METHOD OF CONTROLLING FUNGI

Abstract

A method of controlling or preventing infestation of plants by fungi, wherein a fungicidally effective amount of cyclothiazomycin C, is applied to the plants, to parts thereof or the locus thereof.

Claims

1. A method of controlling or preventing infestation of plants by fungi, wherein a fungicidally effective amount of cyclothiazomycin C, is applied to the plants, to parts thereof or the locus thereof.

2. A method as claimed in claim 1 in which the plants are cereals, groundnut or soybean plants.

3. A method as claimed in claim 1 in which the plants are wheat.

4. A method as claimed in claim 1 in which the fungi are selected from the group consisting of Botrytis cinerea, Cercospora kikuchii, Cercospora sojina, Cochlobolus sativus, Colletotrichum lindemuthianum, Colletotrichum orbiculare, Corynespora cassiicola, Fusarium avenaceum, Fusarium culmorum, Fusarium langsethiae, Fusarium poae, Fusarium sporotrichioides, Fusarium tricinctum, Fusarium virguliforme, Gibberella avenacea, Gibberella fujikuroi, Gibberella zeae, Microdochium majus, Monographella nivalis, Mycosphaerella arachidis, Phakopsora pachyrhizi, Puccinia triticina, Pyrenophora tritici-repentis, Ramularia collo cygni, Rhynchosporium secalis, Septoria glycines, Tilletia tritici, Ustilago segetum var. Tritici, Venturia inaequalis, and Zymoseptoria tritici.

5. A method as claimed in claim 1 in which the fungi are selected from the group consisting of Botrytis cinerea, Fusarium virguliforme, Monographella nivalis, Mycosphaerella arachidis, Phakopsora pachyrhizi, Puccinia triticina or Zymoseptoria tritici.

6. A method as claimed claim 1 in which cyclothiazomycin C is applied to the plants at a rate of 5 g to 6 kg per hectare.

7. An agrochemical composition comprising a fungicidally effective amount of cyclothiazomycin C.

8. An agrochemical composition according to claim 7 which further comprises an agrochemically-acceptable diluent or carrier.

9. A method of controlling or preventing infestation of plants by fungi, wherein a fungicidally effective amount of an agrochemical composition according to claim 6, is applied to the plants, to parts thereof or the locus thereof.

10. The use of cyclothiazomycin C as an anti fungal agent or as a fungicide.

11. The use of cyclothiazomycin C as an anti fungal agent in food, feed, beverages or in cosmetic products.

12. The use according to claim 11, wherein the food is chosen from dairy products or baking products.

13. A method of controlling or preventing infestation of plants by fungi, wherein a fungicidally effective amount of an agrochemical composition according to claim 7, is applied to the plants, to parts thereof or the locus thereof.

Description

EXAMPLES

[0081] The Examples which follow serve to illustrate the invention.

[0082] Throughout this description, temperatures are given in degrees Celsius (° C.) and “mp.” means melting point and rh means relative humidity. LC/MS means Liquid Chromatography Mass Spectrometry and the description of the apparatus and the method is as follows:

Example 1: Isolation of Cyclothiazomycin C from NRRL WC-9803

[0083] NRRL strain WC-3908 obtained from the NRRL culture collection and was cultured in a 100 mL baffled shake flask at 225 rpm in an Innova 44 shaker incubator, stroke 1 inch. The flask contained 25 mL of medium with 10 g/L Merck casein hydrolysate, 8 g/L Difco Bacto tryptone, 2 g/L Difco Bacto Soytone, 1.25 g/L K.sub.2HPO.sub.4, and 0.3 g/L Basildon antifoam. The flask was closed with a foam plug. Prior to autoclaving, the pH was adjusted to 6.8 with 4 N H.sub.2SO.sub.4. From a separately autoclaved 500 g/L glucose.H.sub.2O stock, 15 g/L glucose.H.sub.2O was added. Inoculation was with biomass or spores and incubation at 28° C. was continued until glucose was between 1 and 5 g/L, requiring approx. 2 to 3 days.

[0084] This broth was spotted directly on bioassay plates in 7 μl droplets. Furthermore, extracts were made by freeze drying 1 mL and extraction with 1 mL 80% (w/v) acetonitrile. Of the extracts, 5 μl was spotted. As negative controls, sterile medium was used in the case of the broth containing living cells, or 80% acetonitrile in the case of the extract.

Example 2: Plate Bioassay Against Botrytis cinerea and Zymoseptoria tritici

Preparation of the Bioassay Plates.

[0085] The medium for the bioassay plates was prepared by mixing equal volumes of Difco Plate Count Agar and Difco Potato Dextrose Agar. 40 mL was used in Nunc™ OmniTray™ Single-Well Plates. After solidification and cooling to 20° C., 10 mL top-layer was applied containing equal amounts of sterile water and Difco Potato Dextrose Agar, at 42° C. Just before pouring the top-layer, spores of Fusarium culmorum, Botrytis cinerea or Zymoseptoria tritici were added. The spore concentrations used were 1000 cfu/mL for B. cinerea, and 20,000 cfu/mL for Z. tritici. After pouring the top-layers, the bioassay plates were dried in laminar flow cabinet for 1 hour and used immediately. After applying the samples, the plates were incubated at 22° C. until the fungi allowed visual assessment of the zone of inhibition.

[0086] The broth of example 1 was added to the bioassay plates as 7 μl droplets.

[0087] An example of the zones of inhibition found is given in FIG. 1A showing a zone of inhibition caused by a whole broth sample of NRRL strain WC-3908 on a Botrytis cinerea bioassay plate.

[0088] An example of the zones of inhibition found is given in FIG. 1B showing a zone of inhibition caused by a whole broth sample of NRRL strain WC-3908 on a Zymoseptoria tritici bioassay plate.

[0089] An example of the found zones of inhibition is given in FIG. 1C showing a zone of inhibition caused by a whole broth sample of NRRL strain WC-3908 on a Fusarium culmorum bioassay plate.

Example 3: Efficacy of Purified CtmC on Fusarium Virguliforme

[0090] A Fusarium virguliforme spore suspension of 25′000 spores/ml was produced in PDB (potato dextrose broth) medium supplemented with 0.3% agar. Isolates of the species originate from Syngenta internal collection (CH).

[0091] CtmC was dissolved in DMSO (Dimethylsulfoxid) to a final concentration of 1000 ppm to create a stock solution. Different dilutions of CtmC were created in DMSO: 1000 ppm, 330 ppm, 110 ppm, 37 ppm, 12 ppm and 4.1 ppm. 10 μl of DMSO or CtmC solution were transferred to a 96-well-dilution plate and diluted 10-fold with 90 μl 0.025% Tween 20/H2O solution. Out of the dilution plate, 10 μl were transferred to a 96 well assay plate and 90 μl spore suspension were added to each well.

Wells of 96 well plates contain the following:

TABLE-US-00001 TABLE 1 medium (ul) + 0.0025% DMSO CtmC well Tween/H2O (ul) (ppm) spores A 90 + 9 1 0 no B 90 + 9 1 10 yes C 90 + 9 1 3.3 yes D 90 + 9 1 1.1 yes E 90 + 9 1 0.37 yes F 90 + 9 1 0.12 yes G 90 + 9 1 0.04 yes H 90 + 9 1 0 Yes

[0092] 96 Well plates were then incubated for 72 h at 24° C., 90% relative humidity in the dark. Plates were evaluated by reading the OD at 620 nm.

Assays were run on two independent isolates for the species. Each isolate was tested twice (assay 1 and assay 2) and each isolate×fungicide rate combination was duplicated on the test plates.
The OD620 was averaged over the two wells and used to calculate IC50 values (fungicide concentration resulting in 50% inhibition of growth) with the program GraphPad prism, resulting in 4 independent IC50 values. No significant differences were found between both isolates tested:

TABLE-US-00002 TABLE 2 species and IC50 (ppm) Assay-1 Assay-2 average Fusarium virguliforme isolate 1 0.8 1.6 1.2 Fusarium virguliforme isolate 2 0.7 1.5 1.1
As shown in table 2 above, both tested isolates have a similar IC50 of ca. 1 ppm.

Example 4: Efficacy of Purified CtmC on Fungal Species In-Vitro

[0093] CtmC was dissolved in DMSO (Dimethylsulfoxid) to a final concentration of 1000 ppm to create a stock solution. A second dilutions of 1:10 was made in water+0.025% Tween 20. From this second dilution 10 ul are dispensed into a 96 well plate. To each well, 90 ul of medium with fungal spores are added and mixed, resulting in final CtmC concentrations of 10 ppm. All wells containing CtmC also contain DMSO (1%) and Tween 20 (0.0025%).

Botrytis cinerea (gray mould): Conidia of the fungus from cryogenic storage were directly mixed into nutrient broth (PDB potato dextrose broth). After placing a solution of the test compounds into a microtiter plate (96-well format) the nutrient broth containing the fungal spores was added. The test plates were incubated at 24° C. and the inhibition of growth was determined photometrically after 72 hrs.
Monographella nivalis (snow mould, foot rot of cereals): Conidia of the fungus from cryogenic storage were directly mixed into nutrient broth (PDB potato dextrose broth). After placing a solution of the test compounds into a microtiter plate (96-well format) the nutrient broth containing the fungal spores was added. The test plates were incubated at 24° C. and the inhibition of growth was determined photometrically after 72 hrs at 620 nm.
Mycosphaerella arachidis (Brown leaf spot of groundnut): Conidia of the fungus from cryogenic storage were directly mixed into nutrient broth (PDB potato dextrose broth). After placing a solution of the test compounds into a microtiter plate (96-well format) the nutrient broth containing the fungal spores was added. The test plates were incubated at 24° C. and the inhibition of growth was determined photometrically after approximately 5-6 days at 620 nm.
Zymoseptoria tritici (Septoria leaf blotch): Conidia of the fungus from cryogenic storage were directly mixed into nutrient broth (PDB potato dextrose broth). After placing a solution of the test compounds into a microtiter plate (96-well format) the nutrient broth containing the fungal spores was added. The test plates were incubated at 24° C. and the inhibition of growth was determined photometrically after 72 hrs.

TABLE-US-00003 compounds and control Botrytis Monographella Mycosphaerella Zymoseptoria of fungal growth (%) rate cinerea nivalis arachidis tritici Cyclothiazomycin C 10 ppm 90 100 100 90 Water — 0 0 0 0
Conidial spores of 4 species tested were prevented from growth in presence of 10 ppm of Cyclothiazomycin C (reduction by 90-100%).

Example 5: Efficacy of Purified CtmC on Fungal Species in a Leaf Disc Assay

[0094] A CtmC stock was generated at 1000 ppm in DMSO, then Diluted 1:50 in water+Tween 20 at 0.025% resulting in a 20 ppm solution.

Puccinia triticina (also known as recondite, Brown rust, wheat): Wheat leaf segments are placed on agar in multiwell plates (24-well format) and sprayed with test solutions. After drying, the leaf disks are inoculated with a spore suspension of the fungus. After appropriate incubation the activity of a compound is assessed 8 dpi (days after inoculation) as preventive fungicidal activity.
Phakopsora pachyrhizi (Asian Soybean rust): Soybean leaf disks are placed on agar in multiwell plates (24-well format) and sprayed with test solutions. After drying, the leaf disks are inoculated with a spore suspension of the fungus. After appropriate incubation the activity of a compound is assessed approx. 12 dpi (days after inoculation) as preventive fungicidal activity

TABLE-US-00004 compound and control Puccinia Phakopsora of fungal growth (%) rate triticina pachyrhizi Cyclothiazomycin C 20 ppm 90 50 Water — 0 0
CtmC at 20 ppm was able to reduce development of two tested rust species by 50-90%.

Example 6: Efficacy of Purified CtmC on Fungal Species in the Greenhouse

[0095] Purified Cyclothiazomycin C (CtmC) was formulated as an EC50 formulation. The formulation was diluted in water at rates of 100 ppm and 50 ppm of CtmC. Plants were treated with 400 L/ha of the diluted product supplemented with an adjuvant to improve sticking and spreading, resulting in a treatment of 40 g/ha and 20 g/ha of CtmC, respectively.

Zymoseptoria tritici wheat/preventative (Septoria tritici leaf spot on wheat). 2-week old wheat plants cv. Riband are sprayed in a spray chamber with the formulated test compound diluted in water. The test plants are inoculated by spraying a spore suspension on them one day after application. After an incubation period of 1 day at 22° C./21° C. (day/night) and 95% rh, the inoculated test plants are kept at 22° C./21° C. (day/night) and 70% rh in a greenhouse. Efficacy is assessed directly when an appropriate level of disease appears on untreated check plants (16-19 days after application).
Puccinia triticina (also known as recondata, Brown rust on wheat). 2-week old wheat plants cv. Arina are sprayed in a spray chamber with the formulated test compound diluted in water. The test plants are inoculated by spraying them with a spore suspension one day after application. After an incubation period of 1 day at 20° C. and 95% rh, the inoculated test plants are kept at 20° C./18° C. (day/night) and 60% rh in a greenhouse. The percentage leaf area covered by disease is assessed when an appropriate level of disease appears on untreated check plants (12-14 days after application).
Botrytis cinerea tomato/preventative (Botrytis on tomato) 4-week old tomato plants
cv. Roter Gnom are treated in a spray chamber with the formulated test compound diluted in water. The test plants are inoculated by spraying them with a spore suspension two days after application. The inoculated test plants are incubated at 20° C. and 95% rh in a greenhouse and the percentage leaf area covered by disease is assessed when an appropriate level of disease appears on untreated check plants (5-6 days after application).
Mycosphaerella arachidis peanut/preventative (Brown leaf spot on groundnut) 3-week old peanut plants cv. Georgia Green are sprayed in a spray chamber with the formulated test compound diluted in water. The test plants are inoculated by spraying them with a spore suspension on their lower leaf surface one day after application. After an incubation period of 4 days under a plastic hood at 23° C. and 100% rh, the inoculated test plants are kept at 23° C./20° C. (day/night) and 70% rh in a greenhouse. The percentage leaf area covered by disease is assessed when an appropriate level of disease appears on untreated check plants (12-14 days after application).

TABLE-US-00005 efficacy efficacy 40 g/ha 20 g/ha Disease crop application timing (%) (%) Zymoseptoria tritici wheat 1 day preventative 16 44 Puccinia triticina wheat 2 days preventative 81 81 Botrytis cinerea tomato 2 days preventative 47 30 Mycosphaerella groundnut 1 day preventative 65 56 arachidis
Results indicate a m is active as a fungicide to reduce disease seventy on plants in the greenhouse.

Example 7: The Effect of Tween 20 on the Efficacy of Purified CtmC on Fusarium virguliforme

[0096] Prior art WO2015191789 clearly states that the inventors found no fungal inhibitory action for Cyclothiazomycin C. This is contrary to our results. In order to eliminate the possibility that the fungicidal effect that we observe was down to the presence of Tween 20 we carried out some experiments like those in Example 3 but with various levels of Tween 20, including none.
The experiment at outlined in Example 3 was repeated to evaluate the EC50 values for CtmC control on Fusarium virguliforme in presence of 4 different rates of Tween20: 0% (absence of Tween 20), 0.0025%, 0.0050% (identical to Example 3) and 0.0100%. The plate layout was identical to Example 3, except that the plates were multiplied 4 times to accommodate the different Tween 20 concentrations in the assay plate. All other experimental conditions were kept identical. Fusarium isolates were tested at 25′000 sp/ml.

TABLE-US-00006 TABLE 3 C50 calculation for 2 fungal isolates in presence of different concentrations of Tween20, in ppm. Values are an average over all replica treatments in the experiment. Tween 20 CtmC; EC50 (ppm) 0% 0.0025% 0.0050% 0.0100% Fusarium virguliforme 1.422 1.053 0.8103 1.436 isolate 1 Fusarium virguliforme 1.098 0.889 0.7844 1.035 isolate 2 The results are consistent with Example 3 and indicate that Tween 20 has no significant influence on the EC50 value for Fusarium virguliforme control in-vitro.

Example 8: Efficacy of Purified CtmC on Further Extended List of Fungal Species In-Vitro

[0097] CtmC was dissolved in DMSO (Dimethylsulfoxid) to a final concentration of 1000 ppm to create a stock solution. A second dilutions of 1:10 was made in water+0.025% Tween 20. From this second dilution 10 ul are dispensed into a 96 well plate. To each well, 90 ul of medium (PDB—potato dextrose broth, plus 0.3% agar) with fungal spores are added and mixed, resulting in final CtmC concentrations of 10 ppm. All wells containing CtmC also contain DMSO (1%) and Tween 20 (0.0025%). In the following table 4 some of the species were tested using several distinct isolates (internal strain number indicated) to understand variability among different isolates of the same species sampled in different locations, producing different mycotoxins (genus Fusarium), or expressing various tolerances to fungicides of the SDHI (Succinate Dehydrogenase Inhibitor), SBI (Sterol Biosynthesis Inhibitors) and/or QoI (quinone outside inhibitors) classes (example: Zymoseptoria tritici). Different isolates had variable inoculum density and number of biological replicas, as indicated.

TABLE-US-00007 TABLE 4 Disease control efficacy calculation for additional fungal isolates in presence of different concentrations of Tween20, in ppm. Values are an average overall replica treatments in the experiment. spore optical density at 620 nm efficacy (%) density CtmC CtmC species isolate (per ml) reps blank untreated 10 ppm 10 ppm Cercospora kikuchii K6080 25,000 4 0.044 0.215 0.043 100 Cercospora sojina K5703 25,000 4 0.047 0.357 0.188 54 Cochliobolus sativus K5817 30,000 4 0.044 0.701 0.095 92 Colletotrichum lindemuthianum K5893 25,000 4 0.046 0.373 0.225 45 Colletotrichum orbiculare K5770 25,000 8 0.035 0.449 0.218 56 Corynespora cassiicola K5577 25,000 4 0.047 0.427 0.100 86 Fusarium avenaceum K6940 25,000 3 0.054 0.410 0.225 52 Fusarium culmorum K5488 25,000 4 0.046 0.374 0.203 52 Fusarium culmorum K6937 15,000 3 0.052 0.528 0.319 44 Fusarium culmorum K8196 15,000 3 0.055 0.701 0.388 48 Fusarium langsethiae K5670 25,000 3 0.053 0.346 0.072 94 Fusarium poae K8039 25,000 3 0.050 0.619 0.267 62 Fusarium sporotrichioides K7901 25,000 3 0.052 0.666 0.231 71 Fusarium tricinctum K7452 25,000 3 0.052 0.498 0.197 68 Fusarium tricinctum K7454 20,000 4 0.050 0.463 0.195 65 Gibberella avenacea K6939 25,000 4 0.055 0.675 0.165 82 Gibberella fujikuroi K5299 25,000 3 0.053 0.941 0.528 47 Gibberella zeae K6102 25,000 4 0.044 0.733 0.377 52 Gibberella zeae K6934 25,000 3 0.052 0.641 0.361 48 Microdochium majus K7482 25,000 3 0.050 0.228 0.057 96 Monographella nivalis K7484 25,000 3 0.054 0.233 0.073 90 Pyrenophora tritici-repentis K6186 mycelium 4 0.046 0.508 0.094 90 Ramularia collo-cygni K6218 60,000 4 0.043 0.231 0.119 60 Rhynchosporium secalis K5917 180,000 4 0.046 0.378 0.062 95 Septoria glycines K5204 25,000 4 0.044 0.414 0.115 81 Tilletia tritici K5212 30,000 4 0.046 0.451 0.062 96 Ustilago segetum var. tritici K5349 30,000 4 0.044 0.304 0.051 97 Venturia inaegualis K6222 120,000 4 0.044 0.320 0.082 86 Zymoseptoria tritici K6105 100,000 20 0.044 0.354 0.046 99 Zymoseptoria tritici K6318 100,000 4 0.054 0.253 0.059 98 Zymoseptoria tritici K6420 100,000 4 0.048 0.257 0.064 93 Zymoseptoria tritici K7953 100,000 4 0.055 0.235 0.096 77
The results indicate a large variety of fungal species can at least partially be controlled, and that several isolates of the same species have comparable sensitivity to CtmC.