disclosures of each of these references is herein incorporated by reference.
In plant tissue cultures, stress induced by inactivated fungi or fungal enzymes has been used to enhance production of biologically active secondary metabolites. In several instances it has been reported that this fungal elicitation led to overproduction of pentacyclic triterpenes instead of some other expected metabolites. Suitable example is given by Van der Heijden et al., (1988) Plant Cell Rep. 7, 51-54, where tissue cultures of Tabernaemontana spp., normally producing indole alkaloids, were subjected to stress induced by either fungi, bacteria, or enzyme cellulase or pectinase. When stressed, however, the culture produced up to 3.3 times the normal rate of the ursane-type pentacyclic triterpenes (2% of dry mass) but no increase in the production of indole alkaloids occurred.
Other experiments with Tabernaemontana divaricata treated with Candida albicans elicitor led to production of a series of pentacyclic triterpenes of the ursane and oleane types, and was accompanied by inhibition of both growth and indole alkaloid accumulation (Van der Heijden et al., (1989) Phytochemistry 28, 2981-1988).
Also, the tissue culture of Tripterygium wilfordii, normally a source of potent cytotoxic diterpenes; stressed by fungal Botrytis elicitor dramatically enhanced production of oleane-type pentacyclic triterpenes but not the diterpenes, prompting a conclusion that only triterpenes are inducible anti-microbial phytoalexins. Kutney, et al., (1993) Anti-inflammatory oleane triterpenes from Tripterygium wilfordii cell suspension cultures by fungal elicitation. Plant Cell Rep. 12, 356-359.
The conventional treatments for leafy and grassy plants that have been attacked by fungi and bacteria are usually exercised after an outbreak occurs. The affected plants are then treated with one or more of the commercial synthetic contact antimicrobial sprays at application rates that do not pose phytotoxicity concerns. Effective treatment rates must be balanced against the risks of harming the treated plant with chemicals that are structurally unrelated to those in the plant physiology. It would be desirable to have a protective agent that is effective against microbial pathogens which would also work in a manner analogous to the plant""s natural defense mechanisms to reduce the risk of phytotoxicity. One of the problems associated with treating hydrophobic leaf surfaces is an effective application of the material. Current spraying techniques result in a portion of the sprayed material falling to the ground directly or after being washed off from rain occurring shortly after application. Either event increases concerns for environmental contamination.
Protective agents that are applied by spraying should remain on the plant surface for a time sufficient to serve their intended function. Stability against ultraviolet and visible light is, therefore, a concern for foliar treatments. It would be desirable to develop fungicides for foliar application that resist degradation by exposure to ultraviolet as well as visible light.
Moreover, invading organisms have been known to evade the effects of a treatment agent by mutation and propagation of resistant strains. Such developed resistance is economically detrimental because it forces the discovery of new treatments. It would be helpful to provide plant anti-infective agents which cannot be evaded by mutating pathogens.
It is an object of the invention to provide a composition and process for use thereof for protecting plant surfaces against microbial pathogens with ingredients that are compatible with natural plant defense mechanisms.
It is another object of the invention to provide a composition and method of use that poses little risk to the environment, humans, or beneficial insects.
It is another object of the invention to provide plant anti-infective agents, which cannot be evaded by developing resistance by mutating pathogens.
It is yet another object of the invention to provide a composition having good resistance to degradation by ultraviolet exposure, long storage times, and good inherent sticking ability to plant surfaces.
In accordance with these and other objects of the invention, which will become apparent from the description herein, a composition according to the invention comprises: a pentacyclic triterpene or a mixture of pentacyclic triterpenes, including some plant extracts that are particularly rich in pentacyclic triterpenes, having as main components compounds described by any of formulas I, II, or III.
A method according to the invention comprises applying to plant surfaces an effective amount of a composition comprising a pentacyclic triterpene or a mixture of pentacyclic triterpenes exhibiting any of formulas I, II or III.
Formulas I, II, and III are: 
wherein:
R1=Me, CH2OH, CH2OY1, CH2Oxe2x80x94Xxe2x80x94OH, CH2Oxe2x80x94Xxe2x80x94OY1, CH2Oxe2x80x94Xxe2x80x94Y2, CH2Oxe2x80x94Xxe2x80x94Y3, CH2NHY1, CH2NY12, CH2Y3, CH2NHxe2x80x94Xxe2x80x94OH, CH2NHxe2x80x94Xxe2x80x94Y2, CH2NHxe2x80x94Xxe2x80x94Y3, CH2NHxe2x80x94Xxe2x80x94OY1, CH2OC(O)xe2x80x94OY1, CH2Oxe2x80x94Xxe2x80x94OY1, CO2Y1, COY3, COY2, CHO, CHxe2x95x90N(CH2)m(O(CH2)m)nR4, or CHxe2x95x90N(CH2)m(O(CH2)m)nY2;
R2, R3=H, OH, OY1, Oxe2x80x94Xxe2x80x94OH, Oxe2x80x94Xxe2x80x94OY1, Oxe2x80x94Xxe2x80x94Y2, Y3, NHY1, NY12, Y3, NHxe2x80x94Xxe2x80x94OH, NHxe2x80x94Xxe2x80x94Y2, NHxe2x80x94Xxe2x80x94Y3, NHxe2x80x94Xxe2x80x94OY1, NY1xe2x80x94Xxe2x80x94OH, NY1xe2x80x94Xxe2x80x94Y2, NY1xe2x80x94Xxe2x80x94Y3, or NY1xe2x80x94Xxe2x80x94OY1;
provided that one of R2 and R3 is H or that R2 and R3 together denote carbonyl oxygen;
R4=H, OH, OY1, or Y3;
Y1=H, alkyl of 1-30 carbon atoms, straight chain or branched, cycloalkyl of 3-30 carbon atoms, alkanyl of 3-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms, or phenoxyalkyl of 7-30 carbon atoms;
Y2=NH2, NHY1, or NY12;
Y3=xe2x80x94(O(CH2)m)nR4 or xe2x80x94(O(CH2)m)nY2, where m=2-4 and n=1-230;
X=xe2x80x94OC(CH2)pCOxe2x80x94 where p=1-22.
The present invention provides a composition and method of use that are particularly effective in preventing outbreaks of airborne fungal and bacterial diseases on treated plant surfaces. The pentacyclic triterpenes of the composition are applied to form a film over the plant surface. The film of material prevents the prerequisite attachment of aerial fungal pathogen and penetration of the plant cuticle. In addition, the compounds are not phytotoxic and are compatible with natural plant defenses
The present invention relates to compositions containing and methods of using pentacyclic triterpenes having the following formulas I, II, or III: 
wherein:
R1=Me, CH2OH, CH2OY1, CH2Oxe2x80x94Xxe2x80x94OH, CH2Oxe2x80x94Xxe2x80x94OY1, CH2Oxe2x80x94Xxe2x80x94Y2, CH2Oxe2x80x94Xxe2x80x94Y3, CH2NHY1, CH2NY12, CH2Y3, CH2NHxe2x80x94Xxe2x80x94OH, CH2NHxe2x80x94Xxe2x80x94Y2, CH2NHxe2x80x94Xxe2x80x94Y3, CH2NHxe2x80x94Xxe2x80x94OY1, CH2OC(O)xe2x80x94OY1, CH2Oxe2x80x94Xxe2x80x94OY1, CO2Y1, COY3, COY2, CHO, CHxe2x95x90N(CH2)m(O(CH2)m)nR4, or CHxe2x95x90N(CH2)m(O(CH2)m)nY2;
R2, R3=H, OH, OY1, Oxe2x80x94Xxe2x80x94OH, Oxe2x80x94Xxe2x80x94OY1, Oxe2x80x94Xxe2x80x94Y2, Y3, NHY1, NY12, Y3, NHxe2x80x94Xxe2x80x94OH, NHxe2x80x94Xxe2x80x94Y2, NHxe2x80x94Xxe2x80x94Y3, NHxe2x80x94Xxe2x80x94OY1, NY1xe2x80x94Xxe2x80x94OH, NY1xe2x80x94Xxe2x80x94Y2, NY1xe2x80x94Xxe2x80x94Y3, or NY1xe2x80x94Xxe2x80x94OY1;
provided that one of R2 or R3 is H or that R2 and R3 together denote carbonyl oxygen;
R4=H, OH, OY1, or Y3;
Y1=H, alkyl of 1-30 carbon atoms, straight chain or branched, cycloalkyl of 3-30 carbon atoms, alkanyl of 3-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms, or phenoxyalkyl of 7-30 carbon atoms;
Y2=NH2, NHY1, or NY12;
Y3=xe2x80x94(O(CH2)m)nR4 or xe2x80x94(O(CH2)m)nY2, where m=2-4 and n=1-230;
X=xe2x80x94OC(CH2)pCOxe2x80x94 where p=1-22.
The preferred pentacyclic triterpenes include betulin, betulinic acid, ursolic acid, oleanolic acid, betulin mono- and di-succinate or glutarate, as well as polyethylene glycol derivatives of thereof.
Particularly useful are those pentacyclic triterpenes exhibiting an IC50 value against human leucocyte elastase at concentration less than about 15 micromolar (xcexcM), more preferably an IC50 value of less than about 10, and most preferably an IC50 value at less than about 8 micromolar. The IC50 value represents the concentration of an inhibitor, which can be expressed in micromoles per liter, at which activity of an enzyme is reduced by 50%. Thus, lower IC50 values suggest higher levels of enzyme inhibitory activity.
The pentacyclic triterpenes of the invention are the same as, derived from, synthesized, or otherwise related to those found naturally in the outer surfaces of plants: leaves, fruits, bark, and are subjected to pathogenesis involving enzymatic degradation of cuticle. For the present invention, the pentacyclic triterpenes or their derivatives are applied to the exposed outer plant surfaces in conjunction with a suitable carrier such as water, an aqueous film-forming solution, detergents, emulsion forming additives, suitable polymers to enhance physical properties of the sprayed layer.
Pentacyclic triterpenes can be obtained by extracting the pentacyclic triterpene-containing plant tissues with one or more organic solvents suitable for the triterpenes. Preferred plant tissue sources for PT include bark from white birch trees, apple peels, and the leaves of plants belonging to Vaccinium and Myristica spp. Useful solvents for the extraction include ethyl acetate, acetone, methyl ethyl ketone, ethanol, propanol, isopropanol, methanol, methylene chloride, chloroform, or their mixtures.
The pentacyclic triterpenes or their polyethylene glycol derivatives of the invention should be formed in a non-crystalline state into a well mixed colloidal suspension for application as a uniform coating on the treated plant surfaces. The uniform coating helps to ensure that plant surfaces are well protected against pathogens with the exception of under surfaces or secluded regions unable to be reached by conventional spraying equipment for liquid formulations.
The pentacyclic triterpene compounds of the invention are, however, crystalline solids of hydrophobic character. The solids can be dissolved in a number of solvents suitable for agricultural use. If desired, the solutions or colloidal concentrates of pentacyclic triterpenes can be prepared for shipping and storing. This concentrate can then be further diluted for use by a formulator or applicator.
A preferred solvent for pentacyclic triterpene solids contains about 1-25 wt % acetone, about 0-10 wt % dimethylsulfoxide (DMSO), 0-35% polyethyleneglycol ester of an aliphatic acid, and about 0-25 wt % of a surfactant such as commercially available detergents like Tween 80(trademark) or Palmolive(trademark) dishwashing detergent. Generally, this solvent mixture may carry a concentration of a PT being 1500-4000%, by weight, of that needed for application to the plants.
The concentrate can be further diluted with 100-4000%, preferably 300-1000% by weight of water to make a sprayable composition according to the invention. Particularly useful concentrations are within the range from about 7-30 grams per gallon of water. Adequate mixing requires only low to moderate shearing to ensure adequate mixing of the concentrate during dilution. For example, metering the concentrate into a reservoir attached to a venturi mixer would provide adequate shear to completely mix the concentrate with additional water.
Pentacyclic triterpenes or their polyethylene glycol derivatives according to the invention are applied at a rate sufficient to prevent pathogenic infections. The inhibitory properties of PT are utilized by the plants to inactivate the enzymes excreted by the fungal spore in order to degrade the plant cuticle. Suitable application rates for effective protection include rates within the range from about 0.1-1000 kg/h. Preferably, the application rate is within the range from about 0.1-100 kg/h. The specific application rate that is best for a particular type of plant in a particular region is readily determined by the application of the ordinary skill in the art. The application should, however, be designed to fall on and cover the exposed leaf surfaces of the plants being treated such as it occurs with conventional foliar treatments using conventional foliar spraying equipment.
If desired, the PT or their polyethylene glycol derivatives may be applied in conjunction with one or more inert or active ingredients. Exemplary materials include dyes, additives affecting stability of the concentrate and additives affecting physical properties of the sprayed layer, foliar fertilizers, fungicides, and insecticides.
Virtually any plant that may get infected through the waxy cuticle layer can be beneficially treated with compositions according to the present invention. Commercial plants that would benefit include grain grasses (e.g., rye, wheat, and barley), tomato, bean, pepper, wheat, and peanut plants.
Grain grasses may benefit in particular from the present invention. These plants do not generally produce sufficient levels of pentacyclic triterpenes, if at all, to inhibit enzymatic attacks by invading microorganisms. Natural pathogens of these plants are generally not adapted to produce sufficient amounts of enzymes to overcome the inhibiting effects of the externally applied pentacyclic triterpene compounds.
Plants susceptible to small insect (e.g., aphids) infestation also benefit from treatment according to the invention. Ursolic acid showed toxicity and feeding deterrent effects towards the mites and their survival. The mites"" reproductive indexes decreased in direct proportion to a ursolic acid content in the diet, and in addition, ingestion time on diet containing ursolic acid was reduced about 30% (Varanda et al., 1992). These observations imply a possibility that the insect""s digestive enzymes were compromised by ursolic acid.
The mode of inhibition of enzymes by PT is non-specific and is based primarily on hydrophobic interaction with an enzyme""s hydrophobic domain. This property suggests that the likelihood for developing microbial pathogen resistance through mutation is remote. Pathogens had to deal with the presence of PT in the plant cuticle for as long as the plants existed and it could be assumed that microbial resistance to the PT has been already optimized. The PT based formulations for plant protection should be advantageous against the pathogenic microorganisms that use enzymes to break through the cuticle, such as fungi, bacteria, nematodes and viruses. These formulations should be also advantageous against small insects, acting inhibitory on their digestive enzymes. By covering the leafy surface with a film containing pentacyclic triterpenes, the passive defensive properties of the cuticle are enhanced, which decreases or entirely prevents successful pathogenic penetration.
USPTO Backgrounds
USPTO Backgrounds