Potentiating agents for protecting plants from fungal infections

Abstract

Disclosed is a method for preventing, controlling or treating a fungal infection on a plant. The method includes applying to such plant organ a non-fungicidal amount or a potentiating amount of a composition including a potentiating agent of a plant defense molecule, in association with a phytopharmaceutical vehicle.

Claims

1. A method for controlling or treating a fungal infection by a phytopathogenic fungal strain on a plant organ, said method comprising the steps of: i) in vitro determining the non-fungicidal concentration of at least one potentiating agent of a plant defense molecule selected from the group consisting of chelerythrine, sanguinarine; and Cl.sup.−, HSO.sub.4.sup.−, I.sup.−, HCO.sub.3.sup.− salts thereof, against the phytopathogenic fungal strain; said determination being carried out by comparing the growth of the phytopathogenic fungal strain cultures in contact with increasing concentrations of said at least one potentiating agent of a plant defense molecule, with the growth of a control culture of the phytopathogenic fungal strain, in the absence of said at least one potentiating agent of a plant defense molecule; the last concentration of the increasing concentrations of the at least one potentiating agent of a plant defense molecule resulting in the same fungal culture growth as the control culture being retained as the non-fungicidal concentration of said at least one potentiating agent of a plant defense molecule; then ii) applying on the plant organ a composition comprising said at least one potentiating agent of a plant defense molecule in the determined non-fungicidal concentration, such that the amount applied is a potentiating amount but is not an amount that is fungicidal per se, in association with a phytopharmaceutical vehicle.

2. The method according to claim 1, wherein said in vitro determination of the non-fungicidal amount of at least one potentiating agent of a plant defense molecule is carried-out by spectrophotometry or nephelometry.

3. The method according to claim 1, wherein said phytopathogenic fungal infection is an infection by a phytopathogenic fungus selected from the group consisting of the genera Alternaria, Sclerotinia and Venturia.

4. The method according to claim 1, wherein said fungal infection is an infection by a phytopathogenic fungus selected from the group consisting of Alternaria brassicicola, Alternaria dauci and Ventuna inaequalis.

5. The method according to claim 1, wherein the plant is selected from the group consisting of plants of the Brassicacae, Apiaceae, Vitaceae and Rosaceae families.

6. The method according to claim 1, wherein the plant is selected from the group consisting of Brassica carinata, Brassica juncea, Brassica oleracea, Brassica napus, Brassica nigra and Brassica rapa.

7. The method according to claim 1, wherein the plant is selected from the group consisting of Brassica oleracea, Daucus carota subsp. Sativa and Malus domestica.

8. The method according to claim 1, wherein the plant organ, the phytopathogenic fungus and the non-fungicidal concentration of the at least one potentiating agent of a plant defense molecule in the applied composition is selected from the following combinations: a. said plant organ belonging to a plant of the Brassicaceae family, said phytopathogenic fungus being a strain of the Alternaria genus, and said non-fungicidal concentration of the at least one potentiating agent of a plant defense molecule in the applied composition being from 1 to 25 μM; or b. said plant organ belonging to a plant of the Apiaceae family, said phytopathogenic fungus being a strain of the Alternaria genus, and said non-fungicidal concentration of the at least one potentiating agent of a plant defense molecule in the applied composition being from 1 to 10 μM; or c. said plant organ belonging to a plant of the Rosaceae family, said phytopathogenic fungus being a strain of the Venturia genus, and said non-fungicidal concentration of the at least one potentiating agent of a plant defense molecule in the applied composition being from 1 to 5 μm.

9. The method according to claim 1, wherein the plant organ, the phytopathogenic fungus and the non-fungicidal concentration of the at least one potentiating agent of a plant defense molecule in the applied composition is selected from the following combinations: a. said plant organ belonging to a plant of the Brassicaceae family plant, said phytopathogenic fungus being Alternaria brassicicola, and said non-fungicidal concentration of the at least one potentiating agent of a plant defense molecule in the applied composition being from 1 to 25 μM; or b. said plant organ belonging to the Daucus carota plant, said phytopathogenic fungus being Alternaria dauci, and said non-fungicidal concentration of the at least one potentiating agent of a plant defense molecule in the applied composition being from 1 to 10 μM; or c. said plant organ belonging to the Malus domestica plant, said phytopathogenic fungus being Venturia inaequalis, and said non-fungicidal concentration of the at least one potentiating agent of a plant defense molecule in the applied composition being from 1 to 5 μm.

10. The method according to claim 1, wherein said plant organ belongs to Brassica oleracea plant, said phytopathogenic fungus being Alternaria brassicicola, and said non-fungicidal concentration of the at least one potentiating agent of a plant defense molecule in the applied composition being from 1 to 25 μM.

11. The method according to claim 1, wherein the composition further comprises a plant defense molecule selected from the group consisting of brassinin, camalexin, resveratrol, 3,5-dihydroxybiphenyl, aucuparin and 6-methoxymellein.

12. The method according to claim 1, wherein the composition further comprises an agent for stimulating the production of a plant defense molecule.

13. The method according to claim 1, wherein the composition further comprises at least one agent for stimulating the production of a plant defense molecule; said agent being selected from the group consisting of acibenzolar-S-methyl, chitosan, laminarin, Reynoutria Sachalinensis extract, calcium prohexadione, harpine, yeast wall extracts, oligogalacturonides and calcium phosphite.

14. The method according to claim 1, wherein the composition further comprises an insecticide and/or a herbicide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a growth curve describing the effect of increasing concentrations of chelerythrin on Alternaria brassicicola.

(2) FIG. 2 is a growth curve describing the effect of increasing concentrations of camalexin on Alternaria brassicicola.

(3) FIG. 3 is a growth curve showing the synergistic effect of 25 μM of chelerythrin and of 10 μM of camalexin on growth of Alternaria brassicicola.

(4) FIG. 4 is a combination of pictures of cabbage leaves (Brassica oleracea cv Bartolo) inoculated with Alternaria brassicicola, and treated with a control solution or with the composition of the invention, as stated in Panel C. (A) unwounded leaves. (B) wounded leaves.

(5) FIG. 5 is a growth curve describing the effect of increasing concentrations of Macleaya cordata extract on Alternaria dauci, showing the non-fungistatic concentration of 10 μM, expressed in the equivalent sanguinarine concentration.

(6) FIG. 6 is a graph showing the non-fungistatic effects of the carrot phytoalexin 6-methoxymellein (6-MM) at a concentration of 125 μM and 100 μM on the growth of Alternaria dauci. The graph further shows that the association of non-fungistatic concentrations of Macleaya cordata extract (10 μM, expressed in the equivalent sanguinarine concentration) and 6-methoxymellein (6-MM, at a concentration of 125 μM and 100 μM respectively) presents synergistic effect and effectively inhibits the growth of Alternaria dauci.

(7) FIG. 7 is a growth curve describing the effect of increasing concentrations of Macleaya cordata extract on Venturia inaequalis, showing the non-fungistatic concentration of 5 μM, expressed in the equivalent sanguinarine concentration.

(8) FIG. 8 is a graph showing the effects of the treatment of apple trees (Malus domestica) against apple scab (Venturia inaequalis) with a non-fungicidal amount of Macleaya cordata extract (2 μM, expressed in the equivalent sanguinarine concentration). The treatment groups are i) treatment with the Macleaya cordata extract two days prior to the fungal inoculation ii) treatment with the Macleaya cordata extract simultaneously with the inoculation and iii) treatment with the Macleaya cordata extract two days past the fungal inoculation. The antifungal effects are calculated on the basis of the percentage of the treated infected leaf surface. The results are compared with the percentage of the infected leaf surface of the corresponding non-treated overlying leaves. A control group of apple trees is treated with water.

EXAMPLES

(9) The present invention is further illustrated by the following examples. In these examples, spectrometry was performed using the SPECTROstar nano device commercialized by BMG LABTECH.

Example 1: Determination of the Potentiating Amount of Chelerythrin

(10) a) In Vitro Determination of the Non Fungicidal Amount of Chelerythrin

(11) Alternaria brassicicola strains were cultivated at 24° C. on potato dextrose (PD) medium (Cat. No. 213200; Becton Dickinson, USA).

(12) For inoculum preparation, conidia were collected from 8-days-old solid cultures by adding PD broth followed by gentle scraping of the agar plates. They were counted in a Thoma's chamber and the conidial suspensions were diluted to the concentration of 10.sup.5 conidia/mL.

(13) Growth was automatically recorded for at least 30 hours at 25° C. using a nephelometric reader (NEPHELOstar Galaxy, BMG Labtech, Germany) equipped with a 635-nm laser as radiation source. During incubation, the 96-well plates were subjected to shaking at 175 rpm for 5 minutes every 10 minutes. Measurements were done every hour with a gain value of 90 and a percentage of the maximum value of 20%. Each well was measured for 0.1 second with a laser beam of 2.5 mm.

(14) Chelerythrin (25 μM or 50 μM, test wells) or 10 μL of DMSO (chelerythrin solvent, control wells) were added on wells, and growth curves were drawn (FIG. 1).

(15) As shown in FIG. 1, a concentration of 25 μM of chelerythrin does not inhibit Alternaria brassicicola growth. Said concentration thus corresponds to a non-fungicidal amount.

(16) b) In Vitro Determination of the Non Fungicidal Amount of Camalexin

(17) Alternaria brassicicola growth was measured as described in Example 1a.

(18) Camalexin (10, 20, 40 or 60 μM, test wells) or 10 μL of DMSO (chelerythrin solvent, control wells) were added on wells, and growth curves were drawn (FIG. 2).

(19) As shown in FIG. 2, the concentrations of 10 and 20 μM of camalexin do not inhibit Alternaria brassicicola growth of more than 20%. Said concentrations thus correspond to non-fungicidal amounts.

(20) c) In Vitro Determination of the Potentiating Amount of Chelerythrin

(21) The potentiating effect of chelerythrin is measured in a suspension of conidia treated with a plant defense molecule, camalexin, which is present at a non-fungicidal amount as determined hereinabove. Said non-fungicidal amount is consistent with the amount of camalexin produced by an infected plant organ.

(22) Alternaria brassicicola growth was measured as described hereinabove.

(23) A non-fungicidal amount of chelerythrin (as determined in Example 1a) and/or a non-fungicidal amount of camalexin (as determined in Example 1b) or 10 μL of DMSO are added per well, and growth curves were drawn (FIG. 3).

(24) As shown in FIG. 3, a surprising synergistic effect, which is a potentiating effect, is shown on growth of Alternaria brassicicola. Indeed, the combination of a non-fungicidal amount of chelerythrin and of a non-fungicidal amount of camalexin leads to a drastic inhibition of Alternaria brassicicola growth. The concentration of 25 μM of chelerythrin is thus a potentiating amount.

Example 2: In Vivo Effect of the Composition of the Invention

(25) Example 2 discloses a protocol for determining the in vivo efficacy of the composition of the invention, comprising a potentiating amount of chelerythrin (25 μM), as determined in Example 1.

(26) 5 μL drops of Alternaria brassicicola conidia suspension (10.sup.5 to 10.sup.3 conidia/mL) were inoculated on intact or prewounded (i.e wherein the synthesis of plant defense molecules such as Brassinin was naturally triggered by the aggression) leaves of B. oleracea cv Bartolo plants at stages 4-6 leaves per plant. Inocula were deposited on the left and right sides symmetrically from the central vein: inocula comprising 25 μM of chelerythrin were deposited on the right side, and inocula comprising DMSO were deposited on the left side. The plants were then maintained under saturing humidity (100% relative humidity). Symptoms were observed at day 6 post-infection (6 dpi).

(27) As shown in FIG. 4, the composition of the invention limits in vivo the fungal infection of cabbage leafs.

Example 3: In Vivo Determination of the Inhibition of PKC by Chelerythrin

(28) 1—Test C of the Invention

(29) Construction of a S. cerevisiae strain overexpressing Alternaria brassicicola Pkc1 The cDNA encoding the Pkc1 gene of Alternaria brassicicola (http://genome.jgi-psf.org/Altbr1/Altbr1.home.html; sequence ref: AB07449.1) was amplified by PCR and cloned into a pYES2-CT vector (Invitrogen, Paisley, UK). The resulting vector (pYES-PKC) was inserted in a BY4743 strain of Saccharomyces cerevisiae.

(30) Growth Monitoring

(31) Growth of this strain was monitored in an inducing medium (GS uracil-free medium supplemented with galactose) in presence of increasing concentrations of chelerythrin (0, 25, 50 or 75 μM), and was compared with the growth of a control strain (BY4743 strain transformed with the empty pYES2-CT vector). Growth was measured by spectrometry (Optic density: 600 nm). Inhibition of growth was assessed by comparison of the area under the curves.

(32) Results are shown in Table 3 below.

(33) TABLE-US-00005 TABLE 3 Condition (concentration of Area under the chelerythrin) curve Inhibition pYES2-CT 15.1 — pYES2-CT (25 μM) 8.6 43% pYES2-CT (50 μM) 3.2 79% pYES2-CT (75 μM) 2.5 84% pYES2-PKC 12.6 — pYES2-PKC (25 μM) 10.7 15% pYES2-PKC 50 (50 μM) 9.4 25% pYES2-PKC 75 (75 μM) 8.8 30%

(34) Chelerythrin inhibits the growth of strains expressing normal levels of Pkc1. The inhibition is less efficient in cells overexpressing Pkc1. Therefore, chelerythrin probably is an inhibitor of Pkc1.

(35) 2—Test D of the Invention

(36) The growth of a BY4743 strain of Saccharomyces cerevisiae on a liquid SD medium comprising increasing concentrations of chelerythrin (0, 10, 15, 20 or 25 μM) was monitored and compared to the growth of the same strain on a liquid SD medium containing 1M of sorbitol (high osmotic pressure conditions) in presence of increasing concentrations of chelerythrin.

(37) Growth was measured by spectrometry (optic density: 600 nm) Inhibition of growth was assessed by comparison of the area under the curves. Results are shown in the Table 4 below.

(38) TABLE-US-00006 TABLE 4 Concentration of Area under the Medium chelerythrin (μM) curve Inhibition (%) SD 0 15.7 — 10 14.3 10 15 11.8 26 20 10.4 35 25 8.1 49 SD + Sorbitol 0 5.0 — 10 5.2 −3 15 5.0 1 20 2.0 0

(39) Chelerythrin inhibits the growth of a wild-type strain in normal osmotic pressure conditions, but not in high osmotic pressure conditions. This result seems to confirm the inhibitory action of chelerythrin on Pkc.

Example 4: Triads Compounds/Plant Defense Molecules/Phytopathogenic Fungus

(40) In the Table 5 below are shown compounds (column 1) having a potentiating effect of a plant defense molecule (column 2) for the inhibition of a pathogenic fungus (column 3). The type of the potentiating agent in this particular situation is given in column 4 (homologous or heterologous). Triads were identified according to Test B.

(41) TABLE-US-00007 TABLE 5 Type of Plant defense potentiating Compound molecule Phytopathogenic fungus agent 1 Resveratrol Alternaria brassicicola Heterologous 2 Brassinin Alternaria brassicicola Homologous Brassinin Botrytis cinerea Homologous Camalexin Botrytis cinerea Homologous 6-methoxymellein Alternaria dauci Homologous 3 Resveratrol Alternaria brassicicola Heterologous 4 Brassinin Alternaria brassicicola Homologous Resveratrol Alternaria brassicicola Heterologous 5 Brassinin Alternaria brassicicola Homologous Resveratrol Alternaria brassicicola Heterologous Coptisin Brassinin Alternaria brassicicola Homologous Camalexin Alternaria brassicicola Homologous Chelerythrin Brassinin Alternaria brassicicola Homologous Camalexin Alternaria brassicicola Homologous

Example 5: Potentiating Effect of Macleaya cordata Crude Extract Against Alternaria Dauci

(42) A crude extract of Macleaya cordata was analyzed phytochemical analysis, namely by means of HPLC-UV chromatography and .sup.1H-NMR spectrometry using p-anisaldehyde as internal standard.

(43) The HPLC-UV at 270 nm showed two major peaks corresponding to sanguinarine and chelerythrine having a retention time of 10.3 minutes and 12.45 minutes respectively.

(44) The .sup.1H-NMR spectrum confirmed that sanguinarine is the major alkaloid and indicated that the molar ratio of sanguinarine/chelerythrine is about 2.8 (based on the deshielded H-5 proton of the benzo[c]phenantridine scaffold at 10.14 ppm for the sanguinarine and 10.09 ppm for the chelerythrine).

(45) The global phytochemical analysis showed that the M. cordata extract contains about 40% of sanguinarine, in weight relative to the total extract.

(46) The non-fungicidal amount of the sanguinarine contained in M. cordata extract against A. dauci strain was determined as 12 μM or 4 ppm according to the protocol described in Example 1. The results regarding the growth inhibition of A. dauci by M. cordata extract are presented in FIG. 5.

(47) Thus, the non-fungicidal amount of M. cordata extract against Alternaria dauci is 10 ppm.

(48) Following the aforementioned growth inhibition protocol, the following treatments are assessed: 6-methoxymellein 125 μM, 6-methoxymellein 100 μM, 6-methoxymellein 125 μM and M. cordata extract (10 μM of Sanguinarine), 6-methoxymellein 100 μM and M. cordata extract (10 μM of Sanguinarine).

(49) The results are illustrated in FIG. 6.

(50) Interestingly, 6-methoxymellein, the phytoalexin of the Daucus carotta (carrot) plant species did not inhibit the growth of Alternaria dauci at the concentration of 100 or 125 μM.

(51) Surprisingly, the association of non-fungicide amounts of 6-methoxymellein and M cordata extract showed a synergistic effect resulting in a significant fungal growth inhibition.

Example 6: In Vitro Non-Fungicidal Amount of Macleaya cordata Crude Extract Against Venturia inaequalis

(52) The non-fungicidal amount of the sanguinarine contained in M. cordata extract against Venturia inaequalis 2557 09BC2014 strain was determined as 5 μM or 1.66 ppm according to the protocol described in Example 1.

(53) Thus, the in vitro non-fungicidal amount of M. cordata extract against Venturia inaequalis is 4.15 ppm.

(54) The results of the growth inhibition of V. inaequalis are presented in FIG. 7.

Example 7: In Planta Effects Against Venturia inaequalis of a Non-Fungicidal Amount of Macleaya cordata Crude Extract

(55) A crude extract of Macleaya cordata was diluted to a final concentration of 20 ppm and 5 ppm (corresponding to 8 ppm and 2 ppm of sanguinarine respectively).

(56) The effect of spraying the diluted Macleaya cordata extract against Venturia inaequalis (apple scab) is assessed in planta on apple tree leaves, Malus domestica.

(57) The antifungal effect is assessed by measuring the percentage of the fungal infection surface on the infected leaves. Spraying the leaves with water is used as a negative control treatment. However, given the differences among the infection rate among the apple tree rows, the effect was compared between the treated leaves and the overlying non-treated leaves of the same tree.

(58) The treatment groups are as follows: M. cordata extract (2 μM Sanguinarine equivalent) sprayed on to the leaves two days prior to their inoculation with V. inaequalis, M. cordata extract (2 μM Sanguinarine equivalent) sprayed on to the leaves along with their inoculation with V. inaequalis, M. cordata extract (2 μM Sanguinarine equivalent) sprayed on to the leaves two days after their inoculation with V. inaequalis, Water sprayed on to the leaves two days prior to their inoculation with V. inaequalis.

(59) The percentage of infected leaves was counted 21 days past their inoculation with V. inaequalis. The results are presented in FIG. 8.

(60) Applying a treatment having a sanguinarine at the non-fungicide concentration of 2 prior, simultaneously or after the inoculation with V. inaequalis, showed a considerable re decrease in the infected surface of the treated leaves according to the present method. The treatment of apple tree leaves with the association of M. cordata extract and the plant defense molecule elicitor (SDP) Bion® (acibenzolar-S-methyl, Sygenta, 400 ppm) is currently undergoing.