Method for fungicidal and/or bactericidal treatment of resistant strains using essential oil(s)

Abstract

The present invention relates to a method for fungicidal and/or bactericidal treatment of plants or foodstuffs using one or more essential oil(s) that enable the treatment of strains resistant to synthetic fungicides and/or bactericides.

Claims

1. A method for fungicidal and/or bactericidal treatment of one or more phytopathogenic strain(s) of fungi and/or bacteria resistant to one or more synthetic fungicidal and/or bactericidal agent(s) comprising: applying a composition comprising clove oil on plants or foodstuffs that are infected by said phytopathogenic strain; and applying one or more said synthetic fungicidal and/or bactericidal agent(s) on said plants or said foodstuffs, wherein the clove oil and the fungicidal and/or bactericidal agent(s) are applied in a ratio of the clove oil to the fungicidal and/or bactericidal agent(s) part of between 0.3 and 3, wherein said one or more said synthetic fungicidal and/or bactericidal agent(s) is pyrimethanil, and wherein said phytopathogenic strain is Penicillium digitatum.

2. The method according to claim 1, wherein the fungicidal and/or bactericidal agent(s) is (are) applied simultaneously with the clove oil, or separately.

3. The method according to claim 1, wherein the application is carried out before or after harvesting.

4. The method according to claim 1, wherein the application of the said composition is carried out by means of spraying, dipping and/or drenching.

5. The method according to claim 1, wherein the composition further comprises one or more emulsifiers.

6. The method according to claim 1, wherein said composition before dilution comprises between 50 and 350 g/L with respect to clove oil, before eventual dilution.

7. The method according to claim 5, wherein the composition is in the form of an emulsifiable concentrate and that the method further comprises a preliminary step of dispersing said concentrate in water before application.

8. The method according to claim 1, wherein said composition comprising clove oil and said one or more fungicidal and/or bactericidal agent(s) are both applied, simultaneously or separately, before harvesting said plants or said foodstuffs.

9. The method according to claim 1, wherein said clove oil in combination with said one or more synthetic fungicidal and/or bactericidal agent(s) has a synergistic effect.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the efficacy of the combinations of the invention on strains of Penicillium expansum resistant to pyrimethanil.

(2) FIG. 2 shows the efficacy of the compositions on the strains of Penicillium digitatum that are sensitive to pyrimethanil.

EXAMPLES

Example 1: Example of a Composition According to the Invention

(3) Emulsifiable Concentrate (in g/L)

(4) TABLE-US-00001 Non ionic emulsifier 240 Anionic emulsifier 180 Essential oils 160 Pyrimethanil 190 Lecithin 60 Mono-propylene glycol 200

Example 2: Fungicidal Activity of the Essential Oils Alone or in Combination with a Synthetic Fungicide, on Resistant or Sensitive Strains

(5) The table below shows various fungicidal and/or bactericidal compositions

(6) TABLE-US-00002 Composition A B C Pyrimethanil 400 g/L 192 g/L Essential oil/ 140 g/L of eugenol 180 g/L of eugenol Terpene 20 g/L of agent peppermint oil Lecithin 60 g/L 250 g/L Total 1,000 mL 1,000 mL 1,000 mL

(7) These compositions have been tested on various fungi or bacteria strains, such as illustrated in FIGS. 1 and 2. Unless otherwise indicated, the doses listed in the FIGS. 1 and 2 are given in ppm of active ingredient; the indications in ml/l refer to the quantity of composition C. In FIG. 1 (testing done on Gala apples), the composition B, comprising pyrimethanil, eugenol and peppermint oil exhibits an efficacy of between 90 and 100% while pyrimethanil alone at the same dose showed an efficacy of at most 60%. We also see that clove oil alone (composition C) does not exceed 80% efficacy. Indeed it is observed that there is a synergistic effect between the fungicide and essential oils/terpene agents.

(8) In FIG. 2 (tests carried out on lemon), one observes on strains of Penicillium digitatum sensitive to pyrimethanil, the efficacy of the composition comprising pyrimethanil and essential oils/terpene agents (composition B). In addition, it is clearly observable that the essential oils/terpene agents alone (composition C) are not effective against susceptible strains, with maximum efficacy at about 40%.

Example 3: Assessment of the In-Vitro Efficacy of Essential Oils Against Phytopathogenic Micro-Organisms

(9) Objective of the Study:

(10) The objective of the study was to determine the efficacy of essential oils against phytopathogenic fungi Penicillium Spp., Botrytis Spp., Monilinia fructicola and a bacterium, Pseudonomas syringae, compared to commercial synthetic fungicidal and bactericidal products.

(11) The efficacy of samples of essential oils against these phytopathogenic microorganisms was determined by means of counting colonies (determination of CFU colony forming units), cultured at 25 C.+/1 C. for bacteria and by means of determining the inhibition of the Halo growth for fungi, in accordance with the general guidelines (ISO 6887:2003; ISO 7218:2007; ISO 7954:1987).

(12) Samples Tested:

(13) TABLE-US-00003 Active Content in active Formulation Treatment ingredient(s) ingredient type Fungazil 500 EC Imazalil 50% EC TECTO SC 500 Tiabendazole 42.9% SC Major C 100 Alkyl dimethyl 2.5% SL benzyl ammonium chloride Xedathane 20 Pyrimethanil 20% EC Bioxeda Cloves oil 20% EC Thymol Thymol 18% EC Carvacrol Carvacrol 18% EC Cinnamon oil Cinnamaldehyde 20% EC
Description of the Tests Carried Out:

(14) TABLE-US-00004 Assessment Sample Doses Application Duration of efficacy Phytopathogenic fungus Pennicillum spp. 1) Fungazil Fungazil: By adding into 5 days Inhibition of 100 ml/hl artificial media growth on 2) Thymol Thymol: PDA (Potato Petri dishes 100 ml/hl Dextrose Agar) 300 ml/hl 500 ml/hl 1,000 ml/hl 3) Fungazil + Fungazil + Thymol Thymol: 100 ml/hl + 100 ml/hl 100 ml/hl + 300 ml/hl 100 ml/hl + 500 ml/hl 100 ml/hl + 1000 ml/hl Botrytis spp. 1) Tecto SC Tecto SC By adding 5 days Inhibition of 500 500: into artificial growth on 100 ml/hl media PDA Petri dishes 2) Carvacrol Carvacrol: (Potato 100 ml/hl Dextrose 300 ml/hl Agar) 500 ml/hl 1,000 ml/hl 3) Tecto SC Tecto SC 500 + 500 + Carvacrol Carvacrol: 100 ml/hl + 100 ml/hl 100 ml/hl + 300 ml/hl 100 ml/hl + 500 ml/hl 100 ml/hl + 1000 ml/hl Monilinia 1) Xedathane By adding 5 days Inhibition of fructicola Xedathane 20: into artificial growth on 20 250 ml/hl media PDA Petri dishes 2) Bioxeda Bioxeda: (Potato 100 ml/hl Dextrose 300 ml/hl Agar) 500 ml/hl 1,000 ml/hl 3) Xedathane Xedathane 20 + 20 + Bioxeda: Bioxeda 250 ml/hl + 100 ml/hl 250 ml/hl + 300 ml/hl 250 ml/hl + 500 ml/hl 250 ml/hl + 1000 ml/hl Phytopathogenic bacterium Pseudomonas 1) Major C Major C By adding 24 determination syringae. 100 100: into artificial hours of CFU 250 ml/hl media NA 2) Cinnamon Cinnamon (nutritional oil oil: agar) 100 ml/hl 300 ml/hl 500 ml/hl 1,000 ml/hl 3) Major C Major 100 + C100 + Cinnamon oil Cinnamon oil 250 ml/hl + 100 ml/hl 250 ml/hl + 300 ml/hl 250 ml/hl + 500 ml/hl 250 ml/hl + 1000 ml/hl
Description of the Methods:

(15) TABLE-US-00005 Growth Micro-organism Method Diluent Culture media Replicates Conditions Incubation 1) Penicillium Seeding Physiological PDA, 4 25 C. Fungi: 5/6 spp. solution Pseudomonas 1 C. days 2) Botrytis spp. Agar Bacteria: 3) Monilinia (only for P. syringae) 24 h fructicola 4) Pseudomonas syringae
Analysis:

(16) The samples were added to the artificial media and gently mixed before solidification of the Agar solution.

(17) Efficacy Against Fungi:

(18) A small amount of fungal spores is placed in the centre of the potato dextrose agar (PDA) in the Petri dishes at 251 C. for 5/6 days. At the end of the incubation period, we measure the inhibition of the halo that is formed in comparison with the untreated PDA Petri dishes (control).

(19) Efficacy Against Bacteria:

(20) A suspension of spores of phytopathogenic microorganisms was prepared and an appropriate amount of dilutions of interest (100 microliters) is added to the petri dishes of Pseudomonas Agar and pressed at the agar surface by means of a sterile L spatula.

(21) The petri dishes were incubated at 25 C.+1 C. for 24 hours. At the end of the incubation the colony forming units (CFU/ml of product) were determined.

(22) Evaluation of the Results:

(23) The results are indicated as a percentage of the incidence of the disease and the efficacy percentage of the products tested.

(24) Pennicillum spp.

(25) Samples:

(26) Fungazil Thymol Fungazil+thymol

(27) TABLE-US-00006 Incidence Phytopathogenic of Disease Efficacy fungus Sample (%) (%) Dose ml/hl Pennicillum spp. Untreated 100 0 Fungazil 5 95 100 ml/hl (imazalil) Thymol 80 20 100 ml/hl 50 50 300 ml/hl 30 70 500 ml/hl 30 70 1,000 ml/hl Fungazil + 0 100 100 + 100 ml/hl Thymol 0 100 100 + 300 ml/hl 0 100 100 + 500 ml/hl 0 100 100 + 1,000 ml/hl
Pennicillum spp.Strain Resistant to Imazalil
Samples: Fungazil Thymol Fungazil+thymol

(28) TABLE-US-00007 Incidence Phytopathogenic of Disease Efficacy fungus Sample (%) (%) Dose ml/hl Pennicillum spp. Untreated 100 0 Strain resistant to Fungazil 85 15 100 ml/hl Imazalil Thymol 30 70 100 ml/hl 30 70 300 ml/hl 20 80 500 ml/hl 10 90 1,000 ml/hl Fungazil + 10 90 100 + 100 ml/hl Thymol 10 90 100 + 300 ml/hl 5 95 100 + 500 ml/hl 0 100 100 + 1,000 ml/hl
Botrytis spp.Strain Resistant to Thiabendazole
Samples: Tecto SC 500 Carvacrol Tecto SC 500+Carvacrol

(29) TABLE-US-00008 Incidence Phytopathogenic of Disease Efficacy fungus Sample (%) (%) Dose ml/hl Botrytis spp. Untreated 100 0 Strain resistant to TBZ 100 0 100 ml/hl TBZ Carvacrol 0 100 100 ml/hl 0 100 300 ml/hl 0 100 500 ml/hl 0 100 1,000 ml/hl TBZ + 0 100 100 + 100 ml/hl Carvacrol 0 100 100 + 300 ml/hl 0 100 100 + 500 ml/hl 0 100 100 + 1,000 ml/hl
Monilinia fructicola
Samples: Xedathane 20 Bioxeda Xedathane 20+Bioxeda

(30) TABLE-US-00009 Incidence Phytopathogenic of Disease Efficacy fungus Sample (%) (%) Dose ml/hl Monilinia Untreated 100 0 fructicola Xedathane 5 95 250 ml/hl 20 Bioxeda 75 25 100 ml/hl 70 30 300 ml/hl 30 70 500 ml/hl 20 80 1,000 ml/hl Xedathane 0 100 250 + 100 ml/hl 20 + 0 100 250 + 300 ml/hl Bioxeda 0 100 250 + 500 ml/hl 0 100 250 + 1000 ml/hl
Monilinia fructicolaStrain Resistant to Pyrimethanil
Samples: Xedathane 20 Bioxeda Xedathane 20+Bioxeda

(31) TABLE-US-00010 Incidence Phytopathogenic of Disease Efficacy fungus Sample (%) (%) Dose ml/hl Monilinia Untreated 100 0 fructicola Xedathane 55 45 250 ml/hl Strain resistant 20 to Pyrimethanil Bioxeda 30 70 100 ml/hl 20 80 300 ml/hl 25 75 500 ml/hl 20 80 1,000 ml/hl Xedathane 10 90 250 + 100 ml/hl 20 + 5 95 250 + 300 ml/hl Bioxeda 3 97 250 + 500 ml/hl 5 95 250 + 1000 ml/hl
Pseudomonas syringaeStrain Resistant to Quaternary Ammonium Compounds
Samples: Major C 100 Cinnamon oil Major C 100+Cinnamon oil

(32) TABLE-US-00011 Phyto- Incidence pathogenic of Disease Efficacy fungus Sample (%) (%) Dose ml/hl Pseudomonas Untreated 100 0 syringae Major C 100 75 25 250 ml/hl Strain resistant Cinnamon 70 30 100 ml/hl to quaternary oil 35 65 300 ml/hl ammonium 5 95 500 ml/hl compounds 5 95 1,000 ml/hl Major + 35 65 100 + 100 ml/hl Cinnamon 25 75 100 + 300 ml/hl oil 0 100 100 + 500 ml/hl 0 100 100 + 1,000 ml/hl

CONCLUSION

(33) The plant essential oils show a broad spectrum of activity against fungi, phytopathogenic bacteria, which may be summarised as follows:

(34) Penicillium spp.:

(35) The fungi are present in 100% of the untreated Petri dishes. Thymol shows moderate activity (efficacy comprised between 20% and 70% at a dose of between 100 and 1,000 ml/hl), whereas the combination of thymol and Imazalil enables full control at all the doses tested.

(36) Penicillium spp., Strain Resistant to Imazalil.

(37) The fungi are present in 100% of the untreated Petri dishes. The thymol shows strong activity (efficacy comprised between 70 and 90% at a dose of between 100 and 1,000 ml/hl). Total control may be achieved by the combination of Imazalil and thymol at the dose of 100+1,000 ml/hl.

(38) Botrytis spp., Strain Resistant to Thiabendazole.

(39) The fungi are present in 100% of the untreated Petri dishes.

(40) Thiabendazole shows no efficacy at a dose of 100 ml/hl, while carvacrol allows total control of this strain at all doses tested. The same holds true for the combination of thiabendazole and carvacrol.

(41) Monilinia fructicola:

(42) The fungi are present in 100% of the untreated Petri dishes.

(43) Clove oil enables moderate control of fungi at all doses tested, while clove oil in combination with pyrimethanil allows total control of fungi at all doses tested.

(44) Monilinia fructicola, Strain Resistant to Pyrimethanil:

(45) Pyrimethanil enables only 45% efficacy, while the clove oil allows efficacy of between 70 and 80% at all doses tested.

(46) The combination of clove oil and pyrimethanil allows almost total control at the doses tested.

(47) Pseudomonas syringae, Strain Resistant to Quaternary Ammonium Compounds.

(48) The bacterium is present in 100% of the untreated Petri dishes.

(49) The quaternary ammonium tested shows an efficacy of only 25% whereas the cinnamon oil enables an efficacy of up to 80% at 1,000 ml/hl.

(50) The combination of quaternary ammonium with cinnamon oil allows full control at all doses of cinnamon oil tested and at a dose that is less than half the comparison dose of the quaternary ammonium compound (100 ml/hl vs. 250 ml/hl).