BIOFUNGICIDE BASED ON THE BACTERIUM PSEUDOMONAS LINI STRAIN S57 AND METHOD OF USE FOR THE CONTROL OF PHYTOPATHOGENIC FUNGI

Abstract

The invention relates to a biofungicide based on the bacterium Pseudomonas lini strain S57, RGM 2930 CChRGM (Chilean Collection of Microbial Genetic Resources) for agricultural use, especially in saline-boric environments. The biofungicide can be developed as solid and liquid formulations. Where the strain S57, has fungicidal properties against the phytopathogenic fungi Botrytis cinerea. Fusarium oxysporum, Monilinia fructicola, Alternaria sp., Geotrichum candidum, and Macrophomina phaseolina, both in the presence and absence of saline-bone conditions.

Claims

1. Biofungicide tolerant to saline-boric conditions wherein it comprises the S57 strain of Pseudomonas lini RGM 2930 CChRGM and an appropriate support medium for agriculture.

2. Biofungicide according to claim 1 wherein it comprises the support medium selected from the group consisting of water, aqueous solutions, thick suspensions, granules and powders.

3. Biofungicide according to claim 2, wherein the support is granule or powder, comprising a carrier that is chosen between kaolinite, diatomite, bentonite, carboxymethylcellulose, starch, maltodextrin and mixtures thereof.

4. Biofungicide according to claim 2, wherein it comprises a suspension of Pseudomonas lini strain S57 RGM 2930 CChRGM in a concentration between 1×10.sup.4 and 1×10.sup.27 CFU/mL, or by CFU/g in the case of solids.

5. Biofungicide according to claim 4, wherein it additionally includes additives selected from the group consisting of fertilizer, nematicide, surfactants, UV protection systems and mixtures thereof.

6. Method to prevent or control infestation by phytopategen fungi wherein it includes contacting crops, seeds, plantlets, seedlings, plants or irrigation water, with the biofungicide of claim 1.

7. Method according to claim 6 wherein the crop, seed, plantlet or seedling or plant is chosen between vegetables, fodder plants or ornamental plants such as flowers.

8. Method according to claim 7 wherein the plantlet or seedling or plant crop is chosen from one of the following agronomic species: chili, cotton, rice, pea, eggplant, beetroot, sweet potato, barley, onion, rapeseed/canola, strawberry, sunflower, corn, peanut, apple, lettuce, potato, cucumber, peppers, beans, soybeans, sorghum, tomato, wheat, carrot, alstroemeria, carnation, gerbera, gladiolus, gypsophila.

9. The method of claim 8, wherein the fungal infestation in crops, seeds, plantlets or seedlings, or plants is mediated by fungi of the genera Botrytis, Fusarium, Monilinia, Alternaria, Geotrichum, and Macrophomina.

10. The method of claim 9, wherein the infestation by fungi in crops, seeds, plantlets or seedlings, or plants is mediated by fungi of the species Botrytis cinerea, Fusarium oxysporum, Monilinia fructicola, Alternaria sp., Geotrichum candidum, and Macrophomina phaseolina.

11. The method of claim 6, wherein crops, seeds, plantlets, seedlings, plants or irrigation water, are found in saline-boric environments.

Description

EXAMPLES

[0025] 1. Obtaining Pseudomonas lini

[0026] The bacterium was isolated from samples of Oregano root from Socoroma (S18° 15′ 47.8″, W69° 35′ 46.3″; height 2.892 as). The plants were grown with triple phosphate, and lamb and poultry manure.

[0027] The samples were collected aseptically using a metal shovel disinfected with 70% ethanol, and stored in sterile plastic bags. The samples were kept at 4° C. in a refrigerator and immediately transported to the laboratory for processing.

[0028] The bacteria were obtained by cutting the root into small parts, which were disinfected using 95% ethanol (v/v) for 2 minutes, 2% sodium hypochlorite (v/v) for 2 minutes, and 70% ethanol (v/v) for 2 minutes, followed by two washes with sterile distilled water for 2 minutes each time. The disinfected portions were placed on King's B media plates containing (per liter): 20 g of peptone. 10 ml of glycerol, 1.5 g of K.sub.2HPO.sub.4, 1.5 g of MgSO.sub.4×7H.sub.2O and 15 g of agar (pH 7.0). The plates were incubated at room temperature for 1 week or until microbial growth was observed. The bacteria from enriched cultures were isolated by serial dilution in King's B liquid medium and sown in plates of the same medium supplemented with 15 g/L agar-agar. Incubations were performed at room temperature. The isolated colonies were transferred to liquid medium. These procedures were repeated until a single, homogeneous morphology was observed.

[0029] Once isolated, it was identified. The genomic DNA of the bacterium was obtained using the RNeasy Ultraclean Microbial (QIAGEN) kit according to the procedure described by the manufacturer. The 16S rRNA gene was amplified by PCR using the bacteria-specific primers 27F and 1492R. The results of the sequencing indicated that the isolated strain corresponds to Pseudomonas lini, the internal identification number S57 was assigned and was deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) under the access number RGM 2930 on Feb. 5, 2020, in accordance with the Budapest Treaty.

[0030] 2. Biofungicidal Activity In Vitro

[0031] The inhibition of the growth, in vitro, of phytopathogenic fungi was studied: for this purpose, a dual culture assay was carried out in plates of potato dextrose agar (PDA), which were inoculated with a phytopathogenic fungus (independently with Botrytis cinerea, Geotrichum candidum, fructicola, Fusarium oxysporum, Macrophominia phaseolina and Alternaria sp. in the center of each plate and aliquots of 15 μl of the biofungicide of the invention (1×10 UCF/mL) surrounding the fungus according to the method described by Salvaterra-Martinez et al. (2015). As controls, PDA plates inoculated only with the fungus in the center were used Inhibition of the radial growth of miceli or (ICRM) of the fungus was calculated using the equation: ICRM=[(C−T)/C]×100; where C is the area of fungal growth on the control plate, and T is the area of fungal growth with treatment. The bacterium Pseudomonas lini strain S57 showed antifungal activity against all fungi tested. These results demonstrate the capacity of the strain of the invention as a bio controller of phytopathogenic fungi, or biofungicide.

TABLE-US-00001 TABLE 1 Inhibition, in vitro, of the growth of six phytopathogenic fungi by the bacterium Pseudomonas lini strain S57. Phytopathogenic fungus ICRM (%) Botrytis cinerea 48.3 Fusarium oxysporum 23.5 Geotrichum candidum 58.6 Monilinia fructicola 67.2 Macrophomonia phaseolina 64.6 Alternaria sp. 38.1

[0032] 3. Tolerance, In Vitro, Under Saline-Boric Conditions

[0033] Tolerance to NaCl and H.sub.3BO.sub.3: whether the candidate bacterium was able to grow in the presence of different concentrations of NaCl (8 g/L, 15 g/L and 20 g/L) or H.sub.3BO.sub.3 (10 ppm, 50 ppm and 100 ppm) was determined. In addition, the tolerance of the bacterium to increasing concentrations of NaCl and H.sub.3BO.sub.3 was determined (emulating the concentrations of Lluta irrigation water: 20 ppm boron and 14.64 mmol/L sodium). Whether or not bacterial growth was observed at the different concentrations tested was recorded.

[0034] The inhibition of the growth, in vitro, of fungi in the presence of NaCl and H.sub.3BO.sub.3 was studied: a dual culture test was carried out in PDA plates, which were inoculated with a phytopathogenic fungus (Botrytis cinerea. Geotrichum candidum, Monilinia fructicola or Fusarium oxysporum) the center of each plate and aliquots of 15 μl of the candidate (1×10.sup.8 UCF/mL) surrounding the fungus. The culture medium PDA is amended with 10 g/L NaCl and 110 ppm H.sub.3BO.sub.3. The ICRM was calculated and the results were compared with unamended culture medium.

[0035] The bacterium Pseudomonas strain S57 was able to grow in all the conditions tested, showing abundant growth (+++) similar to growth wider standard conditions (S.C.), in the presence of 8 g/L of NaCl. 10 and 50 ppm H.sub.3BO.sub.3, and in saline-boric conditions similar to the irrigation water of the Lluta River.

[0036] The growth of bacterium Pseudomonas lini strain S57 in the presence of 20 g/L of NaCl was much slower, being able to reach a turbidity similar to that of S.C. in better times. This same effect was observed when the saline-boric concentrations of the Lluta River were emulated 9 times, noting inhibition of bacterial growth at higher concentrations. The results are shown in Table 2.

[0037] It is possible to indicate that the bacterium Pseudomonas lini strain S57 is able to grow and tolerate saline-boric conditions, similar to those measured in the coastal valleys of the Arica and Parinacota Region, where the Lluta valley is the site of the most extreme saline-boric conditions, which reach 20 ppm boron and 14.64 mmol/t, of sodium, which is equivalent to 0.114 g/L of boric acid and 0.86 g/L of NaCl.

[0038] As can be seen in the results, the bacterium of the invention supports even the water of Lluta concentrated 9 times and brine (8 to 20 g/L NaCl). It is interesting to note that seawater has an average salinity of 3.5 g/L of NaCl, and the strain of the invention survives in almost 6 times greater conditions and shows optimal growth at 8 g/L to more than twice the salinity of seawater.

TABLE-US-00002 TABLE 2 Growth of the bacterium Pseudomonas lini strain S57 in the presence of NaCl or H.sub.3BO.sub.3. Condition Growth S.C. +++ 8 g/L NaCl +++ 15 g/L NaCl ++ 20 g/L NaCl + 10 ppm H.sub.3BO.sub.3 +++ 50 ppm H.sub.3BO.sub.3 +++ 100 ppm H.sub.3BO.sub.3 ++ Lluta irrigation water +++ 9X Lluta Irrigation Water +

[0039] 4. Biofungicidal Activity in the Presence and Absence of Saline-Boric Conditions.

[0040] The inhibition of the growth, in vitro, of phytopathogenic fungi under standard and saline-boric conditions was studied: for this purpose, a dual culture test was carried out on plates of potato dextrose agar (PDA), which were inoculated with a phytopathogenic fungus (independently with Botrytis cinerea, Geotrichum candidum, Monilinia fructicola, Fusarium oxysporum) in the center of each plate and aliquots of 15 μl of the biofungicide of the invention (1×10.sup.8 UCF/mL) as in Example 2, both under standard conditions (absence of NaCl and H.sub.3BO.sub.3) and in saline-boric conditions (similar to irrigation water from the Lluta Valley), and the ICRM was determined for each condition, based on 3 repetitions. The results are shown in Table 3.

[0041] The bacterium Pseudomonas lini strain S57 showed antifungal activity against the fungi tested, both in standard conditions and in saline-boric conditions. It is possible to observe that the antifungal activity is maintained in the presence of sodium and boron for both Botrytis cinerea and Fusarium oxysporum, both fungi being an important phytosanitary problem in the Region of Arica and Parinacota and in other localities where intensive horticulture is practiced. However, this effect is minor against Geotrichum candidum and Monilinia fructicola under saline-boric conditions. These results demonstrate the potential as a biofungicide of phytopathogenic fungi for environments where sodium and boron concentrations limit the use of commercial biofungicides.

TABLE-US-00003 TABLE 3 Inhibition, in vitro, of the growth of four phytopathogenic fungi by the bacterium Pseudomonas lini strain S57. ICRM (%) Phytopathogenic fungus Standard conditions Saline-Boric Conditions Botrytis cinerea 48.3 42.8 Fusarium oxysporum 23.5 26.3 Geotrichum candidum 58.6 29.6 Monilinia fructicola 67.2 52.3

[0042] 5. Activity, In Vitro, Anti-Botrytis and Anti-Fusarium in Different Culture Media

[0043] For this purpose, a culture test was carried out on plates of solid medium of PDA or King B medium, which were inoculated with the bacterium Pseudomonas lini cepa S57 in the form of a grass and with the phytopathogenic fungus (Botrytis cinerea or Fusarium oxysporum) in the center of each plate. As a control, respective solid medium plates inoculated only with the fungus in the center were used. The ICRM of the fungus was calculated, as described above. The results are shown in Table 5.

[0044] It is observed that the anti-Botrytis activity is complete in the grass of bacterium Pseudomonas lini strain S57 in both media, and the anti-Fusarium activity greater in King B medium, with respect to the PDA medium.

TABLE-US-00004 TABLE 4 In vitro, anti-Botrytis and anti-Fusarium activity of the bacterium P. lini strain S57. ICRM (%) Culture medium Botrytis cinerea Fusarium oxysporum PDA 100.0 44.0 King's B 100.0 69.0

[0045] These examples should be considered as illustrative and not limiting to the present invention, which is fully defined in the accompanying claims.