Compositions and Methods for the Prevention and Treatment of Diseases in Plants

Abstract

Non-toxic, environmentally friendly, naturally-derived and economical compositions and methods for treating and preventing bacterial or fungal disease in plants, especially disease caused by Xanthomonas species, such as citrus canker, wherein such compositions include d-limonene alone or in combination with wax and/or wax extract, monohydric alcohol, an emulsifier and/or other ingredients are provided. Methods of making such compositions are also provided.

Description

CROSS REFERENCE APPLICATION

This application is a continuation-in-part of U.S. application Ser. No. 11/093,501 filed on Mar. 30, 2005, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for the treatment and prevention of bacterial and fungal disease in plants. Among the important diseases which can be treated and prevented by such compositions and methods is citrus canker.

BACKGROUND OF THE INVENTION

As reported by the University of Florida Plant Pathology Department, fungal and bacterial pathogens can lower yields, reduce quality, negatively affect the aesthetic and economic value and even ultimately destroy plants, crops, pre-harvest fruits, trees, vegetables and grasses. In particular, bacterial and fungal diseases in citrus plants create huge economic burdens on the citrus industry worldwide. Citrus canker alone causes severe economic consequences in the world's citrus growing regions. The bacteria which causes citrus canker, Xanthomonas campestris pv. citri, has been found in southern Asia, Japan, the Middle East, Africa, South America and now North America. Historically, citrus canker outbreaks have occurred in North America in the early 1900s and again in 1986. Fortunately, through the use of aggressive eradication techniques, the disease was eliminated in both instances. Citrus canker, however, can be reintroduced when people inadvertantly transport infected citrus fruits and seedlings into citrus growing areas in the U.S. and elsewhere. In the U.S., Florida is by far the state most at risk from citrus canker given its combination of high humidity throughout the year, seasonal hurricanes and frequent thunderstorms accompanied by high wind gusts. (See The University of Florida Plant Pathology Department Report at vvvvw.biotech.ufl.edu/PlantContainment/canker.htm.)

The University of Florida Extension Service reports that in 1995 citrus canker was again detected in the U.S. in citrus trees in the Miami-Dade County, Fla. area. Citrus canker then spread to the commercial groves in southwest Florida. Since 1998 more than 870,000 trees have been destroyed in conjunction with eradication programs. (See http://edis.ifas.ufl.edu/FE286)

Costs in managing the threat of citrus canker include capital investment for spray equipment, the lost value of affected fruit, costs associated with inspection and eradication, and costs associated with the creation of natural windbreaks and other defensive tactics. Current spraying programs include relatively expensive copper based sprays. The University of Florida Extension Service has estimated that if citrus canker became endemic in Florida, the total cost for countering endemic citrus canker, including the use of copper based sprays, could be more than $300 per acre.

Many types of organic molecules possess antimicrobial properties and can he used to effectively control fungal and bacterial disease in plants. For example, previous attempts to control such disease in plants includes, for example, copper-containing fungicide and streptomycin sprays. Other fungicides, herbicides and pesticides have been used as well.

However, many of the currently used products are toxic to humans, animals and the exposed environment which limits large scale application of such products in, for example, citrus orchards, The Environmental Protection Act was established in 1972, in part, to address concerns over the use of potentially toxic materials to control or treat diseases in crops. In 1996 Congress passed the Food Quality Protection Act requiring the Environmental Protection Agency to reassess the safety and efficacy of every existing pesticide by 2006. Concerns about pesticide residues on plants and the exposure of field workers to such residues severely limits the amounts of pesticide that can be applied and the timing of such application. These limitations decrease the potential effectiveness of commonly used pesticides to prevent and treat bacterial and fungal disease in plants in general and citrus canker in particular.

Accordingly, there exists a tremendous need for effective, economical and non-toxic compositions and methods for preventing and treating bacterial and fungal disease in plants. In particular there is a tremendous need for non-toxic, environmentally friendly, effective and economical compositions and methods for preventing and treating citrus canker.

SUMMARY OF THE INVENTION

The present invention provides non-toxic, environmentally friendly, effective and economical compositions and methods which prevent and treat bacterial and fungal disease in plants. More particularly, the present invention is effective in preventing and treating disease caused by the various species of the bacteria Xanthomonas, including diseases such as citrus canker. Compositions according to the present invention include d-limonene as the primary active ingredient alone or in combination with wax and/or wax extract, monohydric alcohol and other ingredients. Applicants have unexpectedly found that such compositions are capable of preventing and treating bacterial and fungal disease in plants, including citrus canker.

Moreover, compositions and methods according to the present invention are environmentally friendly since they do not include or involve the use of toxic chemicals or chemicals at toxic levels. Accordingly, there are minimal regulatory limitations for the use of such compositions.

The present invention also provides methods of making and using such compositions.

Accordingly, one object of the present invention is to provide compositions for preventing and treating bacterial and fungal disease in plants, especially diseases caused by Xanthomonas species such as citrus canker, comprising d-limonene alone or in combination with wax and/or wax extract, monohydric alcohol and/or other ingredients.

Another object of the present invention is to provide methods of making compositions for preventing and treating bacterial and fungal disease in plants, especially diseases caused by Xanthomonas species such as citrus canker.

A further object of the present invention is to provide methods of preventing and treating disease in plants, including diseases caused by Xanthomonas species such as citrus canker, which include applying compositions described herein to plants.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides non-toxic, naturally-derived, environmentally friendly compositions and methods for treating and preventing bacterial and fungal diseases, especially disease caused by species of the bacteria Xanthomonas such as citrus canker.

The compositions of the present invention may comprise d-limonene alone or in combination with wax and/or wax extract, monohydric alcohol and other ingredients. Applicants have unexpectedly found that such compositions are capable of treating and preventing bacterial and fungal disease in plants, especially disease caused by Xanthomonas such as citrus canker. Moreover, such treatments do not create any perceivable concommitant negative consequences such as, for example, fruit production decreases, leaf drop or leaf wilt.

As used herein, unless otherwise stated, percentage amounts of an ingredient are by total weight of the composition in which that ingredient is included prior to any dilution of the composition with water or other diluent and/or prior to any freeze-drying process.

Compositions of the present invention include d-limonene. D-limonene is the major component of the oil extracted from citrus rind and has been used extensively as an ingredient in cleaning products. D-limonene in compositions according to the present invention may comprise up to about 100% of the non-diluted product. In certain embodiments prior to dilution, d-limonene may comprise by weight from about 0.5% to about 20.0%, in other embodiments from about 5.0% to about 15.0%, and in other embodiments from about 9.0% to about 11.0%.

After dilution the weight percentage of d-limonene preferably drops to less than about 50% of the composition, more preferably less than about 20%, and most preferably less than about 10%. D-limonene may be present in diluted compositions in amounts as low as 0.01% by weight. D-limonene is commercially available from many sources.

Compositions of the present invention also may include wax material, more preferably an extract of wax material. The preferred wax material is beeswax. Prior to any dilution, the wax and/or wax extract may comprise from about 0.005% to about 10.0%, preferably from about 0.01% to about 1.0%, and more preferably from about 0.05% to about 0.5% and even more preferably about 0.1% of the composition. Optionally, as a substitute for, or in addition to wax and/or wax extract, compositions according to the present invention may include benzoic acid, salicylic acid and paraffin, and/or extracts of any of the above, alone or in various combinations, in amounts by weight totaling those given above for wax and/or wax extract alone.

Extracts of wax may be obtained by heating a wax and water mixture and then filtering the solution to obtain the extracts. In certain embodiments about 99% of the solids are filtered out leaving a substantially solid-free wax extract.

After dilution, the wax and/or wax extract may be present in amounts reflecting the degree of dilution. For example, if compositions according to the present invention are diluted prior to use by the inclusion of 3 parts diluent to 1 part concentrated composition, the amount of wax or wax extract will be about 25% of its original concentration by weight.

Wax (such as beeswax), benzoic acid, salicylic acid and paraffin are commercially available from many sources.

Preservative compositions of the present invention also may include emulsifiers such as soy lecithin. Among the advantages of emulsifiers such as soy lecithin are surface-active properties which provide, for example, the reduction in mixing time and the maintenance of stability of dispersions. Emulsifiers such as soy lecithin also provide a desirable homogenous appearance and prevent surface separation. Prior to dilution, emulsifiers used in the present invention may comprise from about 0.05 to about 15%, more preferably from about 0.5 to about 10%, and more preferably from about 1 to about 5%. Lecithins, particularly soy lecithins are preferred in certain embodiments of the present invention. Other ingredients may be used, for example, in amounts described above either alone or in combination with soy lecithin. For example, canola oil and/or caprylic acid may be used as emulsifiers. After dilution, the concentration by weight of emusifiers will drop in proportion to the extent of dilution as discussed above. Emulsifiers such as soy lecithin are commercially available from many sources.

Compositions according to the present invention also may include monohydric alcohols such as methanol, ethanol and butanol, alone or in various combinations. A preferred monohydric alcohol is ethanol. A preferred combination of monohydric alcohols includes ethanol and methanol. Monohydric alcohols according to the present invention may be either denatured or non-denatured. Prior to dilution, monohydric alcohol may comprise from about 0.5% to about 15.0%, more preferably about 2.0 to about 12.0%, and even more preferably from about 4.0 to about 9.0% of the composition. After dilution, the concentration by weight of monohydric alcohol will drop in proportion to the extent of dilution as discussed above. Monohydric alcohols are commercially available from many sources.

Compositions of the present invention also may include other excipients. Other excipients may include potassium hydroxide, xanthan gum, and hydrochloric acid. Potassium hydroxide and hydrochloric acid may be used to control and adjust pH levels. Xanthan gum may be used to control and adjust viscosity and also provide product stability. When included in compositions according to the present invention prior to dilution, potassium hydroxide may comprise from about 0.01% to about 5.0%, more preferably from about 0.05% to about 2.0%, and even more preferably from about 0.05% to about 0.5% of the composition. When included in compositions according to the present invention, xanthan gum may comprise from about 0.01% to about 2.0%, preferably from about 0.01% to about 1.0%, and more preferably from about 0.1% to about 0.5% of the composition. After dilution the concentration by weight of these excipients, if present, will drop in proportion to the extent of dilution as discussed above. All of the above-mentioned excipients are commercially available from many sources.

The balance of undiluted, concentrated forms of compositions according to the present invention may comprise water. The water may be purified and, if so, preferably has a bacterial colony count of less than about 100 colonies per ml. The water may be made alkaline by the addition of potassium hydroxide and preferably has a pH above about 10.0. The preferred water hardness is less than 1 grain per gallon or less than 50 ppm. In certain embodiments water may comprise by weight from about 35% to about 99% of compositions according to the present invention. As discussed previously, extremely concentrated compositions comprising, for example, higher amounts of d-limonene and wax and/or wax extract are intended to be within the scope of the invention. As such, water may be present in amounts lower than 35%.

Methods of Making

The present invention also provides methods of making the compositions described herein. Such methods can be as simple as providing concentrated forms of d-limonene in a suitable container. Methods also include combining d-limonene and water.

In another embodiment, compositions according the present invention may be made by (1) adding monohydric alcohol to water and mixing; (2) heating the alcohol and water mixture; (3) adding d-limonene and mixing; (4) heating the monohydric alcohol, d-limonene and water combination; (5) adding wax and/or wax extract and mixing further; (6) heating the monohydric alcohol, d-limonene, wax and/or wax extract and water mixture; (7) optionally filtering the mixture; and (8) optionally cooling the filtrate.

In another embodiment the method of making compositions includes the steps of (1) adding potassium hydroxide to water and mixing; (2) adding monohydric alcohol (e.g., ethanol) to the water and mixing; (3) heating the potassium hydroxide, alcohol, and water mixture to about 175° F. for five minutes or less; (4) adding d-limonene to the water and mixing, while maintaining the temperature at about 175° F. for about 15 minutes (4) adding beeswax and/or beeswax extract to the water and mixing, while maintaining the temperature at about 170° F. for about 15 minutes; (5) adding xanthan gum and mixing vigorously while allowing the mixture to cool; (6) filtering the cooled mixture.

In another embodiment methods of making compositions according to the present invention may include the steps of (1) mixing water, wax and/or wax extract, and potassium hydroxide in a container; (2) adding to the container d-limonene and an emulsifier, such as soy lecithin, and mixing; and (3) adding to the container a monohydric alcohol and mixing.

In another embodiment, methods of making compositions according to the present invention may include the steps of (1) mixing water, beeswax extract, and potassium hydroxide in a container; (2) adding to the container d-limonene and soy lecithin and mixing; and (3) adding to the container ethanol and mixing.

In yet another embodiment, methods of making compositions according to the present invention may include the steps of (1) mixing in a container water, beeswax extract and potassium hydroxide, wherein water is present in amounts by weight from about 35% to about 99%, wherein the beeswax extract is present in amounts by weight from about 0.005% to about 10.0%, and wherein potassium hydroxide is present in amounts by weight from about 0.01% to about 5.0%; (2) adding to the container d-limonene and soy lecithin and mixing, wherein the d-limonene is present in amounts by weight from about 0.5% to about 20.0%, and wherein soy lecithin is present in amounts by weight from about 0.05 to about 15%; and (3) adding to the container ethanol and mixing, wherein ethanol is present in amounts by weight from 0.5% to about 15.0%.

It should be noted that the order of steps recited above in embodiments of the present invention may be varied to produce compositions according to the present invention. For example, d-limonene may be added prior to adding monohydric alcohol or wax and/or wax extract could be added before either d-limonene or monohydric alcohol.

In certain examples of embodiments of the present invention, water may be heated in any heating step (if the particular method of making the composition includes heating water or water and other components) to between about 110° to about 272° F., preferably to between about 130° to about 210° F., and more preferably to between about 165° to about 180° F.

The liquid mixture may then be pumped through filters at a pressure sufficient to effectively collect the composition. The filtering pressure may be from about 5 to about 100 psi, preferably is from about 10 to about 40 psi, and more preferably is about 30 psi. The composition may be cooled before use. Preferably the composition may be cooled to at least about 95° F. Cooling may be facilitated by the use of a heat exchanger.

Compositions according to the present invention also may be formed into a crystalline powder form to facilitate packaging, storage and use. This may be achieved, for example, by reducing the water content to about 10% of the composition, adding xanthan gum and gum arabic in roughly equal amounts, drying the composition, and then grinding to break up larger particles. Drying may be achieved by, for example, a vacuum drier, a fluidized bed drier, a low temperature roller drier, a vacuum extrusion drier or by other drying means.

Methods of Using

The present invention also includes methods of using compositions which can prevent and treat bacterial and fungal diseases in plants, especially diseases caused by Xanthomonas species, such as citrus canker. An effective amount of the compositions of the present invention can be applied to plants such as citrus trees. In this context an effective amount means an amount that is sufficient to prevent and reduce bacterial or fungal disease affecting plants such as citrus canker. Accordingly, an effective amount can vary depending on whether the primary purpose is prevention or treatment, and if the latter, the severity of the disease state.

In general, however, compositions according to the present invention may be effectively applied to citrus trees in a concentration ranging from about 1% to about 80%. Preferably, the concentration will be between 10% and 50% and more preferably between 20% and 40%. To achieve such concentrations, compositions according to the present invention may be diluted with an appropriate diluent such as water. Other suitable diluents may be used instead of water, although water should be the most economical diluent. Alternatively, compositions according to the present invention may be incorporated into existing (or yet to be developed) formulations of herbicides, pesticides and fungicides, including those currently used by commercial growers. One benefit of including compositions according to the present invention into certain herbicides, pesticides and/or fungicides is that it may reduce the total amount of toxic chemicals that would otherwise be applied to plants, including citrus trees.

Compositions according to the present invention can be sprayed onto plants that do not have any observable disease using conventional spray equipment. Likewise, these compositions can be sprayed onto diseased plants in the same manner. For use in diseased plants, the concentration of compositions according to the present invention generally will be higher than for preventative treatments.

Compositions according to the present invention may reduce or eliminate the growth of a wide-range of disease-causing organisms in plants, including citrus trees. These compositions have been shown to be especially effective in the treatment and prevention of citrus canker caused by Xanthomonas, including Xanthomonas campestris pv. Citri, the causative agent of citrus canker.

In addition to use in citrus species, compositions according to the present invention are also effective in treating and preventing bacterial and fungal infections (and/or infestations) in other types of plants. Such diseases include (but are not limited to) diseases caused by other Xanthomonas species, diseases caused by Pseudomonas, and diseases caused by other bacteria, as well as fungal diseases and disease caused by infestation of certain insects.

In addition to citrus plants, compositions and methods according to the present invention are also effective in preventing and/or treating diseases in tomatoes, bell peppers, grapes and other plants. For example, such compositions will be effective in treating and preventing diseases of tomatoes including bacterial spot, caused by Xanthomonas campestris pv. Vesicatoria. Compositions according to the present invention also will be effective in treating and preventing early blight, late blight and septoria leaf spot.

Similarly, such compositions will be effective in treating and preventing disease in bell peppers (and other peppers) such as, for example, bacterial leaf spot, cercospora leaf spot, southern blight and tobacco mosaic virus.

Compositions according to the present invention also will be effective in treating and/or preventing diseases associated with the wine industry. Such diseases include those effecting grapes or vines caused by fungi, such as mildew and various rot conditions, diseases caused by bacteria, such as bacterial blight and Pierce's disease, grapevine yellows disease, and diseases caused by viruses. Compositions according to the present invention also will reduce the incidence of Pylloxera.

EXAMPLE 1

The composition as shown in Table 1 was used to demonstrate the effectiveness of an exemplary embodiment of the present invention in treating citrus canker:

TABLE 1
monohydric alcohol (ethanol) 6.65%
d-limonene                   10.00%
beeswax extract              1.47%
water                        81.40
potassium hydroxide          0.16%
xanthan gum                  0.16%

Ten citrus trees with existing canker lesions were drench sprayed with a 25% solution of the composition according to Table 1, while ten citrus trees were used as controls. Tree limbs were tagged with strips of caution tape to facilitate precise identification of the treatment trees. Weekly observations were taken to determine the effect of the treatment on the canker lesions. A second treatment was applied approximately two weeks after the first. Over the course of eight weeks of observation, canker lesions had not expanded on the ten treatment trees, while canker lesions had grown substantially on the ten control trees. The treatment trees and control trees both exhibited similar insubstantial levels of leaf wilting and fruit drop.

EXAMPLE 2

In order to more precisely quantify the effectiveness of compositions according to the present invention, compositions in accordance with Table 1 above were tested on cultures of Xanthomonas campestris pv. citrumelo obtained from the Florida Department of Plant Industry, Florida Department of Agriculture & Consumer Services, Gainesville, Fla.

Test Procedures

Xanthomonas was grown via at least two transfers at 30° C. for 48 hr in Tryptic Soy broth (TSB). The final culture was centrifuged at 10,000×g for 10 min., the cells resuspended in sterile Butterfield's phosphate buffer (BPB) to obtain 10 mL of a suspension with an approximate cell density of 1×10⁸ CFU/mL. The final working inoculum suspension was enumerated.

A modification of the AOAC Method 960.09 (Germicidal and Detergent Sanitizing Action of Disinfectants) was used to test the disinfectant efficacy of a solution of the composition.

• • 1) Two flasks containing 99 ml each of the antimicrobial solution were prepared in sterile 250 ml flasks containing sterile stir bars. A single flask of control solution (99 ml of sterile Butterfield's phosphate buffer (BPB) was also prepared. The flasks were equilibrated to room temperature (22-25° C.) before initiating the efficacy tests.
  • 2) One ml of the inoculum suspension (10⁸ CFU/ml) was inoculated into each of the flasks while stirring slowly to yield a flask inoculum level of ca. 10⁶ CFU/ml. The flasks were stirred slowly during the tests.
  • 3) Each flask was sampled 30 sec. after inoculation and subsequently after 1, 5, and 10 min. of incubation/exposure at 25° C. Flasks were sampled by aseptically transferring a one ml aliquot to a tube containing 9.0 ml of Difco neutralizing broth (to yield an initial dilution of 1:10).
    Microbial Analysis

Sample aliquots from the initial (10⁻¹) neutralizing tubes were serially diluted in BPB and enumerated via surface plating on pre-poured plates of Tryptic Soy agar (CYSA) agar, TSA plates were incubated for 48 h at 30° C.

Bacterial counts were expressed as colony forming units per ml of sample solution and converted to log₁₀ transforms. Log₁₀ mean counts were calculated for each solution time variable. The log₁₀ reduction and percentage reduction (versus the control sample after 30 sec.) was also calculated for each exposure time.

Results

Compositions according to the present invention were shown to be highly effective in reducing Xanthomonas counts. The control (BPB buffer) count of Xanthomonas was 6.11, 6.04, 6.11, and 5.94 log₁₀ CFU/ml of solution after 30 sec., 1 min. 5 min., and 10 min., respectively. There was an increasing reduction of Xanthomonas counts in the test solution as the exposure times increased with significant reductions (i.e., >1 log₁₀ CFU/ml) noted after 5 and 10 min. exposure times. There was a 1.98 log₁₀ CFU/ml (98.94%) reduction of mean Xanthomonas counts in the test solution following a 5 min. exposure time and a 4.16 log₁₀ CFU/ml (99.995%) reduction of mean Xanthomonas counts following a 10 min. exposure time.

Claims

1. A composition for treating and preventing disease in plants comprising d-limonene.

2. The composition of claim 1, further comprising wax and/or wax extract.

3. The composition of claim 2, further comprising monohydric alcohol.

4. The composition of claim 3, further comprising potassium hydroxide.

5. The composition of claim 4, further comprising an emulsifier.

6. The composition of claim 5, wherein the emulsifier is soy lecithin.

7. The composition of claim 1, wherein d-limonene is present in an amount up to about 50% by weight and wherein said composition further comprises water.

8. The composition of claim 7, further comprising wax and/or wax extract.

9. The composition of claim 8, further comprising monohydric alcohol.

10. The composition of claim 9, further comprising potassium hydroxide.

11. The composition of claim 10, further comprising an emulsifier.

12. The composition of claim 11, wherein the emulsifier is soy lecithin.

13. The composition of claim 7, comprising from about 0.5% to about 20% d-limonene, from about 0.005% to about 5.0% wax and/or wax extract, from about 0.5% to about 15% monohydric alcohol, from about 0.01% to about 5.0% potassium hydroxide, and from about 0.05% to about 15% emulsifier, all by weight.

14. The composition of claim 13, wherein the monohydric alcohol is selected from the group consisting of ethanol, methanol and butanol, alone or in combination.

15. The composition of claim 13, wherein the wax and/or wax extract comprises beeswax and/or beeswax extract.

16. The composition of claim 13, wherein the emulsifier comprises soy lecithin.

17. The composition of claim 13, wherein the plant is a member of the citrus species.

18. The composition of claim 17, wherein the disease is citrus canker.

19. The composition of claim 13, wherein the disease is caused by any species of Xanthomonas.

20. A method for treating or preventing disease in a plant species comprising applying to said plant species a composition which comprises d-limonene.

21. The method of claim 20, wherein said composition further comprises wax and/or wax extract.

22. The method of claim 21, wherein said composition further comprises monohydric alcohol.

23. The method of claim 22, wherein said composition further comprises potassium hydroxide.

24. The method of claim 23, wherein said composition further comprises an emulsifier.

25. The method of claim 24, wherein the emulsifier is soy lecithin.

26. The method of claim 20, wherein d-limonene is present in an amount up to about 50% by weight and wherein said composition further comprises water.

27. The method of claim 26, wherein said composition further comprises wax and/or wax extract.

28. The method of claim 27, wherein said composition further comprises monohydric alcohol.

29. The method of claim 28, wherein said composition further comprises potassium hydroxide.

30. The method of claim 29, wherein said composition further comprises an emulsifier.

31. The method of claim 30, wherein the emulsifier is soy lecithin.

32. The method of claim 26, wherein said composition comprises from about 0.5% to about 20% d-limonene, from about 0.005% to about 5.0% wax and/or wax extract, from about 0.5% to about 15% monohydric alcohol, from about 0.01% to about 5.0% potassium hydroxide, and from about 0.05% to about 15% emulsifier, all by weight.

33. The method of claim 32, wherein the monohydric alcohol is selected from the group consisting of ethanol, methanol and butanol, alone or in combination.

34. The composition of claim 32, wherein the wax and/or wax extract comprises beeswax and/or beeswax extract.

35. The method of claim 32, wherein the emulsifier comprises soy lecithin.

36. The method of claim 32, wherein the plant is a member of the citrus species.

37. The method of claim 36, wherein the disease is citrus canker.

38. The method of claim 32, wherein the disease is caused by any species of Xanthomonas.