Therapeutic or non-therapeutic use of protozoans of the <i>Willaertia </i>genus as a fungistatic and/or fungicide

Abstract

The invention relates to a therapeutic or non-therapeutic use of protozoans, for examples protozoans of the Willaertia magna amoeba species, as a fungistatic and/or fungicide.

Claims

1. A method for combating proliferation of fungi, excluding treatment methods applied to a human or animal body, comprising a step of bringing protozoa of Willaertia magna species into contact with said fungi, wherein the protozoa of the Willaertia magna species are used in dead cell lysate form.

2. The method as claimed in claim 1, wherein the protozoa correspond to a strain deposited under number PTA-7824 at ATCC.

3. The method as claimed in claim 1, for combating the proliferation of parasitic fungi of plants.

4. The method as claimed in claim 3, for treating fungal diseases of vine, of cereals, of potatoes, of fruit trees and of market garden crops.

5. The method as claimed in claim 1, (i) for disinfecting networks of air treatment facilities, (ii) for combating fungal contaminations and/or formation of fungal biofilms in food products, food product transformation/production equipment, food product transformation/production factories or cosmetic products, surfaces that come into contact with food products or cosmetic products, or (iii) for treating contaminations of interior environments.

6. The method as claimed in claim 3, wherein the parasitic fungi is chosen from B. cinerea, E. necator, F. solani, C. globosum, P. chrysogenum, P. viticola, P. infestans, T. viride, T. harzianum, A. fumigatus, A. alternata, A. flavus, P. variotii, A. niger, C. albicans, U. botrytis, E. nigrum, B. cinerea, P. roqueforti, C. cladosporioides, E. chevalieri, A. pullulans and a combination thereof.

7. The method of claim 4, wherein the fungal disease is selected from the group consisting of vine powdery mildew, vine downy mildew, cereal septoria disease, fusaria disease, cereal net blotch, potato downy mildew, early blight, apple tree scab, sclerotinia stem rot of rape, sclerotinia stem rot of vegetable crops and combinations thereof.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Effect of the amebae on the germination of B. cinerea. Tests in a microplate in the presence of the B. cinerea spores without amebae. Observation after 24 H of incubation. Sensitive strains: B05.10 and M421. Resistant strain: M426. (respiration-inhibiting fungicide)

(2) FIG. 2: Effect of the amebae on the germination of B. cinerea. Tests in a microplate in the presence of the B. cinerea spores+500 amebae in MC medium and B. cinerea spores+. Sensitive strains: B05.10 and M421. Resistant strain: M426. (respiration-inhibiting fungicide). 50 000 amebae in MC medium. Observation after 24 H of incubation.

(3) FIG. 3: Effect of the culture medium (MC) on the germination of B. cinerea sensitive strains. Tests in a microplate in the presence of the B. cinerea spores+culture medium (MC). Sensitive strains: B05.10 and M421. Observation after 24 H of incubation.

EXAMPLES

(4) The examples hereinafter make it possible to illustrate the invention but are not limiting in nature.

Example 1: qPCR Analysis of the Fungal DNA of the Fungus—Willaertia Interactions on 19 Strains

(5) The interaction tests on 19 strains of fungi (table 1) were carried out by bringing the spores into contact with the “Willaertia magna” ameba reference ATCC PTA-7824 according to an ameba/spore ratio=10 amebae/spores, in 24-well plates.

(6) In a first plate, spores alone of each strain were incubated under the same conditions (in 2 ml of MEAc). After 3 H of contact, the solution is centrifuged at 14 000 rpm for 5 minutes. The supernatant is then removed and the pellet taken up in 200 μl for the DNA extraction.

(7) For the tests at 24 H carried out in a second plate, the supernatant is directly removed from the well after 24 hours and the “spores-amebae mixtures” are taken up in 200 μl in order to extract the DNA. Monitoring by microscopic observation is performed at each step. A DNA extraction is carried out on the samples (200 μl) using the “NucleoSpin plant II—Macherey-Nagel” kit according to the manufacturer's instructions after a milling step with fine sand.

(8) The qPCR amplification of the DNA of the fungi alone and of the Willaertia-fungi complexes was carried out on a CFX96-Biorad instrument. The quantification is carried out in a final volume of 20 μl containing 5 μl of DNA, the primers (300 nM) and 10 μl of SybrGreen-Mix. The following program was used: a step of denaturation at 95° C. for 10 minutes with a step of amplification at “95° C. for 15 seconds+60° C. for 1 minute” of 35 cycles.

(9) The tests carried out on the 19 strains of fungi are carried out according to the protocol validated in the preceding step. The plates are observed under a microscope before launching the molecular analysis. The results are qPCR curves obtained in triplicate for each condition (spores-3 H, spores-24 H, spore-ameba3 H and spore-ameba24 H). Given that the qPCR analysis uses universal primers, a range of the P. chrysogenum fungus was used to approximately estimate the amount of DNA amplified for each test. These data make it possible to evaluate the passage from the spore stage to the mycelium in the controls without ameba and also the degree of inhibition of the germination in the presence of the amebae.

Results and Conclusion

(10) TABLE-US-00001 TABLE 1 Difference in qPCR amplification between the tests carried out on the spores alone (difference between 3 and 24 hours) and the spores undergoing interaction with the amebae after 24 H of contact. ΔCq (3 H-24 H): difference in number of cycles between the spores at 3 H and 24 H. ΔCq (T24 H-Willaertia24 H): difference in number of cycles between the spores alone (T24 H) and in presence of amebae (Amebae 24 H) after 24 h of incubation. ΔCq (T24 H- Willaertia Strain ΔCq (3 H-24 H) 24 H)* F. solani 3.3 −4.1 C. globosum 7.6 −3.8 P. chrysogenum 8.4 −7.0 T. viride 13.3 −12.9 T. harzianum 9.3 −8.7 A. fumigatus 7.4 −8.7 A. alternata 3.4 −3.3 A. flavus 4.5 −9.3 P. variotii 6.2 −6.6 A. niger 8.92 −9.63 C. albicans 10.7 −3.3 U. botrytis 4.0 −5.0 E. nigrum 0.1 −4.6 B. cinerea 4.9 −6.2 C. herbarum 0.9 −0.8 P. roqueforti 6.4 −13 C. cladosporioides 12.0 −11.1 E. chevalieri 1.9 −4.5 A. pullulans 7.7 −7.1 *the values of ΔCq (T24 H-Willaertia24 H) are negative since the values of Cq corresponding to the “fungus-ameba 24 H” tests (low fungal biomass) are greater than those of the control tests at 24 H (high biomass).

(11) The qPCR analysis made it possible to quantify the fungal biomass and to distinguish the stages of development of the fungi and to show the fungistatic and/or fungicidal effect on 19 treated strains with very significant differences. The difference in number of Cq cycles between the two tests with and without amebae is very significant and, in certain cases, exceeds 10 cycles. These results reflect the presence of a very low fungal biomass in the presence of the amebae after 24 H of incubation following the inhibition of the spore germination. The difference between the spore stage at 3 H and the mycelium developed after 24 H is clearly observed by the difference in amplification between the spores at 3 H and 24 H on tested strains. The qPCR results perfectly reflect the visual observations carried out under a microscope for developing this protocol.

Example 2: Study of the Effect of Willaertia magna on the Growth of Botrytis cinerea

(12) Principle: the tests carried out are based on the tests used in the laboratory to evaluate the efficacy of conventional fungicidal molecules. The effect of the Willaertia magna ameba (strain ATCC PTA-7824) on the germination of the B. cinerea spores is analyzed by bringing various concentrations of amebae into contact with spores of the fungus. After 24 hours of incubation, the germination of the B. cinerea spores in the presence of ameba is compared to the control without amebae.

(13) Protocol: the B. cinerea strains are maintained on culture medium (based on oat flakes) under alternating day-UV light (12 H/12 H) at 21° C. The strains have phenotypes sensitive or resistant to a fungicide belonging to the cell respiration inhibitors. The references of the strains tested are: strain B05.10 (sensitive), M421 (sensitive isolated from the field) and M426 (resistant isolated from the field). In order to test the effect of the Willaertia magna ameba on the germination of B. cinerea conidia, the spores of the fungus are incubated with various concentrations of amebae. In a 96-well plate, 500 B. cinerea spores are brought int contact with 5×10.sup.2 and 5×10.sup.4 amebae per well in a final volume of 50 μl. The amebae tested are prepared in culture medium (MC).

(14) The following controls are also prepared under the same conditions: B. cinerea spores in a YBA liquid culture medium B. cinerea spores in culture medium (MC) B. cinerea spores with the respiration-inhibiting fungicide

(15) Each condition is tested in triplicate. The plates are incubated in the dark at 21° C. for 24 H or 48 H. The germination of the B. cinerea conidia is observed under an inverted microscope and a series of photos is taken for each condition.

(16) Results and conclusion: The observation of the spores after 24 H made it possible to demonstrate a slowing of the germination of the fungus at high amebae concentrations. Regardless of the B. cinerea strain, this effect is much more visible when the fungus is in contact with amounts of amebae forming a confluent layer (FIG. 2). With lower ratios (500 amebae) the effect is less visible compared to the controls (FIG. 1 and FIG. 2). The B05.10 strain is a collection strain and it is less vigorous/lively. Compared to the wild-type strains (taken in the field), it will take more time to develop. The culture medium (MC) alone does not affect the growth of the fungus, the culture medium even appears to stimulate the germination of the B. cinerea spores (FIG. 3).

(17) These results show an efficacy of Willaertia magna on the germination of B. cinerea under the experimental conditions, with a qualitative dose effect observed.

Example 3: Efficacy of Willaertia magna on Vine (P. viticola) Downy Mildew

(18) Principle: the tests are carried out on young Cabernet sauvignon plants. Each group of plants is treated under control conditions with a pump sprayer. After treatment of the plants with the solutions tested, an intentional infection of the plants with vine downy mildew is carried out. After 7 to 10 days of incubation, the seriousness of the disease is evaluated in order to evaluate the efficacy of various products with respect to the seriousness of the disease on the nontreated plants.

(19) Protocol:

(20) The cuttings of Cabernet sauvignon plants are produced in a greenhouse (temperature 15° C.-30° C.) and six groups of six plants are formed for the study of each test condition.

(21) The Willaertia magna cells are cultured in a culture medium until the desired concentration of 9×10.sup.9 cells per liter is obtained. Various samples are tested: 1. Negative control, condition 1: without treatment. 2. Control of the medium, condition 2: dilution medium 3. Fungicide, condition 3: Willaertia magna in culture medium 4. Fungicide, condition 4: sample of condition 3, diluted in dilution medium 5. Fungicide, condition 5: a sample of condition 3 is placed under mechanical stress to kill the Willaertia cells. The sample is passed under pressure through a virtually closed, strong-vacuum valve by means of a pump, leading to the death of some of the cells and the lysis of some of the dead cells. The sample obtained is observed under a microscope, with a non-viability label (Trypan blue) in order to count the remaining live cells (not labeled with Trypan blue) 6. Fungicide, condition 6: a sample of condition 4 is placed under mechanical stress to kill the Willaertia cells. The sample is passed under pressure through a virtually closed, strong-vacuum valve by means of a pump, leading to the death of some of the cells and the lysis of some of the dead cells. The sample obtained is observed under a microscope, with a non-viability label (Trypan blue) in order to count the remaining live cells (not labeled with Trypan blue) 7. Positive control, condition 6: bordeaux mixture equivalent to 3 kg/ha.
Each sample is applied on one of the groups of six plants by spraying on the whole plant.
24 hours later, a suspension of P. viticola sporanges is sprayed on the lower face of the leaves. The plants are then incubated at 24±5° C., illuminated for 14 hours per day, in individual chambers at a humidity favorable to the development of the disease. After 7 to 10 days, an evaluation of the fungicide efficacy under each condition is carried out, for each plant and each leaf stage. The evaluation is based on visual observations of the fungal lesion, consisting of two elements: the infected surface area and the intensity of the sporulation. A scale of 0 to 100% (in 5% increments) is used; each leaf being observed individually. The efficacy is calculated according to the following formula: efficacy=100×[(NT−T)/NT] (NT being the average degree of attack on the nontreated plants and T the average degree of attack on the treated plants). The results are presented in table 2.

(22) TABLE-US-00002 TABLE 2 Test conditions and efficacy results Average degree of attack Willaertia Volume of the magna sprayed downy Fungicide Condition Treatment with: concentration (ml) mildew efficacy 1 Negative control: 0 175 56.3%   0% nontreated 2 Control: 0 160 55.0%  2.2% Willaertia magna dilution medium 3 9.02 × 10.sup.9 live 9.02 × 10.sup.9 live 190 1.3% 97.8% Willaertia magna/ Willaertia magna/ liter in culture liter medium 4 Condition 3 2.80 × 10.sup.9 live 200 2.5% 95.6% diluted in Willaertia magna/ Willaertia magna liter dilution medium 5 Condition 3 + At least 80% of 175 1.3% 97.8% mechanical dead cells inactivation compared to condition 3 6 Condition 4 + At least 96% of 185 30.0% 46.7% mechanical dead cells inactivation compared to condition 4 7 Positive control: 0 180 0.0%  100% bordeaux mixture (equivalent 3 kg/ha)
Results and conclusion: the severity of the disease is high (56.3%) on the nontreated plants. This value is also high (55%) after spraying the dilution medium.
A very small amount of disease is observed on the plants treated with Willaertia magna, in live form: the infection with P. viticola is 2.5 to 1.3% of the surface area of the leaves (compared with 56% for the nontreated negative control). That is to say an efficacy of more than 95%, close to the efficacy of the reference fungicide (bordeaux mixture at 3 kg/ha) which enables a complete control of vine downy mildew with an efficacy of 100% (no downy mildew attack).
A small amount of disease is observed on the plants treated with Willaertia magna in dead form, inactivated by mechanical action by passing through a pump and strong-vacuum valve: the infection with P. viticola is from 30% to only 1.3% of the surface area of the leaves (compared with 56% for the nontreated negative control).
In conclusion, Willaertia magna, in live form and in dead form, provides a high activity against vine downy mildew under the experimental conditions.