Composition comprising chitosan and a fungicide

Abstract

The present invention provides a composition comprising (i) chitosan or chitopoly- or chitooligo-saccharides thereof, wherein said chitosan or chitopoly- or chitooligo-saccharides thereof comprise -(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine monomers and have a degree of acetylation between 0.05 and 0.20 and an average degree of polymerization 250 (molecular weight 42,000 Da), and (ii) a fungicide not containing chitopoly- or chitooligo-saccharides.

Claims

1. A composition comprising (i) chitooligo-saccharides, wherein said chitooligo-saccharides comprise -(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine monomers and have a degree of acetylation between 0.01 and 0.40 and an average degree of polymerization of 20-60 as assessed by measurement with .sup.1H NMR spectroscopy, and (ii) a fungicide not containing chitopoly- or chitooligo-saccharides; wherein said fungicide is not an inorganic fungicide; wherein the fungicidal activity of said chitooligo-saccharides and said fungicide is synergistic; wherein the chitooligo-saccharides are present in the composition at a concentration of 1-1000 g/ml; wherein the chitooligo-saccharides and said fungicide are present in a ratio of 1000:1 to 1:2 (w/v); and wherein the fungicide is selected from the group consisting of an anilide fungicide; an anilinopyrimidine fungicide; a pyrrole fungicide; a methoxyacrylate strobilurin fungicide; a carbanilate fungicide; a pyrazole fungicide; a pyridine fungicide; a methoxycarbanilate strobilurin fungicide, a naphthoquinone fungicide, a benzamide fungicide, a strobilurin fungicide, a conazole fungicide, a carboxamide fungicide, a triazole fungicide, and a dicarboximide fungicide, and combinations thereof.

2. The composition as claimed in claim 1 having an average degree of polymerization (DP.sub.n) of between 20 to 40.

3. The composition as claimed in claim 1 wherein the DP.sub.n is 23, 30, or 40.

4. The composition as claimed in claim 1 wherein 50% of the polymer chains in the chitooligo-saccharides have a D-glucosamine sugar unit at their reducing ends.

5. The composition as claimed in claim 1 where said chitooligo-saccharides are prepared by acid hydrolysis.

6. The composition as claimed in claim 1 where said chitooligo-saccharides are prepared by a method comprising dissolving chitosan in water or a weak acidic solution followed by enzymatic cleavage using an enzyme capable of catalysing degradation of chitosan into chitooligo-saccharides.

7. The composition as claimed in claim 5 wherein the resultant chitooligo-saccharide product mix is separated by size exclusion chromatography.

8. The composition as claimed in claim 1 wherein the chitooligo-saccharides are present in the composition at a concentration between 10 and 1000 g/ml.

9. The composition as claimed in claim 1 wherein the fungicide is selected from the group consisting of fenhexamid, cyprodinil, fludioxonil, azoxystrobin, boscalid, pyraclostrobin and dithianon, and combinations thereof.

10. The composition as claimed in claim 1 wherein at least 2 fungicides not containing chitopoly- or chitooligo-saccharides are present.

11. The composition as claimed in claim 1 wherein the fungicide is present in the composition at a suboptimal concentration.

12. The composition as claimed in claim 11 wherein the concentration of the fungicide in the composition is 1-20% of the optimal concentration of the fungicide.

13. The composition as claimed in claim 1 wherein the fungicide is selected from (i) fenhexamid; (ii) cyprodinil and fludioxonil; (iii) azoxystrobin; (iv) boscalid and pyraclostrobin and (v) dithianon and the chitooligo-saccharides have a DP.sub.n of between 20 and 60.

14. A kit comprising (i) chitooligo-saccharides and (ii) a fungicide, wherein each component is as defined in claim 1.

15. A method for treating fungal disease, damage or infection in a plant caused by a fungus, comprising contacting the plant or part thereof which is affected by the fungus with an effective amount of the composition of claim 1.

16. The method as claimed in claim 15 wherein the method is carried out on plants and the fungus is selected from Botrytis cineria, Alternaria brassicicola, Mucor piriformis, Microdochium sp and Venturia inaequalis.

17. The method as claimed in claim 15 wherein the plant is a cereal, turf grass, rice, grape or root, tuber, fruit, berry, vegetable or pulse crop plant.

18. A method for treating fungal disease, damage or infection in a plant caused by a fungus comprising contacting the plant or part thereof which is affected by the fungus with an effective amount of the composition of claim 13.

19. The composition of claim 1 wherein said chitooligo-saccharides have a degree of acetylation between 0.05 and 0.20.

20. The composition of claim 2 having an average degree of polymerization (DP.sub.n) of between 20 to 35.

21. The composition of claim 12 wherein the concentration of the fungicide in the composition is <10% of the optimal concentration.

22. The composition of claim 13 wherein the chitooligo-saccharides have a DP.sub.n of 20 to 40.

23. The composition of claim 13 wherein the chitooligo-saccharides have a DP.sub.n of 20 to 35.

24. The composition of claim 13 wherein the chitooligo-saccharides have a DP.sub.n of 23, 30, or 37.

Description

(1) The invention will now be described by way of the following Examples in which:

(2) FIG. 1 shows a size exclusion chromatogram of chitopoly- or chitooligo-saccharides (CHOS) with DP.sub.n 30. The material in the DP>12 areas was fractionated into several samples. The DP.sub.n of the fractions was found using .sup.1H-NMR and the material was further used in the biological assays.

(3) FIG. 2 shows the effect on germination of Botrytis cinerea 101 of CHOS mixtures with a DP.sub.n of 30, produced by hydrolysis of chitosan DP.sub.n 206 (F.sub.A 0.15) with ScCsn46A, (95% deacetylated reducing ends). Subsequently, subfractions were prepared by separating this hydrolysis mixture using a SEC column as described in the Materials and Methods section. The resulting sub-fractions had DP.sub.n values ranging from 34 to 163.

(4) FIG. 3 shows the effect of acetylation of reducing end sugars on the ability of CHOS to inhibit germination of B. cinerea 101. CHOS with 95% deacetylated reducing ends was obtained by hydrolyzing chitosan (F.sub.A 0.15, DP.sub.n=206) with ScCsn46A, whereas CHOS with 35% deacetylated reducing ends was obtained by hydrolyzing the same chitosan with ChiA.

(5) FIG. 4 shows the results of an experiment to assess dose-response relationships of CHOS DP.sub.n 37 (prepared by hydrolyzing with ScCsn46A) on germination of B. cinerea 101, Alternaria brassicicola A 328 and Mucor piriformis M 119.

(6) FIG. 5 shows the results of an experiment to assess the effect of combinations of chitosan DP.sub.n 206, CHOS DP.sub.n 30 (F.sub.A 0.15 and 95% deacetylated reducing ends) and Switch on the cumulative infection of detached chickpea leaves by B. cinerea 101.

(7) FIG. 6 shows the results of an experiment to assess the effect of the combination of chitosan DP.sub.n 206, CHOS DP.sub.n 30 (F.sub.A 0.15 and 95% deacetylated reducing ends) and Signum (Sig) on infection of detached chickpea leaves by B. cinerea 101.

(8) FIG. 7 shows the results of an experiment to assess the effect of the combination of chitosan DP.sub.n 206, CHOS DP.sub.n 30 and Switch on B. cinerea 101 infection of bean leaves.

(9) FIG. 8 shows the results of an experiment to assess the effect of chitosan DP.sub.n 206, the CHOS DP.sub.n 30 and Signum (Sig) on B. cinerea 101 infections of bean leaves.

EXAMPLES

(10) Materials and Methods

(11) Fungal Cultures

(12) Botrytis cinerea isolate BC 101, B. cinerea BD, Alternaria brassicicola A 328, Microdochium majus and Mucor piriformis M 119 were obtained from the culture collection at the Norwegian University of Life Sciences (UMB). For the in vitro and in vivo bioassays, conidia were collected from cultures grown on potato dextrose agar (FDA) (Difco Laboratories, Detroit, Mich.) under regular laboratory light for 2 weeks at 231 C. Concentrations of conidia in aqueous suspensions were determined by haemocytometer count at 400 magnification and adjusted to 410.sup.4 conidia/ml with sterile water.

(13) Fungicides

(14) Five fungicides not containing chitopoly- or chitooligo-saccharides were used: 1. Teldor@ WG 50 (Bayer Crop Science Pty Ltd.): 500 g/kg fenhexamid. Recommended concentration of Teldor; 150 g/100 L water 2. Switch 62.5 WG (Syngenta Crop Protection Pty Ltd.): 375 g/kg cyprodinil, 250 g/kg fludioxonil. Recommended concentration of Switch 50 g/100 L 3. Amistar (Syngenta Crop Protection Pty Ltd.): 500 g/kg azoxystrobin. Recommended concentration of Amistar 100 g/100 L 4. Signum WG (BASF): 26.7% w/w boscalid and 6.7% w/w pyraclostrobin. Recommended concentration of Signum 100 g/100 L 5. Delan WG (BASF): 70% w/w dithianon. Recommended concentration of Delan 80 g/100 L.

(15) Chitosan and Chitooligosaccharide (CHOS) Production

(16) Chitosan KitoNor (DP.sub.n 206, F.sub.A 0.15, M.sub.Wn 34 kDa); was produced by acid hydrolysis from chitin from Snow crab (Chionoecetes opilio) by Norwegian Chitosan, Gardermoen, Norway. The average molecular weight and DP.sub.n was calculated from viscosimetric measurements. CHOS with lower DP.sub.n were produced by enzymatic hydrolysis of chitosan (DP.sub.n 206) using a chitosanase, ScCsn46A (Heggset et al., Biomacromolecules, 2010, p 2487-2497), or a chitinase, Chi A (Brurberg et al., FEMS Microbiol Lett., 1994, p 399-404; Horn et al., FEBS J., 2006, p 491-503.).

(17) KitoNor (20 mg/mL) was dissolved in water with 0.5% (v/v) acetic acid. After all of the chitosan was dissolved the pH was adjusted with 0.1N NaOH to 5.5. Recombinant chitosanase ScCsn46A from Streptomyces coelicolor A3(2) (Heggset et al., Biomacromolecules, 2010, p 2487-2497) or chitinase A (ChiA) from Serratia marcescens (Brurberg et al., FEMS Microbiol Lett., 1994, p 399-404) was added to the chitosan solutions to a final concentration of 0.5 g/mg chitosan and the reaction was incubated with shaking (225 rpm) at 37 C. The reaction was stopped by decreasing the pH to 2.5 with 0.1N HCl and by keeping the tube with the reaction mixture at boiling temperature for ten minutes to permanently inactivate the enzymes. The DP.sub.n of the resulting CHOS sample was determined by .sup.1H-NMR analysis on a Varian 300 mHz instrument, as described in Srbotten et al. FEBS J., 2005, p 538-549.

(18) Separation of CHOS by Size Exclusion Chromatography (SEC)

(19) The CHOS were separated by SEC on three XK 26 columns packed with Superdex 30 prep grade (GE Healthcare) coupled in series with an overall dimension of 2.6 cm180 cm. The mobile phase (150 mM NH.sub.4Ac pH 4.6) was run at a constant flow of 0.4 mL/min (Srbotten et al. 2005, FEBS J., p 538-549). The signals were read on a RI detector (Gilson model 133). In each run 100 mg of CHOS was applied (i.e. 5 mL) and fractions were collected. Identification of DP.sub.n of the fractions was performed with .sup.1H-NMR. The fractions were not baseline separated.

(20) The fractions were dialyzed with Float-A-Lyzers (MWCO 100-500 Da, SpectrumLabs) to remove salts, sterile filtrated through Filtropur S 0.2 m sterile filters (Sarstedt, Germany) and lyophilized, prior to use.

(21) In Vitro Bioassay of Chitosan and Fungicides Not Containing Chitopoly- or Chitooligo-Saccharides

(22) The antifungal effects of chitosan and CHOS samples and fungicides not containing chitopoly- or chitooligo-saccharides were investigated in a synthetic medium (2.5 mM NH.sub.4NO.sub.3; 0.28 mM CaCl.sub.2.2H.sub.2O; 0.16 mM MgSO.sub.4.7H.sub.2O; 0.002 mM MnSO.sub.4.4H.sub.2O; 0.002 mM ZnSO.sub.4.7H.sub.2O; 1 mM KH.sub.2PO.sub.4; 0.06 mM FeC.sub.6H.sub.SO.sub.7.5H.sub.2O and 55.5 mM glucose, pH 5.2-5.3) in a flat-bottom 96-well microtiter plate (Nunc, Roskilde, Denmark), 200 L/well with 210.sup.4 conidia/mL. There were 4 replicate wells of each treatment. The microtiter plates were incubated at 231 C. for up to 72 h. An invert microscope (Fluovert FU, Ernst Leitz Wetzlar GmbH, Wetzlar, Germany) was used to visually estimate the germination percentage at 400 magnification after 24 h and these estimates were used to express anti-microbial activity as half maximal inhibitory concentrations (IC.sub.50) or minimum inhibitory concentrations (MIC). Mycelial growth following germination was measured as absorbance at 595 nm in a microtiter plate reader 72 h after inoculation.

(23) Synergistic effects were calculated as the ratio between observed efficacy, E.sub.obs (% inhibition) and the expected efficacy, E.sub.exp (calculated by Abbott's formula) (Levy et al. Eppo Bulletin, 1986, p 651-657) % E.sub.exp=(ab/100). Here a=% germination inhibition by that concentration of the fungicide alone, b=% germination inhibition by that concentration of the chitosan alone. An E.sub.obs/E.sub.exp ratio of 1 indicates additivity, ratios >1 indicates synergy and ratios <1 indicates antagonistic interactions.

(24) In Vivo Bioassay of Chitosan and Fungicides Not Containing Chitopoly- or Chitooligo-Saccharides on Plants

(25) The flower infection test was performed on detached, newly opened strawberry (Fragariaananassa) flowers (cv. Corona) from the greenhouse (Hjeljord et al. Phytopathology, 2001, p 1172-1180). Eighteen flowers per treatment (six replications of three flowers) were cut off with a 1-2 cm stem and placed in empty pipette tip racks set in plastic containers filled with 1-2 cm water. Conidia suspensions of the pathogen (final concentration: 110.sup.6 conidia/ml) were mixed with test ingredients (i.e. fungicides, CHOS, chitosan or mixtures thereof, as well as control ingredients) and 10 l drops of each mixture were placed at the base of three petals on each flower using an automatic pipette (Finnpipette 4027, Thermo Labsystems, Finland). All flowers were then incubated at 231 C. in large trays covered with aluminium foil. The relative humidity around the flowers was 90-95%, measured by a thermo-hygrometer (Lambrecht, Germany).

(26) The necrotic regions on the abaxial surface of the flowers under the inoculation point were recorded daily for 8 days and the area under the disease progress curve (AUDPC) was calculated on the basis of the cumulative infection percentage by the following equation:
AUDPC=[(D.sub.iD.sub.i1){S.sub.i1+0.5(S.sub.iS.sub.i1)}]

(27) Where, D.sub.i=Days of the i.sup.th assessment, S.sub.i=Proportion of the i.sup.th infected inoculation point.

(28) The protection index was calculated by using the AUDPC values in the following equation (Bardin et al., Biological Control, 2008, p 476-483).
100(AUDPC.sub.controlAUDPC.sub.treatment)/AUDPC.sub.control

(29) where AUDPC.sub.control represents flowers inoculated with only B. cinerea conidia and AUDPC.sub.treatment represents flowers treated with the conidia applied in solutions containing pesticides and/or chitooligosaccharides to be tested. The interaction (synergy) between fungicides and the chitosan products in the flower assay was determined by Abbott's equation as above.

(30) Similar tests were performed on detached chickpea leaves (Cicer arientinum) (36 leaves per treatment and 3 parallels), or on 30 day-old bean leaves (Vicia faba) (6 inoculation drops on each leaf and 3 parallels). The leaves were inoculated with 10 L drops with 210.sup.6/ml B. cinerea 101 conidia. The development of the disease and the amount of sporulation were recorded every 24 hours up to 8 days. The experiment was repeated at least twice.

(31) Field Trials with Chitosan and the Fungicides Not Containing Chitopoly- or Chitooligo-Saccharides

(32) Apple trees (Malus domestica Broch) of the cultivar Aaker in the apple orchard at the Norwegian University of Life Sciences, Aas, Norway were used. There were three replicates of each treatment and three trees in each replicate. The trees were sprayed to runoff once in the flowering period (28.sup.Th of May) and three times in the fruiting season (24.sup.th of June, 7.sup.th of July and 17.sup.th of August). At harvest (3.sup.rd of September) the number of apples with infection of apple scab (Venturia inaequalis) was recorded.

(33) Determination of Average Degree of Polymerization (DP.sub.n) with .sup.1H-NMR Spectroscopy

(34) The chitooligosaccharide (CHOS) samples were analysed by .sup.1H-NMR spectroscopy on a Varian Gemini 300 MHz instrument. The average degree of polymerization (DP.sub.n) was calculated by the equation (D+D+D+A+A+A)/(D+D+A+A), where D, D, A and A are the integral of the reducing end signals of the and anomers of the deacetylated (D) and acetylated (A) units, D is the integral of the signals from the internal and nonreducing end deacetylated units and A is the integral of the signals from the internal and nonreducing end acetylated units (Srbotten et al. FEBS J., 2005, p 538-549).

(35) Data Analysis

(36) The % inhibition data for the fungal germination were transformed using arcsine transformation and tested by one-way ANOVA (analysis of variance). Non-transformed data are presented. When appropriate, means were separated by Tukey's method (Fenech, J Am. Stat. Ass., 1979, p 881-884). All calculations were done using Microsoft Office Excel 2007 and Minitab 16 (MINITAB, USA).

Example 1: Production of Chitooligosaccharides (CHOS)

(37) Chitosan KitoNor (DP.sub.n 206, F.sub.A 0.15 and M.sub.W 34 kDa) was hydrolyzed with chitosanase ScCsn46A, which primarily cleaves after deacetylated units and under the conditions of the assay produced 95% deacetylated reducing ends, as determined by .sup.1H-NMR). The chitooligomers were separated by SEC (see Materials and Methods) and the results are shown in FIG. 1. These chitooligosaccharides of varying DP.sub.n were used in Example 2. For details see Materials and Methods: Chitosan and chitooligosaccharide (CHOS) production.

Example 2: Effect of DPn of CHOS on Fungicidal Activity (Inhibition of Germination)

(38) The effect of chitosan (F.sub.A 0.15, DP.sub.n 206), or chitooligosaccharides (CHOS), obtained by hydrolysis of this chitosan with ScCsn46A to DP.sub.n values varying from 9.5 to 96 (95% deacetylated reducing ends, as determined by .sup.1H-NMR) on spore germination of Botrytis cinerea 101 was evaluated. The experiment was conducted as described in the Materials and Methods section under In vitro bioassay.

(39) The DP.sub.n of the chitosan/CHOS influences the inhibitory effect on the germination of B. cinerea 101. The most active fractions have DP.sub.n values in the order of 30, but the data also show that DP.sub.n values in the range of 10-40 have useful activities (Table 1).

(40) TABLE-US-00001 TABLE 1 Effect of DP.sub.n of CHOS on fungicidal activity (inhibition of germination) DP.sub.n of chitosan/CHOS MIC (g ml.sup.1) IC.sub.50 (g ml.sup.1) 206 5000 2500 96 2500 1230 62 2500 630 49 2500 630 40 1200 250 37 310 80 28 310 80 15 310 120 9.5 >2500 2500 IC.sub.50 = half maximal inhibitory concentrations. MIC = minimum inhibitory concentrations.

Example 3: Effect of Deacetylated Reducing Ends in CHOS on Fungicidal Activity (Inhibition of Germination)

(41) An in vitro assay of the fungicidal activity of chitosan and CHOS with >95% (chitosan hydrolysed by chitosanase ScCsn46A from Streptomyces coelicolor) or 35% (chitosan hydrolysed by chitinase A from Serratia marcescens) deacetylated reducing ends was carried out as described in the Materials and Methods section under In vitro bioassay. The results are shown in FIG. 3.

(42) Given the same DP.sub.n, CHOS with deacetylated reducing ends are much more inhibitory to spore germination than CHOS with acetylated reducing ends (FIG. 3). For example, 80 g ml.sup.1 CHOS with 95% deacetylated reducing ends prevented further hyphal growth, whereas 310 g ml.sup.1 CHOS with 35% deacetylated reducing ends was needed to attain the same effect (data not shown).

Example 4: Effect of Partial Purification of CHOS with Various DPn on Fungicidal Activity (Inhibition of Germination)

(43) This experiment assessed the effect of partially purified chitooligosaccharides (CHOS) with varying chain lengths on the germination inhibition of B. cinerea 101, assessed 24 hours after inoculation. Purification was by SEC, see the Materials and Methods section. The results are shown in FIG. 2 and demonstrate that sub-fractions with high DP.sub.n (DP.sub.n 78-163) are less inhibitory than the original non-hydrolyzed chitosan DP.sub.n 206, probably due to the removal of low molecular weight oligomers from those fractions. The CHOS DP.sub.n 30 hydrolysate that had not been separated further on the SEC column had about the same inhibitory effect as the DP.sub.n 34 SEC fraction. The non-purified DP.sub.n 30 and the purified DP.sub.n 34 fractions were the most inhibitory of the fractions tested.

Example 5: Effect of Chitosan or CHOS with Various DPn on Germination or Growth of Different Fungi

(44) An experiment was designed to assess effects of chitosan and CHOS with various DP.sub.n on germination and mycelial growth of two strains of B. cinerea and a strain of Mucor piriformis. This experiment assessed germination inhibition (GI) and growth inhibition (Gr.I) of B. cinerea 101 (BC 101), B. cinerea BD (BC BD) and Mucor piriformis M 119 caused by 80 g ml.sup.1 non-hydrolyzed chitosan and chitooligosaccharides (CHOS) (F.sub.A 0.15) with different DP.sub.n, produced by enzymatic hydrolysis with ScCsn46A (95% deacetylated reducing ends). Growth inhibition was measured by absorbance reading at 595 nm 72 hours after inoculation.

(45) The results, depicted in Table 2, show that CHOS with average DP.sub.n between 23 and 40 were generally more inhibitory to spore germination and growth than CHOS with higher or lower average DP.sub.n. The data further show that the inhibitory effect of the compounds varies depending on the target organism.

(46) TABLE-US-00002 TABLE 2 Effect of chitosan or CHOS with various DP.sub.n on germination inhibi- tion (GI %) or growth inhibition (Gr.I %) of different fungi Chitosan/CHOS BC 101 BC BD M. piriformis GI % Gr.I % GI % Gr.I % GI % Gr.I % DP.sub.n 24 hrs 72 hrs 24 hrs 72 hrs 24 hrs 72 hrs 206 0 c 14 e 0 c 7e 0 g 23 c 75 6.8 4 c 36 d 0 c 25 d 26 e 48 b 58 2.7 0 c 50 cd 0 c 42 c 50 d 53 ab 48 3.0 4 c 54 c 0 c 46 c 48 d 51 ab 40 1.4 77 a 99 a 100 a 99 a 90 a 57 a 23 1.3 72 b 76 b 100 a 78 b 81 b 57 a 15 1.4 0 c ND 100 a ND 58 c ND 9 0.8 0 c 6 e 0 c 3 e 0 g 22 c Means in columns without common letters are significantly different according to Tukey's method at P 0.01. ND = not determined.

Example 6: Effect of Chitosan or CHOS with Various DPn on Disease Severity Caused by Two Strains of B. cinerea

(47) A bioassay was designed to assess effects of non-hydrolyzed chitosan and CHOS with various DP.sub.n on infection of strawberry flowers by two strains of B. cinerea. In this experiment the effect on the disease severity caused by B. cinerea 101 and B. cinerea BD on detached strawberry flowers treated with 500 g/mL chitosan or chitooligosaccharides (CHOS) (F.sub.A 0.15 and 95% deacetylated reducing ends) with different DP.sub.n was assessed. The CHOS fractions with lower DP.sub.n were produced by enzymatic hydrolysis of chitosan (DP.sub.n 206) using a chitosanase, ScCsn46A. For details see Materials and Methods section: Chitosan and chitooligosaccharide (CHOS) production. The disease severity was assessed as area under the disease progress curve (AUDPC), calculated from cumulative disease incidence at 231 C., 1 to 8 days after inoculation.

(48) The results in Table 3 show that CHOS with DP.sub.n 23 are more protective against B. cinerea infection of strawberry flowers than CHOS with higher and lower DP.sub.n. Of the other products tested, the DP.sub.n 40 material clearly shows the best results.

(49) TABLE-US-00003 TABLE 3 Effect of chitosan or CHOS with various DP.sub.n on disease severity caused by two strains of B. cinerea on strawberry flowers B. cinerea 101 Chitosan/ Protection B. cinerea BD CHOS AUDPC index (%) AUDPC Protection index (%) Control 4.3 a 4.3 a DP.sub.n 206 4.0 ab 7.8 c 3.5 a 19.8 d DP.sub.n 48 3.4 b 20.3 c 2.8 c 34.5 c DP.sub.n 40 1.9 c 55.2 b 1.7 d 61.5 b DP.sub.n 23 0.9 d 79.8 a 0.7 e 83.6 a DP.sub.n 9 4.0 ab 7.0 c 3.5 a 19.0 d Means in columns without common letters are significantly different according to Tukey's method at P 0.01.

Example 7: Effect of CHOS with DPn 37 on Germination of Different Fungi

(50) An experiment was designed to compare effects of CHOS (DP.sub.n 37) on three genera of plant pathogenic fungi. As seen in FIG. 4, the results show a dose-response relationship for all genera tested, but these did respond somewhat differently. B. cinerea and M. piriformis showed decreasing germination over a broad CHOS concentration range (0.002-0.25%), whereas A. brassicicola showed complete germination at 0.002% and none at 0.008%.

Example 8: Fungicidal Activity of Chitosan and the Fungicide Switch (Inhibition of Germination) on Two Strains of B. cinerea

(51) Non-hydrolyzed chitosan was mixed with a fungicide (Switch) to compare the effects of the mixture and the components separately on germination of two strains of B. cinerea. This experiment evaluated germination inhibition of B. cinerea 101 or B. cinerea BD recorded 24 hours after inoculation with chitosan (DP.sub.n 206) and/or the fungicide Switch.

(52) The results are provided in Table 4 and show that Switch applied at 25 g ml.sup.1, a concentration that is only 1/20 of the recommended concentration (the recommended concentration, 500 g ml.sup.1, are according to the standard product leaflets provided by the manufacturers of this product) together with 640 g/ml chitosan DP.sub.n 206 completely inhibited spore germination of both Botrytis strains. The combinations were clearly synergistic.

(53) TABLE-US-00004 TABLE 4 Fungicidal activity of chitosan and the fungicide Switch (inhibition of germination) on two strains of B. cinerea % inhibition of % inhibition of Treatment B. cinerea 101 B. cinerea BD Chitosan 640 g ml.sup.1 32 d 17 d Chitosan 80 g ml.sup.1 11 c 5 e Switch 25 gml.sup.1 82 b 43 c Chitosan 640 g ml.sup.1 + 100 a 100 a Switch 25 g ml.sup.1 Chitosan 80 g ml.sup.1 + 100 a 90 b Switch 25 g ml.sup.1 Means in columns without common letters and related to the same fungicide, are significantly different according to Tukey's method at P 0.01.

Example 9: Fungicidal Activity of Chitosan and the Fungicides Switch or Signum (Inhibition of Germination) on Microdochium majus

(54) Further experiments assessed the effects of combining chitosan with fungicides (Switch or Signum) on germination of the plant pathogenic fungus Microdochium majus. This experiment evaluated germination inhibition of M. majus recorded 24 h after inoculation with chitosan (DP.sub.n 206) and fungicides. For details see Materials and Methods: In vitro bioassay of chitosan and fungicides not containing chitopoly- or chitooligo-saccharides.

(55) The results in Table 5 show that combination of 640 g ml.sup.1 chitosan DP.sub.n 206 with Switch at 1/50 of recommended concentration (recommended concentration is 500 g ml.sup.1) or Signum at 1/1000 of recommended concentration (recommended concentration is 1000 g ml.sup.1), completely inhibited spore germination of M. majus. Recommended concentrations of Switch and Signum are according to the standard product leaflets provided by the manufacturers of these products.

(56) TABLE-US-00005 TABLE 5 Fungicidal activity of chitosan and the fungicides Switch or Signum (inhibition of germination) on Microdochium majus Treatment % inhibition of M. majus Chitosan 640 g ml.sup.1 3 d Switch 10 g ml.sup.1 71 b Signum 1 g ml.sup.1 26 c Chitosan 640 g ml.sup.1 + Switch 10 g ml.sup.1 100 a Chitosan 640 g ml.sup.1 + Signum 1 g ml.sup.1 100 a Means in columns without common letters and related to the same fungicide, are significantly different according to Tukey's method at P 0.01.

Example 10: Fungicidal Activity of CHOS with DPn 23 and the Fungicides Teldor, Switch, Amistar and Signum (Inhibition of Germination) on B. cinerea

(57) Possible synergism between CHOS (DP.sub.n 23) and fungicides (Teldor, Switch, Amistar and Signum) in inhibition of germination of B. cinerea was investigated in this experiment. The effect of combination of chitooligosaccharides (CHOS) DP.sub.n 23 (F.sub.A 0.15 and 95% deacetylated reducing ends) and fungicides in inhibiting germination (assessed 24 hours after inoculation) of B. cinerea BC 101 was assessed.

(58) The results are shown in Table 6 which shows that high synergistic effects are seen when combining 5 g DP.sub.n 23 with 1% of the recommended concentration of Switch, Amistar and Signum and 10% of the recommended concentration of Teldor. Comparison of the data in Table 6 with the data in Table 4 further shows that the DP.sub.n 23 product is more powerful than the non-hydrolyzed chitosan with DP.sub.n 206.

(59) TABLE-US-00006 TABLE 6 Fungicidal activity of CHOS with DP.sub.n 23 and the fungicides Teldor, Switch, Amistar and Signum (inhibition of germination) on B. cinerea 101 Treatment Germination (g/ml) inhibition (%) E.sub.obs/E.sub.exp Control 0 b DP.sub.n 23 5 g ml.sup.1 1 b Teldor 150 g ml.sup.1 0 b DP.sub.n 23 5 g ml.sup.1 + Teldor 150 g ml.sup.1 20 a 20 DP.sub.n 23 5 g ml.sup.1 1 c Switch 5 g ml.sup.1 30 b DP.sub.n 23 5 g ml.sup.1 + Switch 5 g ml.sup.1 94 a 3 DP.sub.n 23 5 g ml.sup.1 1 b Amistar 10 g ml.sup.1 2 b DP.sub.n 23 5 g ml.sup.1 + Amistar 10 g ml.sup.1 92 a 31 DP.sub.n 23 5 g ml.sup.1 1 b Signum 10 g ml.sup.1 1 b DP.sub.n 23 5 g ml.sup.1 + Signum 10 g ml.sup.1 98 a 49 Means in columns without common letters and related to the same fungicide, are significantly different according to Tukey's method at P 0.01. Synergism is calculated by the E.sub.obs/E.sub.exp ratio, 1 indicates additivity, ratios >1 indicate synergy and ratios <1 indicate antagonistic interactions. The recommended concentrations for application of Teldor, Switch, Amistar and Signum are 1500, 500, 1000, 1000 g/ml, respectively, according to the standard product leaflets provided by the manufacturers of these products.

Example 11: Fungicidal Activity of Chitosan and the Fungicides Teldor, Switch, Amistar and Signum (Inhibition of Infection of Strawberry Flowers) on B. cinerea

(60) A bioassay was designed to evaluate effects of the combination of chitosan and fungicides (Teldor, Switch, Amistar or Signum) on flower infection by B. cinerea 101. For experimental details see: In vivo bioassay of chitosan and fungicides on plants, infection on strawberry flowers in Materials and Methods section. This experiment assessed the effect of combination of chitosan with DP.sub.n 206 and fungicides in g ml.sup.1 in inhibiting B. cinerea BC 101 infection of detached strawberry flowers. Disease severity was measured as the area under the disease progress curves (AUDPC), with AUDPC values calculated from the cumulative disease incidence at 231 C., recorded up to 8 days after inoculation. Percent protection index is % reduction in AUDPC by the treatment compared with the control.

(61) The results are shown in Table 7 which shows that 1% of the recommended concentration of Teldor, Switch and Amistar (15, 5 and 10 g/ml respectively) in combination with 1000 g chitosan DP.sub.n 206 and 5000 g ml.sup.1 chitosan in combination with 0.2% of the recommended concentration of Signum (2 g/ml), gave the same level of protection against infection by B. cinerea as the recommended concentration of the respective fungicides alone.

(62) TABLE-US-00007 TABLE 7 Fungicidal activity of chitosan and the fungicides Teldor, Switch, Amistar and Signum (inhibition of infection of strawberry flowers) on B. cinerea Treatment Protection (all concentrations in g ml.sup.1) AUDPC index (%) E.sub.obs/E.sub.exp Untreated control 4.8 a Chitosan 5000 2.5 de 49 cd Chitosan 1000 2.9cd 39 de Chitosan 400 3.7 bc 24 ef Teldor 1500 1.5 fg 69 ab Teldor 15 4.4 ab 9 f Chitosan 1000 + Teldor 15 1.5 fg 70 ab 2 Chitosan 400 + Teldor 15 1.9 ef 61 bc 2 Switch 500 1.0 g 79 a Switch 5 4.5 a 7 f Chitosan 1000 + Switch 5 1.5 fg 70 ab 2 Chitosan 400 + Switch 5 1.8 efg 63 abc 2 Amistar 1000 2.0 ef 59 bc Amistar 10 4.5 a 7 f Chitosan 1000 + Amistar 10 1.8 efg 63 abc 2 Chitosan 400 + Amistar 10 1.8 efg 63 abc 2 Signum 1000 1.3 fg 72 ab Signum 10 4.2 ab 13 f Signum 2 4.5 a 7 f Chitosan 400 + Signum 10 2.1 ef 56 bc 2 Chitosan 400 + Signum 2 2.5 de 49 cd 2 Means in columns without common letters and related to the same fungicide, are significantly different according to Tukey's method at P 0.01. The recommended concentrations for application of Teldor, Switch, Amistar and Signum are 1500, 500, 1000 and 1000 g/ml respectively, according to the standard product leaflets provided by the manufacturers of these products.

Example 12: Fungicidal Activity of CHOS with DPn23 and the Fungicides Teldor, Switch, Amistar and Signum (Reduction in Disease Severity on Strawberry Flowers) on B. cinerea

(63) Possible synergism between CHOS (DP.sub.n 23) and fungicides (Teldor, Switch, Amistar and Signum) in reduction of disease severity in inoculated strawberry flowers was assessed in this experiment. For experimental details see: In vivo bioassay of chitosan and fungicides not containing chitopoly- or chitooligo-saccharides on plants, infection on strawberry flowers in the Materials and Methods section. This experiment assessed the effect of a chitooligosaccharide (CHOS) DP.sub.n 23 (F.sub.A 0.15 and 95% deacetylated reducing ends) alone and in combination with fungicides in inhibiting B. cinerea BC 101 infection of detached strawberry flowers, assessed using the area under the disease progress curve (AUDPC), to calculate the protection index and synergy. Disease severity was measured as the area under disease progress curve (AUDPC), with AUDPC values calculated from the cumulative disease incidence at 231 C., recorded up to 8 days after inoculation.

(64) The results in Table 8 shows that 10 g ml.sup.1 of CHOS DP.sub.n 23 in combination with 10% of the recommended dose (150 g ml.sup.1) of Teldor, 5% of the recommended dose (25 g ml.sup.1) of Switch, 1% of the recommended dose (10 g ml.sup.1) of Amistar and 1% of the recommended dose (10 g ml.sup.1) of Signum gave the same or better protection against infection by B. cinerea than the fungicides alone, applied at their recommended concentrations. Comparison of the data in Table 8 with the data in Table 7 further shows that the DP.sub.n 23 product is more powerful than the non-hydrolyzed chitosan with DP.sub.n 206. For example 10 g/ml DP.sub.n 23+10 g/ml Signum gave better protection than 400 g/ml DP.sub.n 206+10 g/ml Signum.

(65) TABLE-US-00008 TABLE 8 Fungicidal activity of CHOS with DP.sub.n 23 and the fungicides Teldor, Switch, Amistar and Signum (reduction in disease severity on strawberry flowers) on B. cinerea Protection E.sub.obs/ Treatment (g ml.sup.1) AUDPC Index (%) E.sub.exp Untreated control 4.6 a DP.sub.n 23 10 g ml.sup.1 4.5 a 2 f Teldor 1500 g ml.sup.1 1.5 68 cd Teldor 150 g ml.sup.1 2.3 b 51 e Switch 500 g ml.sup.1 1.0 78 Switch 25 g ml.sup.1 4.2 a 9 f Amistar 1000 g ml.sup.1 2.0 57 de Amistar 10 g ml.sup.1 4.5 a 2 f Signum 1000 g ml.sup.1 1.3 d 71 bc Signum 10 g ml.sup.1 4.5 a 1 f DP.sub.n 23 10 g ml.sup.1 + Teldor 150 g ml.sup.1 0.5 e 89 a 2 DP.sub.n 23 10 g ml.sup.1 + Switch 25 g ml.sup.1 0.7 e 84 a 8 DP.sub.n 23 10 g ml.sup.1 + Amistar 10 g ml.sup.1 0.8 e 82 ab 21 DP.sub.n 23 10 g ml.sup.1 + Signum 10 g ml.sup.1 0.6 e 87 a 30 Percent protection index is % reduction in AUDPC by the treatment compared with the control. Synergism is calculated by the E.sub.obs/E.sub.exp ratio, 1 indicates additivity, ratios >1 indicates synergy and ratios <1 indicates antagonistic interactions. Means in columns without common letters and related to the same fungicide, are significantly different according to Tukey's method at P 0.01. The recommended concentrations for application of Teldor, Switch, Amistar and Signum are 1500, 500, 1000 and 1000 g ml.sup.1 respectively, according to the standard product leaflets provided by the manufacturers of these products.

Example 13: Fungicidal Activity of Chitosan or CHOS with DPn 30 and the Fungicide Switch (Reduction of Infection on Chickpea Leaves) on B. cinerea

(66) Possible synergism between non-hydrolyzed chitosan, CHOS (DP.sub.n 30) and Switch in reducing infection of chickpea leaves by B. cinerea 101 was investigated. For experimental details see: In vivo bioassay of chitosan and fungicides not containing chitopoly- or chitooligo-saccharides on plants, infection on chickpea leaves in the Materials and Methods section.

(67) The results, depicted in FIG. 5, show that, when combined with Switch, CHOS (DP.sub.n 30) was much more effective than the non-hydrolyzed chitosan (DP.sub.n 206). The figure also illustrates the power of CHOS: the combination of 1/50 of the recommended Switch concentration (10 g ml.sup.1) (recommended concentration is 500 g ml.sup.1) with 320 g ml.sup.1 CHOS (DP.sub.n 30) was as protective as the recommended concentration of Switch.

Example 14: Fungicidal activity of chitosan or CHOS with DPn 30 and the fungicide Signum (reduction of infection on chickpea leaves) on B. cinerea 101

(68) This experiment was similar to that described in Example 13, except that the fungicide Signum was tested. For experimental details see: In vivo bioassay of chitosan and fungicides not containing chitopoly- or chitooligo-saccharides on plants, infection on chickpea leaves in the Materials and Methods section.

(69) The results are shown in FIG. 6. FIG. 6 shows that all combinations of chitosan DP.sub.n 206 and the CHOS DP.sub.n 30 with Signum showed a better effect in reducing disease severity than each component alone. 1000 g ml.sup.1 Signum, 2500 g ml.sup.1 chitosan DP.sub.n 206 and 2500 g ml.sup.1 DP.sub.n 30 completely controlled infection, whereas the combination of 10 g ml.sup.1 Signum and 320 g ml.sup.1 CHOS DP.sub.n 30 resulted in only 5% infection after 8 days. These results illustrate the synergistic effect of CHOS with a reduced concentration of Signum.

Example 15: Fungicidal Activity of Chitosan or CHOS with DPn 30 and the Fungicide Signum (Reduction of Sporulation on Infected Chickpea Leaves) on B. cinerea 101

(70) Sporulation of the plant pathogenic fungus on infected plant parts is a source of secondary inoculum and an important factor in disease epidemiology. An experiment was designed to assess the effects of the combination of chitosan or CHOS with the fungicide Signum on sporulation of B. cinerea on infected chickpea leaves. This experiment assessed the effect of combination of chitosan DP.sub.n 206, or chitooligosaccharide (CHOS) DP.sub.n 30 and Signum on the average number of spores produced after 8 days by B. cinerea 101 in each inoculation spot on chickpea leaves. For experimental details see: In vivo bioassay of chitosan and fungicides on plants not containing chitopoly- or chitooligo-saccharides, infection on chickpea leaves in the Materials and Methods section.

(71) The results in Table 9 show that the combination of chitosan and Signum reduced sporulation of B. cinerea 101 more than each component alone and CHOS DP.sub.n 30 was more effective than chitosan DP.sub.n 206 when each were combined with Signum in reducing the sporulation of B. cinerea 101.

(72) TABLE-US-00009 TABLE 9 Fungicidal activity of chitosan or CHOS with DP.sub.n 30 and the fungicide Signum (reduction of sporulation on infected chickpea leaves) on B. cinerea 101 Average number of conidia produced in Treatment inoculation point Untreated control 2.1 10.sup.5 Signum 10 g ml.sup.1 3.6 10.sup.4 Chitosan DP.sub.n 206, 320 g ml.sup.1 7.8 10.sup.4 CHOS DP.sub.n 30, 320 g ml.sup.1 4.1 10.sup.4 Signum 10 g ml.sup.1 + Chitosan DP.sub.n 206, 320 g ml.sup.1 7.8 10.sup.3 Signum 10 g ml.sup.1 + CHOS DP.sub.n 30, 320 g ml.sup.1 2.9 10.sup.2

Example 16: Fungicidal Activity of Chitosan or CHOS with DPn 30 and the Fungicide Switch (Reduction of Infection on Bean Leaves) on B. cinerea

(73) An experiment was designed to assess the effects of the combination of chitosan or CHOS with the fungicide Switch on B. cinerea 101 infection of bean leaves. For experimental details see: In vivo bioassay of chitosan and fungicides not containing chitopoly- or chitooligo-saccharides on plants, infection on bean leaves in the Materials and Methods section.

(74) The results are shown in FIG. 7. In this assay 500 g ml.sup.1 Switch (recommended concentration) and the combination of 2.5 g ml.sup.1 Switch and 160 g ml.sup.1 of DP.sub.n 30 or DP.sub.n 206 chitosan completely controlled infection, while 2.5 g ml.sup.1 Switch, or 160 g ml.sup.1 chitosan DP.sub.n 206 or CHOS DP.sub.n 30 separately were less effective.

Example 17: Fungicidal Activity of Chitosan or CHOS with DPn 30 and the Fungicide Signum (Reduction of Infection on Bean Leaves) on B. cinerea 101

(75) This experiment was similar to that described in Example 16, except that the fungicide Signum was tested. For experimental details see: In vivo bioassay of chitosan and fungicides not containing chitopoly- or chitooligo-saccharides on plants, infection on bean leaves in the Materials and Methods section.

(76) The results are shown in FIG. 8 which shows that in this assay, complete control of infection was obtained by 1000 g ml.sup.1 Signum (recommended concentration), or the combination of 5 g ml.sup.1 Signum+160 g ml.sup.1 chitosan DP.sub.n 206.

Example 18: Field Experiment for Testing the Fungicidal Activity of CHOS with DPn 30 and the Fungicide Delan Against Apple Scab (Venturia inaequalis)

(77) In a field trial the effect of 0.1% CHOS and recommended (0.8%) and 1/10 concentration (0.08%) of Delan on the infection of Venturia inaequalis on apples was investigated. For experimental details see: Field trials with chitosan and the fungicides not containing chitopoly- or chitooligo-saccharides in the Materials and Methods section.

(78) The results in Table 10 shows that the combination of CHOS and 1/10 of the recommended concentration of Delan was more effective than the recommended concentration of Delan

(79) TABLE-US-00010 TABLE 10 Fungicidal activity of CHOS with DP.sub.n 30 and the fungicide Delan (reduction of apples with apple scab) on Venturia inaequalis. % apple with apple Treatment scab Untreated control 31.2 9.7.sup.a Delan 0.8 g/L (800 g ml.sup.1) 20.9 9.5 Delan 0.08 g/L (80 g ml.sup.1) 27.5 12.0 CHOS DP.sub.n 30, 1.0 g/L (1000 g ml.sup.1) 25.9 13.3 Delan 80 g ml.sup.1 + Chitosan DP.sub.n 30, 1000 g ml.sup.1 16.7 5.2 .sup.aStandard deviation