XENORHABDUS SZENTIRMAII METABOLITES, TRANS-CINNAMIC ACID, AND ANALOGS THEREOF AS ENHANCERS OF FUNGICIDAL ACTIVITY
20180049444 ยท 2018-02-22
Inventors
Cpc classification
A01N63/20
HUMAN NECESSITIES
A01N65/30
HUMAN NECESSITIES
A01N65/30
HUMAN NECESSITIES
A01N63/20
HUMAN NECESSITIES
A01N35/02
HUMAN NECESSITIES
A01N37/10
HUMAN NECESSITIES
A01N37/10
HUMAN NECESSITIES
A01N35/02
HUMAN NECESSITIES
International classification
A01N63/00
HUMAN NECESSITIES
Abstract
The fungicidal activity of TCA and cell-free extracts of X. szentirmaii is superior compared to other Xenorhabdus and Photorhabdus species. The effectiveness of TCA and X. szentirmaii metabolites to synergize and enhance fungicidal efficacy of commercial fungicides when combined with them at particular concentrations to suppress growth of Monilinia fructicola and Rhizoctonia solani was evaluated. Fungicidal activity was measured by phytopathogen growth on PDA plates with and without treatments. For suppression of M. fructicola, synergy was observed between TCA when combined with certain concentrations of Elast, PropiMax, Regalia or Serenade, and for combinations of X. szentirmaii, strain 17C+E, when combined with Abound. For suppression of R. solani, synergy was observed between TCA combined with Regalia or Serenade. TCA combined with X. szentirmaii resulted in synergistic levels of suppression of M. fructicola. Utilizing TCA and X. szentirmaii for enhancing fungicidal activity improves control of phytopathogenic diseases and reduces environmental impact.
Claims
1. A synergistic fungicidal composition comprising: a fungicide; and a fungicidal enhancer, wherein said fungicidal enhancer is at least one of (a) the fungicidal metabolite of Xenorhabdus szentirmaii, strain 17C+E (NRRL B-67309), (b) trans-Cinnamic acid (TCA), or (c) an analog of TCA, and wherein the amount of the fungicidal enhancer is in an amount effective to synergistically enhance a fungicidal effect of said fungicide to suppress the growth and sporulation of phytopathogenic fungi.
2. The composition of claim 1, wherein said phytopathogenic fungi are any one of Venturia carpophila, V. effusa, Monilinia fructicola, Glomerella cingulata, Rhizoctonia solani, Alternaria solani, and Phytophthora capsici.
3. The composition of claim 1, wherein said fungicidal enhancer is one of TCA or an analog of TCA, and said fungicide is any one of the commercial fungicides Prophyt, Regalia, Serenade, Elast, PropiMax, Abound, and Super Tin.
4. The composition of claim 3, wherein the fungicidal enhancer has a chemical structure of the form: ##STR00002## wherein R.sup.1 is O or N; R.sup.2 is OH, H, OCH.sub.3, or an akyl chain of length 1-12, wherein each of R.sup.3, R.sup.4, and R.sup.5 are one of H, OH, OCH.sub.3, NH.sub.2, CN, NO.sub.2, Cl, or NR.sup.6R.sup.7, and each of R.sup.6 and R.sup.7 are one of H or an alkyl chain of length 1-12, and wherein the bond designated by (a) is either a double bond or a single bond, and in the case of (a) being a double bond, the compound may be either the trans or cis isomer.
5. The composition of claim 1, wherein said fungicidal enhancer is one of TCA or an analog of TCA, and said fungicide is any one of azoxystrobin, potassium phosphite, a plant extract from Reynoutria sachalinensis, the bacterium Bacillus subtilis, dodine, propiconazole, and triphenyltin hydroxide.
6. The composition of claim 5, wherein the fungicidal enhancer has a chemical structure of the form: ##STR00003## wherein R.sup.1 is O or N; R.sup.2 is OH, H, OCH.sub.3, or an akyl chain of length 1-12, wherein each of R.sup.3, R.sup.4, and R.sup.5 are one of H, OH, OCH.sub.3, NH.sub.2, CN, NO.sub.2, Cl, or NR.sup.6R.sup.7, and each of R.sup.6 and R.sup.7 are one of H or an alkyl chain of length 1-12, and wherein the bond designated by (a) is either a double bond or a single bond, and in the case of (a) being a double bond, the compound may be either the trans or cis isomer.
7. The composition of claim 1, wherein said fungicidal enhancer is the fungicidal metabolite of X. szentirmaii, strain 17C+E, and said fungicide is Abound.
8. The composition of claim 1, wherein said fungicidal enhancer is the fungicidal metabolite of X. szentirmaii, strain 17C+E, and said fungicide is azoxystrobin.
9. The composition of claim 1, wherein a ratio of the amount of the fungicide and the amount of the fungicidal enhancer is such that the fungicidal activity is synergistically enhanced.
10. The composition of claim 1, wherein the fungicide is one of TCA or an analog of TCA, and the fungicidal enhancer is the fungicidal metabolite of X. szentirmaii, strain 17C+E.
11. The composition of claim 1, wherein the fungicide is the fungicidal metabolite of X. szentirmaii, strain 17C+E, and the fungicidal enhancer is one of TCA or an analog of TCA.
12. A method for reducing the amount of growth or sporulation of one or more phytopathogenic fungal populations on a living plant or tree comprising: (a) applying to a plant, tree, seed, or to soil surrounding said plant, tree, or seed, the synergistic fungicidal composition of claim 1 in an amount effective to reduce the growth or sporulation of the phytopathogenic fungal populations; and (b) thereby reducing the amount of growth or sporulation of one or more phytopathogenic fungal populations on the living plant or tree.
13. The method of claim 12, wherein the fungicidal enhancer is at least one of TCA or an analog of TCA, and wherein the fungicide is at least one of the commercial fungicides Prophyt, Regalia, Serenade, Elast, PropiMax, Abound, and Super Tin.
14. The method of claim 12, wherein the fungicidal enhancer is at least one of TCA or an analog of TCA, and wherein the fungicide is at least one of azoxystrobin, potassium phosphite, a plant extract from Reynoutria sachalinensis, the bacterium Bacillus subtilis, dodine, propiconazole, and triphenyltin hydroxide.
15. The method of claim 12, wherein the fungicidal enhancer is the fungicidal metabolite of X. szentirmaii, strain 17C+E (NRRL B-67309), and wherein the fungicide is Abound.
16. The method of claim 12, wherein the fungicidal enhancer is the fungicidal metabolite of X. szentirmaii, strain 17C+E (NRRL B-67309), and wherein the fungicide is azoxystrobin.
17. The method of claim 12, wherein the fungicide is one of TCA or an analog of TCA, and wherein the fungicidal enhancer is the fungicidal metabolite of X. szentirmaii, strain 17C+E.
18. The method of claim 12, wherein the fungicide is the fungicidal metabolite of X. szentirmaii, strain 17C+E, and wherein the fungicidal enhancer is one of TCA or an analog of TCA.
19. The method of claim 12, wherein the phytopathogenic fungal populations are any one of Venturia carpophila, V. effusa, Monilinia fructicola, Glomerella cingulata, Rhizoctonia solani, Alternaria solani, and Phytophthora capsici.
20. A biocontrol method for treating at least one of peach scab, pecan scab, brown rot and anthracnose by reducing the amount of growth or sporulation of one or more phytopathogenic fungal populations on a living plant or tree according to claim 12.
21. The biocontrol method of claim 20, comprising reducing the amount of growth or sporulation of one of Venturia carpophila, V. effusa, Monilinia fructicola, Glomerella cingulata, Rhizoctonia solani, Alternaria solani, and Phytophthora capsici.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0022] Advantages of embodiments of the present invention will be apparent from the following detailed description of the exemplary embodiments. The following detailed description should be considered in conjunction with the accompanying figures in which:
[0023] Exemplary
[0024] Exemplary
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[0026] Different letters above bars indicate statistical significance (Tukey's HSD test, =0.05). Exemplary
[0027] Exemplary
[0028] Exemplary
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DETAILED DESCRIPTION
[0036] Aspects of the invention are disclosed in the following description and related drawings directed to specific embodiments of the invention. Alternate embodiments may be devised without departing from the spirit or the scope of the invention. Additionally, well-known elements of exemplary embodiments of the invention will not be described in detail or will be omitted so as not to obscure the relevant details of the invention. Further, to facilitate an understanding of the description discussion of several terms used herein follows.
[0037] As used herein, the word exemplary means serving as an example, instance or illustration. The embodiments described herein are not limiting, but rather are exemplary only. It should be understood that the described embodiment are not necessarily to be construed as preferred or advantageous over other embodiments. Moreover, the terms embodiments of the invention, embodiments, or invention do not require that all embodiments of the invention include the discussed feature, advantage or mode of operation.
[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. As used herein, the term about refers to a quantity, level, value, or amount that varies by as much as 30%, preferably by as much as 20%, and more preferably by as much as 10% to a reference quantity, level, value, or amount. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
[0039] Other compounds may be added to the composition provided they do not substantially interfere with the intended activity and efficacy of the composition; whether or not a compound interferes with activity and/or efficacy can be determined, for example, by the procedures utilized below.
[0040] The amounts, percentages, and ranges disclosed herein are not meant to be limiting, and increments between the recited amounts, percentages, and ranges are specifically envisioned as part of the invention.
[0041] The terms effective amount and an amount effective [to] of a compound or property as provided herein is meant such amount as is capable of performing the function of the compound or property for which an effective amount is expressed. As will be pointed out below, the exact amount required will vary from process to process, depending on recognized variables such as the compounds employed and the processing conditions observed. Thus, it may not be possible to specify an exact effective amount. However, an appropriate effective amount may be determined by one of ordinary skill in the art using only routine experimentation based on the disclosures herein.
[0042] Fungicidal activity or fungicidal effect refers to the ability of a compound or composition to (i) prevent the growth of a fungus, including the prevention of sporulation, (ii) inhibit the growth of a fungus or its sporulation, or (ii) substantially kill or eliminate a fungus population. A fungicide is a compound or composition which exhibits substantial fungicidal activity.
[0043] The term treat, treating, or treatment, as used herein, refers to the use of a fungicide to reduce or prevent a condition, symptom, or disease caused by a fungus by (i) preventing the growth of a fungus, including the prevention of sporulation, (ii) inhibiting the growth of a fungus or its sporulation, or (ii) substantially killing or eliminating a fungus population.
[0044] A fungicidal enhancer is a compound or composition which, when combined with a fungicide, substantially enhances the fungicidal activity of the fungicide. In particular, a fungicidal enhancer is a compound or composition which has a synergistic, rather than an additive, ability to increase the fungicidal activity of the fungicide.
[0045] As used herein, analog or chemical variant of a compound refers to a structural analog of the identified compound having a similar structure and similar activity. For example, one analog of trans-Cinnamic acid is cinnamaldehyde.
[0046] Combined application of different anti-fungal compounds can result in synergism resulting in improved efficacy and reduced potential for resistance development (Moreno-Martinez et al. 2015. Sci. Reports 5:16700 DOI: 10. 1038/srep16700). However, combination of pesticidal agents can also result in additive or antagonistic interactions (Shapiro-Ilan et al. 2004. Biol. Control 30:119-126; Shapiro-Ilan et al. 2017). One of the bioactive compounds found in Photorhabdus species is TCA. TCA by itself seems unlikely to be marketed as a fungicide, but an enhanced version based on combination with other materials could be quite potent.
[0047] TCA and metabolites from X. szentirmaii, strain 17C+E, possess higher antifungal activity than metabolites derived from a variety of other Xenorhabdus or Photorhabdus species (Examples 3 and 4; Hazir et al. 2017a. Eur. J. Plant Pathol. In Press, DOI 10.1007/s10658-017-1277-7). This finding held true when comparing potency against a variety of phytopathogens including major pathogens of peach, Armillaria tabescens (the causal agent of root rot), Venturia carpophila (causal agent of peach scab) and Monilinia fructicola (causal agent of brown rot), and pecan pathogens V. effusa (causal agent of pecan scab), and Glomerella cingulata (causal agent of anthracnose=Colletotrichum gloeosporioides). Some of these pathogens species or genera cause disease in a variety of other crops as well. Therefore, there is strong justification to explore further the potential role of Xenorhabdus and Photorhabdus metabolites in the control of plant diseases.
[0048] The inventors found that TCA activity can be greatly enhanced (synergistically) when combined with crude culture broth of other entomopathogenic bacteria such as Xenorhabdus szentirmaii, in particular, X. szentirmaii, strain 17C+E. Synergistic levels of suppression were observed against brown rot (Monilinia fructicola) when TCA was combined with cell-free broths of X. szentirmaii, strain 17C+E. Development of these combinations (or similar ones) as novel fungicides could have substantial impact on control of important plant diseases.
[0049] Synergy between fungicidal agents has been sought and observed previously (Moreno-Martinez 2015). For example, Baider and Cohen (2003. Phytoparasitica 31:399-409) reported BABA (DL-3-aminobutyric acid) and the protectant fungicide mancozeb were more effective than either agent applied alone in controlling late blight (Phytophthora infestans) in potato and tomato, and downy mildew (Pseudoperonospora cubensis) in cucumber. However, there is a dearth of examples that extend to field results. Furthermore, no reports to date have examined fungicidal interactions between metabolites of Xenorhabdus or Photorhabdus bacteria and commercial fungicides.
[0050] Interactions between TCA, TCA analogs, and metabolites from X. szentirmaii, strain 17C+E, when combined with commercial fungicides were evaluated. Phytopathogens included Monilinia fructicola and Rhizoctonia solani. For suppression of M. fructicola, synergy was observed between TCA when combined with particular concentrations of the commercial fungicides Elast, PropiMax, Regalia or Serenade, and for combinations of X. szentirmaii with Abound. For suppression of R. solani, synergy was observed between TCA combined with Regalia or Serenade. Additionally, when TCA was combined with X. szentirmaii synergistic levels of suppression to M. fructicola were observed.
[0051] TCA analogs which may also be used in the present invention include cis-Cinnamic acid, cinnamaldehyde, hydrocinnamic acid. In general, TCA and analogs thereof may include any compound of the form:
##STR00001##
wherein R.sup.1 is O or N; R.sup.2 is OH, H, OCH.sub.3, or an akyl chain of length 1-12; each of R.sup.3, R.sup.4, and R.sup.5 are one of H, OH, OCH.sub.3, NH.sub.2, CN, NO.sub.2, Cl, or NR.sup.6R.sup.7, where each of R.sup.6 and R.sup.7 are one of H or an alkyl chain of length 1-12; and the bond designated by (a) is either a double bond or a single bond, and in the case of (a) being a double bond, the compound may be either the trans or cis isomer.
[0052] The present findings may hold promise for enhancing control of key phytopathogens in an effective and environmentally sound manner. Specifically, the use of bio-based fungicidal agents such as bacterial metabolites is likely to have lower environmental impact relative to broad spectrum chemicals. Thus, combinations using TCA or X. szentirmaii, strain 17C+E, in synergistic combinations with other fungicides considered biorational, e.g., Regalia and Serenade, could be particularly beneficial as efficacy is increased while minimizing environmental impact. However, synergistic combinations of TCA or X. szentirmaii, strain 17C+E, with conventional chemical fungicides will also be beneficial because higher efficacy may be achieved while the amount of chemical input into the system is reduced. Some of the synergistic interactions observed in the present study involved lower rates of the commercial fungicide, and therefore may have more potential for reduced environmental impact, e.g., TCA combined with Elast, Prophyt or Regalia. Moreover, in one case the fungicide (Serenade) did not produce any effect when applied alone for suppression of R. solani, yet synergistic suppression was achieved when the high rate was combined with TCA; thus, a fungicide that was ineffective may be made efficacious by adding TCA as an enhancer. Additional research is needed to explore the potential for TCA or X. szentirmaii, strain 17C+E supernatants (or other Xenorhabdus spp. or Photorhabdus spp. metabolites) as fungicide synergists in combination with other fungicides, and against other phytopathogens not tested in this study. Furthermore, research will be needed to confirm and optimize the synergistic interactions under field conditions.
[0053] The highly antagonistic metabolites of X. szentirmaii, strain 17C+E, are applied to fungi in amounts effective to reduce population levels of said fungi. As used herein reduce population levels refers to a reduction in numbers of M. fructicola propagules compared to that which would be expected in soil which was not treated according to the methods of the present invention. Any accurate method of measuring and comparing population levels may be used for such comparisons, as would be apparent to those skilled in the art.
EXAMPLES
[0054] Having now generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.
[0055] Methods and Materials
[0056] Bacterial Extracts and Fungicides.
[0057] Four Xenorhabdus spp., two strains of Photorhabdus luminescens and one strain of P. temperata (7 bacterial isolates total; Table 1) were used in the assays. TCA was also included. The bacterial exudates and cell-free supernatants were isolated from the hemolymph of nematode-infected Galleria mellonella (Lepidoptera: Pyralidae) larvae according to Kaya and Stock (1997. Techniques in Nematology. In: Manuel of Techniques in Insect Pathology, L. A. Lacey (Ed.) Academic Press, San Diego, pp. 281-324). Photorhabdus spp. and Xenorhabdus spp. produce two colony form variants (phase-1 and phase-II). Phase-I has been reported to provide better support for nematode growth compared with phase-II and only phase-I produces antimicrobial compounds. Therefore, the phase-I form of the bacteria was used. The phase of the bacteria was determined by their cell and colony morphology on NBTA plates and by using the catalase test (Boemare and Akhurst 2006). Bacterial stock suspensions were kept in Tryptic Soy Broth (TSYB; Difco, Detroit, Mich.) with 20% glycerol at 80 C. until they were used in experiments (Boemare and Akhurst, 2006). As needed, the bacterial cells were taken from the frozen stock cultures and transferred directly to NBTA medium (nutrient agar with 0.004% (w/v) triphenyltetrazolium chloride and 0.025% (w/v) bromothymol blue). The growth of the bacteria and colony morphology were checked after 48 hours incubation to ensure that there was no contamination. A loopful of bacteria from NBTA medium was transferred to 100 ml TSYB plus 0.5% yeast extract (Sigma, St. Louis, Mo.) in an Erlenmeyer flask (300 ml capacity). The liquid cultures were incubated on a rotary incubator shaker at 130 or 200 rpm for 24 hours in the dark at 25 C. (Shapiro-Ilan et al. 2014; Hazir et al. 2017a). Subsequently, the bacterial broth was centrifuged at 10,000 rpm for 20 min at 4 C. The supernatant was filtered through a 0.22 m filter (Millipore, Thermo Scientific, NY). The bacterial exudates were poured into the 50 ml sterile centrifuge tubes (Corning, N.Y.) and kept at 4 C. for up to two weeks prior to use. TCA (98%+purity) was obtained from Acros Organics (New Jersey, USA). In addition, trans-Cinnamaldehyde (98%+purity) was obtained from Arcos Organics (New Jersey, USA).
TABLE-US-00001 TABLE 1 Mutualistic bacteria and their isolated nematode species. Entomopathogenic Strain Mutualistic bacteria Nematode Spp. (EPN) Identifier* Xenorhabdus bovienii Steinernema feltiae SN Xenorhabdus nematophila Steinernema carpocapsae Cxrd Xenorhabdus cabanillasii Steinernema riobrave 7-12 Xenorhabdus szentirmaii Steinernema rarum 17c+e Photorhabdus temperata Heterorhabditis megidis UK211 Photorhabdus luminescens Heterorhabditis bacteriophora VS Photorhabdus luminescens Heterorhabditis floridensis K-22 *all strains used were from the Entomopathogenic Nematode Collection, a US Federal Scientific Collection maintained by the US Department of Agriculture
[0058] The following fungicides were used in experiments: azoxystrobin (Abound, Syngenta, Greensboro, N.C.), dodine (Elast, Greensboro Port Washington, N.Y.), potassium phosphite (ProPhyt, Helena Chemical Company, Collierville, Tenn.), propiconazole (PropiMax, Dow, Indianapolis Ind.), a plant extract from Reynoutria sachalinensis (Regalia, Marrone Bio Innovations, Davis Calif.), the bacterium Bacillus subtilis (Serenade, Bayer CropScience LP Research Triangle Park, NC), and triphenyltin hydroxide (Super Tin, United Phosphorus, King of Prussia, Pa.). To simulate applications and combinations that growers would apply, formulated commercial products were used in all experiments (rather than using the technical grade active ingredient alone). In addition, for ease of reference, in the following experiments and Examples, the names of the commercial formulations are used. However, it is envisioned that one of ordinary skill in the art could use the active ingredients, given above, in a composition other than the commercial products referred to in the practice of this invention using the present disclosure.
[0059] Phytopathogen Cultures.
[0060] The fungal cultures of Venturia carpophila (peach scab) and V. effusa (pecan scab) were isolated from peach and pecan trees, respectively, at the USDA-ARS research station in Byron, Ga., USA. Both were cultured on potato dextrose agar (PDA). The cultures of Monilinia fructicola (brown rot) and Glomerella cingulata (anthracnose) were isolated from peach trees at the USDA-ARS research station in Byron, Ga., USA, and were also cultured on PDA. Rhizoctonia solani was isolated from the roots of cotton seedlings in Aydin, Turkey. The isolate of Alternaria solani was obtained from a blighted tomato plant in Gray, Ga. and was cultured on PDA. The isolate of Phytophthora capsici was obtained from a collection at the University of Georgia, Tifton, Ga., and was cultured on V8 agar. All fungi were grown in Petri dishes at 25 C.-27 C. and were stored for up to one week at 4 C. prior to experimentation. Depending on the experiment, either a 5 mm diameter mycelia plug from a fungal culture plate was transferred to the center of each experimental agar dish and growth subsequently monitored, or suspensions of conidia the fungus were prepared in sterile distilled water for subsequent experimental use. Suspensions of V. effusa (210.sup.4 conidia/ml) and of V. carpophila (110.sup.3 conidia/ml) were prepared from cultures after 3 weeks of incubation. Suspensions of spores of A. solani (110.sup.3 conidia/ml) were prepared after 1 week of incubation. Inoculum of Phytophthora capsici was prepared after approximately 1 week of incubation as macerated mycelium with approximately 110.sup.4 mycelial fragments/ml. For R. Solani, only mycelial plugs were used. Mycelial plugs were taken from similarly aged cultures of each species, respectively.
Example 1: Suppression of Spore Germination in Peach and Pecan Scab
[0061] The spores of V. carpophila and V. effusa were used to determine the inhibitory effect of the bacterial supernatants on germination. Supernatants tested were from X. nematophila, X. cabanillasii, X. bovienii, X. szentirmaii, strain 17C+E, P. temperate, P. luminescens (VS) and P. luminescens (K-22). The conidia suspension (0.1 ml) of V. effusa or V. carpophila was added to 0.8 ml potato dextrose broth (PDB) in 2 ml eppendorf tubes containing 0.1 ml (10% v/v) bacterial supernatant. Controls were PDB alone and conidial suspension without supernatant. Because TCA derived from Photorhabdus spp. was identified as an active compound with significant antifungal activity (Bock et al. 2014), TCA was added (1.27 mg/ml) in all experiments for comparison as well. The stock solution of TCA (99% purity, Sigma, St. Louis, Mo.) was prepared with 12.7 g dissolved in 100 ml ethanol (96%). Each treatment was replicated three times, and the experiment was repeated once on a different date. The tubes were incubated at 25 C. for 48 hours and the first 100 conidia were counted under the microscope (400) to determine percentage spore germination (which was used to determine treatment effects). The other fungi (M. fructicola, and G. cingulata) did not produce spores as readily as V. carpophila and V. effusa, so these other phytopathogens were not assessed for effects of supernatant on spore germination. An inhibition rate was determined according to the formula: 100[(the average percentage of spores germinated in the controlthe average percentage of spores germinated in the treatment)/the average percentage of spores germinated in the control] (Fang et al. 2011). Analysis of Variance (ANOVA) (SAS V9.3, SAS Institute Inc., Cary, N.C.) was also applied to the data for V. carpophila.
[0062] Results of spore germination are shown in Table 2 and
TABLE-US-00002 TABLE 2 Inhibition rate of spore germination (%) of different plant pathogens by supernatants of different EPN bacteria.sup.a cultures. P. lum P. lum X. nem X. cab X. bov X. sze P. tem (VS) (K-22) TCA V. carpophila 18.99 44.65 31.35 53.2 3.32 1.66 7.59 87.65 V. effusa 60.39 64.85 60.69 61.02 34.81 45.68 35.14 76.68 .sup.aX. nem = Xenorhabdus nematophila; X. cab = Xenorhabdus cabanillasii; X. bov = Xenorhabdus bovienii; X. sze = Xenorhabdus szentirmaii, strain 17C+E; P. tem = Photorhabdus temperate; P. lum (VS) Photorhabdus luminescens (VS); P. lum (K-22) = Photorhabdus luminescens (K-22); TCA = trans-Cinnamic acid.
Example 2: Suppression of Vegetative Growth in Peach and Pecan Scab
[0063] Treatment effects were compared by measuring in vitro vegetative growth of the phytopathogens V. carpophila and V. effusa when exposed to the same bacterial exudates evaluated above in the germination study. Solutions of the fungicidal treatments were incorporated into PDA and autoclaved. Prior to autoclaving the PDA, the prescribed volume of distilled water was reduced to allow addition of treatment suspensions. Controls consisted of PDA without any amendment or PDA with acetone only (the solvent for TCA). The media were vortexed when cooled to 40-42 C. and poured to Petri dishes.
[0064] A 5 mm diameter mycelia plug from a fungal culture plate was transferred onto the center surface of each agar dish (Fang et al. 2011; Hazir et al. 2017a) and incubated at 25 C. Vegetative growth of the fungus was measured after 7 days. Growth was measured in two perpendicular directions to obtain the approximate area of the fungal mat.
[0065] For G. cingulata, TCA caused the most suppression followed by X. szentirmaii, strain 17C+E (
TABLE-US-00003 TABLE 3 Ranking of top bacterial metabolites in suppression (1.sup.st, 2.sup.nd, 3.sup.rd) for each phytopathogen. The lowest overall score indicates superiority in fungal suppression. G. scingulata M. fructicola V. effusa V. carpophila SCORE X. cabanillasii 2 3 3 1 9 X. nematophila 3 1 1 3 8 X. szentirmaii, 1 2 2 2 7 strain 17C+E
[0066] TCA caused the greatest level of suppression in all experiments. Among the bacterial supernatants, X. szentirmaii, strain 17C+E, was superior in suppressing spore germination and vegetative growth and therefore is superior in suppressing phytopathogenic fungi.
Example 3: Effects of Fungicidal Agents Applied Alone or in Combination
[0067] TCA and the bacterial exudates of X. szentirmaii, strain 17C+E (associated with the nematode, Steinernema rarum), were tested alone and in combination with commercially available fungicides. Treatment effects were compared by measuring in vitro vegetative growth of the phytopathogens when exposed to fungicidal agents applied alone or in combination; the approach was based on methods described above. Solutions of the fungicidal treatments were incorporated into PDA and autoclaved. Prior to autoclaving the PDA, the prescribed volume of distilled water was reduced to allow addition of treatment suspensions. Controls consisted of PDA without any amendment or PDA with acetone only (the solvent for TCA). The media were vortexed when cooled to 40-42 C. and poured to Petri dishes.
[0068] A 5 mm diameter mycelia plug from a fungal culture plate was transferred onto the center surface of each agar dish and incubated at 25 C. Vegetative growth of the fungus was measured after 7 days. Growth was measured in two perpendicular directions to obtain the approximate area of the fungal mat.
[0069] Prior to experimentation, a dose-response test was conducted for each fungicidal treatment and each phytopathogen (data not shown). Various rates of the fungicidal treatments (TCA, X. szentirmaii, strain 17C+E, and commercial fungicides) were applied individually and assessed for fungicidal activity against each phytopathogen as described above. The goal was to obtain a level of fungal suppression that was intermediate for the individual treatment, i.e., approximately 30 to 60% suppression, so that synergistic or additive effects would be clearly apparent when tested together with other fungicidal treatments.
[0070] A series of experiments were designed based on the dose-response tests, and selection of fungicide concentrations. The list of experiments, single treatments and their rates of application (and treatment abbreviations) is provided in Table 4. All commercial fungicides were applied at two rates: high rate (hi) and low rate (lo); and, in one case, a medium rate (me) was also used. Multiple rates were applied because application rate can impact the type of interaction between two agents (Koppenhfer and Grewal. 2005. Compatibility and interactions with agrochemicals and other biocontrol agents. In: Nematodes as Biological Control Agents, Grewal et al. (Eds.), CABI Publishing, Wallingford, UK, pp. 363-381). Within each experiment, all of the fungicidal treatments (TCA, X. szentirmaii, strain 17C+E, and commercial fungicides) were applied individually. Moreover, TCA or the X. szentirmaii, strain 17C+E, supernatant was combined with each of the commercial fungicides at each concentration within an experiment. Two experiments with TCA and the commercial fungicides were run against M. fructicola (Experiments 1 & 2), one experiment with M. fructicola and X. szentirmaii, strain 17C+E (Experiment 3), and one experiment with TCA and R. solani (Experiment 4) (see Table 4). Within each experiment, there were nine replicate plates for each treatment, and each experiment was repeated once in time (hence two trials). Additionally, one trial was conducted to explore the combination of TCA with X. szentirmaii, strain 17C+E, as potential synergists of each other; this experiment also targeted M. fructicola (Experiment 5).
TABLE-US-00004 TABLE 4 List of experiments and treatments used to assess suppression of vegetative growth in fungal phytopathogens.sup.a Expt. # Pathogen + Treatment Treatment % Conc.sup.b % Field rate.sup.c Abbre.sup.d 1 M. fructicola + TCA Acetone 0.2 NA Ace ProPhyt 0.5 100 Prohi ProPhyt 0.25 50 Prolo Regalia 0.5 100 Reghi Regalia 1 50 Reglo Serenade 0.0003 0.02 Serhi Serenade 0.00015 0.01 Serlo TCA 0.2 NA TCA 2 M. fructicola + TCA Abound 0.01 0.05 Abohi Abound 0.001 0.005 Abolo Acetone 0.2 NA Ace Elast 0.0001 0.0005 Ehi Elast 0.00001 0.00005 Elo Propimax 0.0001 0.0005 Pxhi Propimax 0.00001 0.00005 Pxlo TCA 0.2 NA TCA 3 M. fructicola + X. szentermaii Abound 0.01 0.05 Abohi Abound 0.001 0.005 Abolo Elast 0.001 0.005 Ehi Elast 0.0001 0.0005 Eme Elast 0.00001 0.00005 Elo ProPhyt 0.5 100 Prohi ProPhyt 0.25 50 Prolo Propimax 0.001 0.005 Pxhi Propimax 0.0001 0.0005 Pxme Propimax 0.00001 0.00005 Pxlo Regalia 0.5 100 Reghi Regalia 1 50 Reglo Serenade 0.0003 0.02 Serhi Serenade 0.00015 0.01 Serlo TSYB 10 NA Tsy X. szentirmaii 10% NA Xs 4 Rhizoctonia solani + TCA Acetone 0.2 NA Ace Regalia 0.5 100 Reghi Regalia 1 50 Reglo Serenade 0.0003 0.02 Serhi Serenade 0.00015 0.01 Serlo TCA 0.2 NA TCA 5 M. fructicola with TCA 0.25 NA TCAhi TCA + X. szentirmaii, TCA 0.03 NA TCAlo strain 17C+E X. szentirmaii 5 NA Xshi X. szentirmaii 1 NA Xslo .sup.aPhytopathogens included Monilinia fructicola, or Rhizoctonia solani. Within each experiment, trans-Cinnamic-acid (TCA) or Xenorhabdus szentirmaii, strain 17C+E, was combined with each of the other treatments, and all fungicidal treatments were also applied alone. All treatments were incorporated into potato dextrose agar (PDA). Acetone (the solvent of TCA) was used as a control, and an untreated control (no amendment) was also included in all experiments. .sup.bPercentage (v/v) per 500 ml potato dextrose agar. Prior to incorporation, TCA was dissolved in acetone at 10 mg/ml. .sup.cPercentage (v/v) of recommended field rate. .sup.dAbbreviation used in the text and figures.
[0071] Treatment effects for experiments testing in vitro antimycotic activity (based on area of fungal growth) were analyzed using ANOVA. Data from repeated trials were pooled and trial was considered as a block effect. If a significant treatment effect was detected in the ANOVA (=0.05), a means separation was performed using Tukey's HSD test. The area of vegetative growth (cm.sup.2) was log transformed prior to analysis; non-transformed means are presented in the results.
[0072] The nature of interactions (antagonism, additivity, or synergy) between fungicidal agents was determined through a comparison of expected and observed fungal growth (Koppenhfer and Kaya. 1997. Biol. Control 8:131-137; Shapiro-Ilan et al. 2011. J. Econ. Entomol. 104:14-20). Corrected fungicidal suppression was calculated based on Abbott's formula, i.e., (growth in the controlgrowth in the treatment)/growth in the control (Abbott, W. S. 1925. J. Econ. Entomol. 18:265-267). Subsequently, the expected additive proportional suppression S.sub.E for combination treatments was calculated by S.sub.E=S.sub.C+S.sub.M (1S.sub.C), where S.sub.C and S.sub.M are proportional suppression caused by commercial fungicide and bacterial metabolites (TCA or X. szentirmaii, strain 17C+E) applied alone, respectively. A chi-square (.sup.2) value was calculated as (S.sub.CMS.sub.E)2/S.sub.E, where S.sub.CM is the observed suppression for the combination of commercial fungicide and bacterial metabolites. If the calculated value was >3.84 (as specified for 1 degrees of freedom (df)), a non-additive effect of the two control agents was indicated. A positive value for the difference S.sub.CMS.sub.E indicated synergy, whereas a negative value indicated antagonism.
[0073] In the first experiment with M. fructicola and TCA, differential in fungal suppression and interactions were observed among the treatments (F=31.27; df=14,240; P<0.0001) (
TABLE-US-00005 TABLE 5 List of chi-square values and interactions for combined fungicidal treatments..sup.a Treatment Expt. Pathogen + Treatment Combination.sup.b Chi-square Interaction 1 Monilinia fructicola + TCA TCA + Serenade-lo 0.753588252 Additive TCA + Serenade-hi 29.96186631 Synergistic TCA + Prophyte-hi 0.499013839 Additive TCA + Prophyte-lo 7.826168042 Synergistic TCA + Regalia-hi 4.120990928 Synergistic TCA + Regalia-lo 0.029198133 Additive 2 Monilinia fructicola + TCA TCA + Elast-hi 1.14034E05 Additive TCA + Elast-med 66.77938759 Synergistic TCA + Elast-lo 2.423059442 Additive TCA + PropiMax-hi 15.28139511 Antagonistic TCA + PropiMax-med 11.75188694 Antagonistic TCA + Propimax-lo 9.227071724 Synergistic TCA + Abound-hi 8.879275233 Antagonistic TCA + Abound-lo 5.620861631 Antagonistic 3 Monilinia fructicola + Xs Xs + Abound-hi 1.009025 Additive Xs + Abound-lo 3.946475 Synergistic Xs + Elast-hi 49.10311 Antagonistic Xs + Elast-lo 6.588946 Antagonistic Xs + PropiMax-hi 0.027503 Additive Xs + Propimax lo 2.458579 Additive 4 Rhizoctonia solani + TCA TCA + Regalia-lo 83.75610867 Synergistic TCA + Regalia-hi 0 Additive TCA + Serenade-lo 0.03692118 Additive TCA + Serenade-hi 10.59377153 Synergistic 5 M. fructicola with TCA + Xs-hi + TCA-hi 3.167351 Additive X. szentirmaii, strain 17C+E Xs-hi + TCA-lo 22.90543 Antagonistic Xs-lo + TCA-hi 80.04454 Synergistic Xs-lo + TCA-lo 21.200562 Antagonistic .sup.aChi-square values were generated by comparing observed and expected fungicidal activity of treatments applied alone or in combination; >3.84 indicates significance. .sup.blo = lower rate of application, hi = higher rate of application, see text and Table 4.
[0074] Similarly in the second experiment with M. fructicola and TCA, differences in fungal suppression and interactions among fungicidal agents were observed (F=1008.43; df=18,321; P<0.0001) (
[0075] In Experiment 3, treatment effects were observed when cell-free X. szentirmaii, strain 17C+E, supernatant and commercial fungicides were applied alone or in combination for suppression of M. fructicola (F=304.82; df=14,254; P<0.0001) (
[0076] Treatment differences and differential interactions were observed when TCA was combined with commercial fungicides against R. solani (Experiment 4, F=10752.9; df=10,186; P<0.0001) (
[0077] In Experiment 5, when TCA was applied with X. szentirmaii, strain 17C+E, only one of the four rate combinations synergistically suppressed M. fructicola (F=198.94; df=8, 72; P<0.0001) (
[0078] In conclusion, overall, the data indicated a broad spectrum of synergistic interactions among the fungicidal agents. Toward M. fructicola suppression, TCA was synergistic with concentrations of all six of the commercial fungicides tested except Abound. However, the cell-free supernatant of X. szentirmaii, strain 17C+E, was synergistic with Abound (though not synergistic with the other two fungicides tested). For suppression of R. solani, TCA was synergistic with both of the fungicides tested, Serenade and Regalia. Also, TCA was synergistic with X. szentirmaii, strain 17C+E, (for M. fructicola suppression) albeit at only one of the four combined concentrations. Thus, it is clear that an array of synergistic interactions can be achieved for fungicidal activity, yet the outcome is dependent on concentration of the fungicide. Therefore, concentrations to achieve maximum control of the targeted phytopathogens will have to be optimized specifically for each combination. The impact of concentration on level of interaction (synergy, additivity or antagonism) has been observed in other systems such as combinations of entomopathogens with each other or with chemical pesticides.
Example 4: Other Related Compounds to TCA Also Display Synergistic Effects
[0079] Following the procedures above and those in Hazir et al (2017b, J. Invertebrate Path. 145: 1-8), synergy assessments were implemented to compare trans-Cinnamic acid to an exemplary analog, trans-Cinnamaldehyde. The target phytopathogen was Monilinia fructicola (brown rot).
[0080] Treatment effects were compared by measuring vegetative growth of the phytopathogens in vitro when they were exposed to the fungicidal agents applied alone or in combination. Solutions of the fungicidal treatments were incorporated into potato dextrose agar (PDA) to achieve the desired concentrations, and autoclaved. Prior to autoclaving the PDA, the prescribed volume of distilled water was reduced to allow addition of the fungicide solutions. Controls consisted of PDA without any amendment or PDA with acetone only (the solvent used for TCA). Trans-Cinnamic acid and trans-Cinnamaldehyde were applied alone or with commercial fungicides Regalia (extract of Reynoutria sachalinensis) or Serenade (Bacillus subtilis). After 4 days the area of M. fructicola growth was determined.
[0081] A full list of treatments is displayed in Table 6 below.
TABLE-US-00006 TABLE 6 List of treatments comparing synergistic effects of trans- Cinnamic acid (TCA) and trans-Cinnamaldehyde (CA). Treatment #/Name Treatment type 1 control water 2 acetone PDA with .5 ml old acetone added to 499 mls 3 TCA 499.5 ml PDA + 0.5 ml TCA (500 mg TCA in 50 ml Acetone) 4 CA 499.5 ml PDA + 0.5 ml CA (500 mg CA in 50 ml Acetone) 5 Regalia lo PDA with 5 ml added to 495 mls 6 Regalia hi PDA with 2.5 ml Regalia added to 497.5 mls 7 Serenade hi PDA with .016 serenade added to 499.984 mls 8 Serenade lo PDA with.008 serenade added to 499.992 mls 9 TCA-Serenade hi PDA with .5 ml TCA and .016 serenade to 499.484 mls 10 TCA-Serenade lo PDA with .5 ml TCA and .008 serenade to 499.492 mls 11 TCA -Regalia hi PDA with .5 ml TCA and 5 ml regalia to 494.5 mls 12 TCA-Regalia lo PDA with .5 ml TCA and 2.5 ml regalia to 497 mls 13 CA-Serenade hi PDA with .5 ml CA and .016 serenade to 499.484 mls 14 CA-Serenade lo PDA with .5 ml CA and .008 serenade to 499.492 mls 15 CA -Regalia hi PDA with .5 ml CA and 5 ml regalia to 494.5 mls 16 CA-Regalia lo PDA with .5 ml CA and 2.5 ml regalia to 497 mls
[0082]
Example 5: Synergistic Suppression Between TCA and Other Fungicides Targeting Additional Fungi
[0083] A. Solani.
[0084] Following the above procedures, interactions between trans-Cinnamic acid (TCA) and commercial fungicides azoxystrobin (Abound), dodine (Elast), and triphenyltin hydroxide (Super Tin) were evaluated for suppression of A. solani, a major pathogen of tomatoes and potatoes.
[0085]
[0086] P. capsici.
[0087] Following the same bioassay procedures as described above, interactions between TCA and fungicides Prophyte, Regalia and Serenade were evaluated.
[0088]
[0089] The foregoing description and accompanying figures illustrate the principles, preferred embodiments and modes of operation of the invention. However, the invention should not be construed as being limited to the particular embodiments discussed above. Additional variations of the embodiments discussed above will be appreciated by those skilled in the art.
[0090] Therefore, the above-described embodiments should be regarded as illustrative rather than restrictive. Accordingly, it should be appreciated that variations to those embodiments can be made by those skilled in the art without departing from the scope of the invention as defined by the following claims.