Use of Volatile Organic Compounds as Pesticides

Abstract

The present invention relates to the use of 1-octen-3-ol, 3-octanone, isoamyl formate, or a mixture thereof, as a nematicide.

Claims

1. Use of 3-octanone, 1-octen-3-ol, isoamyl formate, or a mixture thereof, as a nematicide.

2. The use according to claim 1, wherein 1-octen-3-ol is the nematicide.

3. The use according to claim 1, wherein 3-octanone is the nematicide.

4. The use according to claim 1, wherein isoamyl formate is the nematicide.

5. The use according to claim 1, wherein said 1-octen-3-ol, 3-octanone isoamyl formate, or mixture thereof is used separately, sequentially and/or in combination with a further pesticide.

6. The use according to claim 5, wherein said further pesticide is a further nematicide.

7. The use according to claim 6, wherein said further nematicide is selected from methyl bromide, 1,3-dichloropropene, ethylene dibromide, metam-sodium, dazomet, methyl isothiocyanate, chloropicrin, thionazin, ethoprophos, fenamiphos, fensulfothion, terbufos, isazofos, ebufos, aldicarb, aldoxycarb, oxamyl, carbofuran, cleothocarb and mixtures thereof.

8. The use according to claim 1, wherein said 1-octen-3-ol, 3-octanone, isoamyl formate, or mixture thereof is used in the form of a composition, and said composition comprises one or more additional ingredients.

9. The use according to claim 8, wherein said composition is a solid composition.

10. The use according to claim 9, wherein said sold composition is in the form of pellets, granules (e.g. emulsifiable granules, water dispersible granules or granules for broadcast application), powders (e.g. wettable powders, soluble powders), briquettes, blocks, dusts or mixtures thereof.

11. The use according to claim 8, wherein said composition is a liquid composition.

12. The use according to claim 8, wherein said composition is a slurry composition.

13. The use according to claim 12, wherein said slurry composition is applied to seeds.

14. The use according to claim 1, wherein said 1-octen-3-ol, 3-octanone, isoamyl formate, or mixture thereof is a nematicide in a fumigant.

15. The use according to claim 1 to control plant parasitic nematodes.

16. (canceled)

17. The use according to claim 1 to treat soil, seeds, bulbs, crops, tubers, trees, stored produce or timber.

18-23. (canceled)

24. The use according to claim 1, wherein said 1-octen-3-ol, 3-octanone, isoamyl formate, or mixture thereof is applied to an area to be treated by band application, basal application, broadcast application, crack and crevice application, directed-spray application, foliar application, soil application, soil incorporation, soil injection, rope-wick treatment, wiper treatment, space treatment, spot treatment, tree injection, spraying and/or fumigation.

25. The use according to claim 1 as a nematicide in a fumigation process.

26. A method of controlling nematodes, comprising applying an effective amount of 3-octanone, 1-octen-3-ol, isoamyl formate, or a mixture thereof, to an area requiring nematode control.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] These and further aspects of the invention will now be further described, by way of example only, with reference to the accompanying figures in which:

[0050] FIG. 1 is a schematic diagram showing the experimental set up used in the studies described in the Examples below. FIG. 1A is a cross-section of the experimental arena; FIG. 1B is a plan view of the sealed arena, indicating the circles (central, inner and outer) used for the transect (solid line) and a visual representation of the expected VOC concentration gradient (dashed lines);

[0051] FIG. 2 is a series of graphs showing the percentage mortality of EPNs exposed to VOCs at 5, 10, 15 and 20 l, 3 hours post treatment. 0 l results are the results for the control group (no VOC). Boxes denote interquartile range, bisected horizontally by median values; whiskers extend to 1.5interquartile range beyond boxes; outliers are marked as dots beyond whiskers. The graph marked a) (top row, left hand side) shows the results for the EPN S. feltiae (Sf) treated with 3-octanone. The graph marked b) (top row, middle) shows the results for Sf treated with 1-octen-3-ol. The graph marked c) (top row, right hand side) shows the results for Sf treated with 1-octene. The graph marked e) (middle row, left hand side) shows the results for the EPN H. bacteriophora (Hb) treated with 3-octanone. The graph marked f) (middle row, middle) shows the results for Hb treated with 1-octen-3-ol. The graph marked g) (middle row, right hand side) shows the results for Hb treated with 1-octene. The graph marked i) (bottom row, left hand side) shows the results for the EPN S. carpocapsae (Sc), treated with 3-octanone. The graph marked j) (bottom row, middle) shows the results for Sc treated with 1-octen-3-ol. The graph marked k) (bottom row, right hand side) shows the results for Sc treated with 1-octene;

[0052] FIG. 3 is a series of graphs showing the percentage mortality of EPNs exposed to VOCs at 5, 10, 15 and 20 l, 6 hours post treatment. 0 l results are the results for the control group (no VOC). Boxes denote interquartile range, bisected horizontally by median values; whiskers extend to 1.5interquartile range beyond boxes; outliers are marked as dots beyond whiskers. The ordering of the graphs, and the EPN/VOC combinations the results of which are depicted in the graphs, are as described for FIG. 2 above;

[0053] FIG. 4 is a series of graphs showing the percentage mortality of EPNs exposed to VOCs at 5, 10, 15 and 20 l, 12 hours post treatment. 0 l results are the results for the control group (no VOC). Boxes denote interquartile range, bisected horizontally by median values; whiskers extend to 1.5interquartile range beyond boxes; outliers are marked as dots beyond whiskers. The ordering of the graphs, and the EPN/VOC combinations the results of which are depicted in the graphs, are as described for FIG. 2 above;

[0054] FIG. 5 is a series of graphs showing the percentage mortality of EPNs exposed to VOCs at 5, 10, 15 and 20 l, 24 hours post treatment. 0 l results are the results for the control group (no VOC). Boxes denote interquartile range, bisected horizontally by median values; whiskers extend to 1.5interquartile range beyond boxes; outliers are marked as dots beyond whiskers. The ordering of the graphs, and the EPN/VOC combinations the results of which are depicted in the graphs, are as described for FIG. 2 above;

[0055] FIG. 6 is a series of graphs showing the percentage mortality of the root knot nematode M. hapla exposed to VOCs at 5 and 10 l, 3, 6 and 24 hours post treatment. 0 l results are the results for the control group (no VOC). Boxes denote interquartile range, bisected horizontally by median values; whiskers extend to 1.5interquartile range beyond boxes; outliers are marked as dots beyond whiskers. The graph marked a) (top row, left hand side) shows the results for M. hapla treated with 3-octanone 3 hours post treatment. The graph marked b) (top row, middle) shows the results for M. hapla treated with 3-octanone 6 hours post treatment. The graph marked c) (top row, right hand side) shows the results for M. hapla treated with 3-octanone 24 hours post treatment. The graph marked d) (bottom row, left hand side) shows the results for M. hapla treated with 1-octen-3-ol 3 hours post treatment. The graph marked e) (bottom row, middle) shows the results for M. hapla treated with 1-octen-3-ol 6 hours post treatment. The graph marked f) (bottom row, right hand side) shows the results for M. hapla treated with 1-octen-3-ol 24 hours post treatment.

[0056] FIG. 7a shows the decrease in adult and juvenile population of parasitic plant nematodes versus the concentration of a composition comprising 1-octen-3-ol and microcrystalline cellulose. The % decrease in concentration was measured one day (blue bar) and five days (orange bars) post treatment in the top (A), middle (B) and bottom (C) layers of soil.

[0057] FIG. 7b shows the decrease in eggs from parasitic plant nematodes versus the concentration of a composition comprising 1-octen-3-ol and microcrystalline cellulose. The % decrease in concentration was measured one day (blue bar) and five days (orange bars) post treatment in the top (A), middle (B) and bottom (C) layers of soil.

EXAMPLES

Analysis Methods

[0058] All statistical analyses were carried out using RStudio statistics package (RStudio, Inc. version 1.0.153), using MASS and multcomp packages. For all mortality data, differential sensitivity between EPN species and/or insect species and differential compound potency were determined using a generalised linear model (GLM) and Tukey's range test in post-hoc analysis. LD.sub.50 values were calculated using a GLM and the dose.p function within the MASS package. An ANOVA was carried out on the chemotactic responses of EPNs to VOCs, with the strength of response compared at each time and concentration. Welch's t-tests were used to compare means of mortality results in the final EPNs efficacy assay.

Extraction of VOCs from Metarhizium brunneum Metarhizium brunneum isolates V275 (Origin: Cydia pomonella, Austria) were maintained on Sabouraud Dextrose Agar, pH 5.6 (SDA) and the conidia from these cultures used in subsequent studies. VOCs were collected from M. brunneum cultures produced on several substrates including: (1) Osmotic Stress Medium (OSM, 8% glucose, 2% peptone, 5.5% agar, 5.5% KCl), (2) High C:N (75:1) medium (HCN, 9.1% glucose, 1% peptone, 2% agar), (3) Intermediate C:N (35:1) medium (ICN, 4% glucose, 1% peptone, 2% agar), and (4) low C:N (10:1) medium (LCN, 0.6% glucose, 1% peptone, 2% agar). VOCs were collected at two time points: 7 days and 14 days post-inoculation, which correspond with the mycelial and sporulating stages of the fungus. All media were obtained from Sigma Aldrich (Poole, UK) except peptone, which was obtained from Oxoid Ltd. Cultures were produced on 10 ml medium in 25 ml glass vials incubated in the dark at 25 C. There were three replicates per treatment (isolate, culture medium) with the whole experiment being repeated twice.

[0059] Headspace VOCs were collected from the above treatments using a 50/30 mm Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) solid phase microextraction (SPME) fibre (Supelco, Bellefonte, Pa., USA) and analyzed using an Agilent 6890N Gas Chromatograph equipped with an HP-5MS fused capillary column (30 m0.25 mm0.25 m film thickness), interfaced directly with an Agilent 5975 mass spectrometer. Helium was used as the carrier gas with a constant flow of 1.0 ml/min. Immediately after collection of VOCs, the SPME needle was inserted manually into the injection port (230 C.; splitless mode) of the GC-MS for thermal desorption and held for 2 minutes. After desorption, the oven was held at 40 C. for 2 min, then the temperature raised to 200 C. at a rate of 3 C./min. Finally, the temperature was raised to 270 C. at a rate of 8 C./min and held at 270 C. for 10 min. Mass spectra were scanned repeatedly over 35-650 amu. Ionization was performed in electron impact (EI) mode at 70 eV. A blank run was performed after each analysis in order to confirm that no residual compound was polluting the fibre or column. Total ion current (TIC) chromatograms were integrated without any correction for co-elution and results were expressed as percent of the total peak area. All peaks were identified from their mass spectra by comparison with spectra in Wiley Registry (9.sup.th edition) and NIST11 (National Institute of Standards and Technology, Gaithersburg, Md., USA) libraries. Identifications were confirmed by comparing the retention time (R.sub.1), molecular ions, and fragmentation pattern with authentic standard samples. The peaks observed in the control (blank media without fungus) were excluded from the samples during the sample analysis. All reference compounds used for identification were purchased from Sigma Aldrich (Poole, UK).

[0060] The results are shown in Table 1 below.

TABLE-US-00001 TABLE 1 VOCs produced by M. brunneum V275 on different substrates. The values are percentage values and are proportional to the compound with the highest peak (100%) in the chromatogram. Values are means of 5 replicates. indicates the VOC was not observed. Tr. indicates a trace amount of the VOC was observed. V275 (After 7 days) V275 (After 14 days) Compound R.sub.t (min) MW OSM HCN ICN LCN OSM HCN ICN LCN Ethyl acetate 88 43.04 37.95 16.42 Acetic acid 60 100 85.76 3-hydroxy-2-butanone 2.015 88 49.38 33.58 32.25 57.9 73.73 100 Isoamyl alcohol 2.075 88 2.30 20.44 100 100 Isoamyl formate 2.770 116 4.88 Methyl isovalerate 2.945 116 2.01 1-Octene 3.520 112 11.13 2.67 10.02 3.89 2,3-butanediol 4.168 90 Tr. Tr. 43.43 44.49 4.16 (R or S isomer) 2,3-butanediol 4.479 90 100 65.96 100 100 19.48 (R or S isomer) 1,3-Octadiene 4.662 110 56.20 14.92 88.05 34.14 Isovaleric acid 4.667 102 8.46 20.40 Isopentyl acetate 4.708 130 10.05 9.40 7.5 1-Octene-3-ol 7.099 128 100 12.39 32.89 18.03 (R,S isomer) 3-Octanone 7.101 128 20.53 Methyl 2- 11.636 158 8.40 11.1 12.93 29.68 1.79 1.40 5.85 ethylhexanoate Methyl 3-hydroxy 11.869 158 5.87 cyclopentyl acetate 1-Undecene 14.090 154 9.28 56.61 3.66 19.25 2-Phenylethanol 15.780 122 6.52 Cedrene 16.454 204 14.2 Tr. 4.85 17.64 9.30 5.53 13.20

[0061] The results show that isoamyl formate, 1-octene-3-ol and 3-octanone are all produced by the V275 strain of M. brunneum on different agar media.

[0062] The VOCs used in the examples below were purchased from Sigma Aldrich.

Maintenance of Test Entomopathogenic Nematode (EPN) and Insects

[0063] Third instar infective juveniles (IJs) of the EPN Steinernema carpocapsae, S. feltiae and Heterorhabditis bacteriophora were kindly provided by BASF Ltd. (UK). The EPNs were stored at 4 C. until required. At least >90% viability was determined for the control in all the experiments. Nematodes that did not move even after prodding were considered dead. Late instar larvae of G. mellonella and yellow mealworm beetle (T. molitor) were obtained from Livefood UK Ltd. (Somerset, UK). Both were reared in 0.7 L glass jars at 28 C., 50-60% RH with a 12:12 light:dark cycle. Mealworm were fed bran while wax moth larvae were provided an artificial diet composed of 22.5% corn meal, 12.5% honey, 12.5% glycerol, 12.5% beeswax, 10% wheat flour, 12.5% milk solids, 5% yeast and 12.5% distilled water.

[0064] EPNs were used in Examples 1 and 2 because they were readily obtainable. It is expected that plant parasitic nematodes are at least as susceptible to the VOCs as the EPNs tested.

Example 1: Screening of 12 VOCs for Nematicidal Activity

[0065] Authenticated M. brunneum VOCs, along with other selected VOCs, were screened for nematicidal activity against S. feltiae and H. bacteriophora. 750 l of nematode suspension containing approximately 5000 Us in tap water, was spread uniformly over the water agar surface (2.6% w/v agar, 5 mM potassium phosphate pH 6, 1 mM CaCl.sub.2 and 1 mM MgSO.sub.4) of a Petri dish. Each Petri dish (90 mm) contained 20 ml of the water agar medium. The inoculated plates were kept in darkness for 3 hr at room temperature to acclimatize. The plates were then exposed to 20 l of the VOC dispensed from a 8 mm paper disc (Whatman, 0.34 mm thickness) positioned on a 2525 mm glass coverslip placed in the centre of the Petri dish lid (FIG. 1A). The plates were sealed with a double layer of Parafilm and were kept in the dark at 21 C. and checked after 24 hr. The number of live and dead nematodes were counted with the aid of a stereo binocular microscope (30).

[0066] The transect (FIG. 1B) allowed for recording of dead, debilitated and live EPN whose distribution correlated with VOC deposition. The deposition was predetermined using bacterial cultures highly sensitive to these VOCs. The edge of the circular zone of inhibition delineated the area of activity. The highest concentration of the VOC was in the centre (immediately beneath loaded filter paper) then gradually decreased to low levels at the outer zone. As a result, a transect was used to take into account any differences in compound concentration within the agar.

[0067] Mean of mortality was calculated from the total number of alive and dead individuals across all circles. There were five replicates per treatment and the whole experiment was repeated twice. The results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Screening of M. brunneum VOCs for nematicidal activities against two EPN species. Mortality (%) (Mean SE) Compound (purity) S. feltiae (Sf) H. bacteriophora (Hb) Isoamyl alcohol (99%) 20.8 3 9.7 2.1 Isoamyl formate (95%) 58 2.2 46.6 1.8 Methyl isovalerate (98%) 38.1 7 55 2.6 3-Octanone (98%) 100 0 100 0 (R)-(+)-Limonene (97%) 16.7 3.2 5.9 1.9 Isovaleric acid (99%) 16.1 3.8 13 2.8 1-Octene-3-ol (98%) 100 0 91.6 2.1 Farnesene 43 1.1 7.8 3.1 (mixture of isomers) 2,3-Butanediol (98%) 12.5 7.2 11.4 2.3 1-Octene (98%) 20.1 3 19.4 3.8 Undecane (99%) 25.4 5.6 7.5 2.7 Tridecane (99%) 32.3 5 4.1 1.6 Control (EPN only) 7.5 0.8 7.9 0.9

[0068] Of the 12 compounds tested, the M. brunneum VOCs 3-octanone and 1-octen-3-ol were highly toxic, causing 85-100% mortality (Table 2). 3-octanone caused 100% mortality in both EPN species while the M. brunneum VOC 1-octen-3-ol caused 100% and 91.62.1% mortality of S. feltiae and H. bacteriophora, respectively (Table 2). Isoamyl formate caused 58% and 46.61.8 mortality of S. feltiae and H. bacteriophora, respectively (Table 2).

Example 2: Dose Mortality Assay for the EPNs

[0069] Dose mortality assays were performed using 3-octanone and 1-octen-3-ol. The assays were performed as described in Example 1, except that the EPNs (S. carpocapsae (Sc), S. feltiae (Sf) and H. bacteriophora (Hb)) were exposed to different doses of the VOCs (5, 10, 15 and 20 l). The mortality was recorded as described previously at 3, 6, 12 and 24 hr post treatment. Controls included EPNs only (no VOC) and EPNs+1-octene. The results are shown in FIG. 2 (results 3 hr post treatment), 3 (results 6 hr post treatment), 4 (results 12 hr post treatment) & 5 (results 24 hr post treatment).

[0070] The EPNs exhibited somewhat differential sensitivity to the nematicidal compounds at shorter exposure times (FIG. 2, FIG. 3). For example, following 6 hr exposure time S. feltiae was most sensitive, with S. carpocapsae being the most tolerant (FIG. 3). However, mortality was very high for all three EPNs exposed to 3-octanone or 1-octen-3-ol after 24 hr (FIG. 5). By contrast, mortalities for the EPNs exposed to 1-octene were extremely low and remained low over time.

[0071] It was observed that the mortality was generally higher in the centre of the Petri dish and decreased with the distance from the centre.

[0072] For each species, an LD.sub.50 value was calculated at the most appropriate time point. The results are set out in Table 3 below.

TABLE-US-00003 TABLE 3 A summary of the LD.sub.50 values for the three nematode species exposed to each compound after an appropriate time period (SE). Where a = >99% mortality from 3 hr and b = mortality too low across all time periods to calculate an accurate LD.sub.50, therefore no nematicidal effect assumed. Species Compound Time (hr) LD.sub.50 (l) (SE) S. carpocapsae 1-octen-3-ol 24 41.01 1.06 S. feltiae 6 10.56 1.04 H. bacteriophora 6 21.72 1.05 S. carpocapsae 3-octanone 12 94.88 8.27 S. feltiae a a a H. bacteriophora 3 5.68 1.03 S. carpocapsae 1-octene b b b S. feltiae b b b H. bacteriophora b b b

[0073] LD.sub.50 values could not be obtained for EPN exposed to 1-octene because the mortality was too low or for S. feltiae exposed to 3-octanone because mortality was >99% at all time points. The LD.sub.50 of 3-octanone against H. bacteriophora was 5.68 l1.03 after 3 hr, but could not be calculated at later time points because the mortality exceeded 90%. The LD.sub.50 of H. bacteriophora exposed to 1-octen-3-ol was 21.72 l1.05 after 6 hr. For S. carpocapsae, the LD.sub.50 values of 3-octanone and 1-octen-3-ol were 94.88 l8.27 after 12 hr and 41.01 l1.06 after 24 hr, respectively. The LD.sub.50 values could not be calculated for S. carpocapsae exposed to 3-octanone after 24 hr because all nematodes were dead at all doses. For 1-octene, both Steinernema species showed low mortality with no statistical difference (Est.=0.33, s.e.=0.43, z=0.76, p<0.721). However, after 24 hr, the mortality was slightly higher for both species, but did not exceed 50% with no significant difference found between EPN species.

Example 3: Susceptibility of Meloidogyne hapla to VOCs

[0074] A study was conducted to determine the susceptibility of the root knot nematode M. hapla, a major crop pest, to 3-octanone and 1-octen-3-ol. Single egg-mass populations of M. hapla were kindly provided by Dr Ivan Grove (Harper Adams University/Crop and Environment Sciences). Four-week-old tomato plants (Solanum lycopersicum cv. Rutgers) were grown in a greenhouse. Soil in each pot was infested by pipetting egg masses of M. hapla suspended in tap water and cultivated at approximately 252 C. in a greenhouse. Six months following inoculation, tomato plants were uprooted and root systems were carefully washed with running tap water to remove adhered soil. The roots were cut into small pieces and extracted by the Baermann funnel method for 3-5 days to obtain live nematodes. The inoculum concentration included a range of life stages and was adjusted to approximately 2000 individuals using tap water by the aid of a haemocytometer. A mortality assay was carried out as described in Examples 1 and 2 above. The assay was carried out at two doses of the VOCs (5 l and 10 l) and the mortality was recorded at 3, 6 and 24 hr post treatment. There were three replicates per treatment and the whole experiment was repeated twice. The results are shown in FIG. 6.

[0075] Both 3-octanone and 1-octen-3-ol showed strong nematicidal activity against M. hapla. 3-octanone caused 100% mortality exposed to 10 l and 5 l within 3 hr and 6 hr, respectively. 1-octen-3-ol exhibited a dose dependent response after 3 hr and 6 hr. When the nematodes were examined after 24 hr, over 90% were dead at both doses. The control showed no mortality at 3 hr and 6 hr and less than 2% at 24 hr.

Example 4: Nematicidal Effect of VOC Formulation

[0076] 1-octen-3-ol (5% v/w) was mixed with microcrystalline cellulose and the wettable powder was mixed into soil containing a plant parasitic nematode at different concentrations (1, 5 and 10% w/w). The % decline in the population of adult and juveniles as well as eggs was recorded one day (black bars) and five days (grey bars) post treatment in the top (A), middle (B) and bottom (C) layers of soil. The results are shown in FIGS. 7a and 7b.

[0077] It can be seen from FIGS. 7a and 7b that the presence of the composition comprising 1-octen-3-ol and microcrystalline cellulose significantly decreases both the amount of adult and juvenile parasitic nematodes as well as the number of eggs. The compositions comprising a higher amount of 1-octen-3-ol generally achieved a higher level of decrease, i.e. achieved a higher level of mortality. The level of decrease measured was also generally higher at 5 days compared to 1 day showing that the nematodes were killed.