Isolation, Antimicrobial Effect and Metabolite Analysis of Bacillus amyloliquefaciens ZJLMBA1908 against Citrus Canker Caused by Xanthomonas citri subsp. citri
Abstract: Citrus canker caused by Xanthomonas citri subsp. citri is a devastating bacterial disease with severe implications for the citrus industry. Microorganisms possessing biocontrol capabilities against X. citri subsp. citri offer a highly promising strategy for healthy citrus management. In the present study, a broad-spectrum antagonist strain ZJLMBA1908 with potent antibacterial activity against X. citri subsp. citri was isolated from symptomatic lemon leaves, and identified as Bacillus amyloliquefaciens. Cell-fr… Show more
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Bacteria-antagonists of the agents of soryz bacterial diseases
Aim. To isolate and identify bacteria with antagonist properties for biocontrol of the agents of bacterial diseases of soryz (Sorghum oryzoidum) and sorghum crops. Methods. The studies were conducted in 2021-2023. Spore-forming bacteria were isolated from the soryz samples, collected in the fields of the experimental farm of the Uman National Horticulture University (Cherkasy region, Uman). Lactic acid bacteria were isolated from soryz plants, collected in the private land plot, located between the villages of Teolyn, Vladyslavchyk, Kniazhyky in Monastyryshche com- munity, Uman district, where Pershotravneve hamlet used to be situated. A total of 1,250 samples were analyzed. The experiment had three repeats. Spore-forming and lactic acid bacteria were isolated from the surface of soryz plants on the firm ripe stage in summer while isolating phytopathogenic bacteria. The isolates of lactic acid bacteria- antagonists were also isolated from the inner part of winter stubble stalk of soryz, collected from the tilled field. The antagonistic activity of the strains of lactic acid bacteria and spore-forming bacteria, isolated from different ecological niches, to phytopathogens of soryz and sorghum crops was determined in vitro. The strains of Pseudomonas syringae, the agents of soryz bacterial spots, were used as test-cultures: 211141a, 211141, 210341, 21034, and 210521, along with the collection strains of phytopathogens: Pseudomonas syringae 8299, Pseudomonas syringae subsp. syringae UKM B-1021, X. oryzae 8375, Dickeya chrysanthemi 8683, Diskeya chryzanthemi 8683. The antagonistic activity of the extracted isolates of spore-forming and lactic acid bacteria was studied using the method of radial strokes (joint cultivation of the antagonist and the strains under investigation). The bacterial isolates were deemed inactive if the growth delay zone was 0–5 mm (–), from 5 to 10 mm (+) – low activity, 11–20 mm (++) – moderate activity, over 20 mm (+++) – high activity regarding the test-cultures. To check the effect of the isolate-antagonist of phytopathogenic bacteria, artificial infecting was conducted in the field conditions. For this purpose, a diurnal culture of the antagonist was introduced into the stalk of plants in the concentration of 1×108 colony-forming units, and 24 h later, a culture of test-strain of the phytopathogen was administered above the previous puncture. The results were evaluated 7–14 days after the artificial infection. The experiment had three repeats. The isolates of bacteria which demonstrated their an- tagonistic properties regarding the phytopathogenic bacteria were identified by their morphological properties, Gram staining, catalase test, profile of carbohydrate fermentation and mass-spectrometry (MALDI-TOF – Matrix Assisted Laser Desorption/Ionization) using VITEK MS mass-spectrometer. Results. Thirty-eight spore-forming bacterial iso- lates were extracted from soryz; among these, 21030, 21095, 21040, ASV1, ASV3, B4 demonstrated their antagonistic activity towards the investigated phytopathogenic bacteria. Isolate 21040 showed high antagonistic activity to most test-strains of P. syringae from soryz (the zone of negative culture – 23–30 mm) and lower activity regarding the collection cultures. Isolates B4 and AVS3 demonstrated their selective activity regarding the investigated phytopatho- gens. Twenty isolates of lactic acid bacteria were extracted. Higher antagonistic activity was noted for the isolates of lactic acid bacteria 8/1 and F1 to the strains of P. syringae, isolated from soryz and collection cultures. The highest antagonistic activity of isolate 8/1 was noted regarding test-strains of P. syringae 210521 and X. oryzae 8375 (the zone of negative culture – 40–35 mm). In the field conditions, the treatment of sorghum plants with F1 affected the pathological process that developed due to the impact of the phytopathogenic bacteria P. syringae, which led to the reduction in disease symptoms. The taxonomic position of the isolates of bacteria, which seem to be promising for the control of disease agents, was determined. In terms of morphology of cells and colonies, the biochemical profile, and mass-spectrometry MALDI-TOF, the spore-forming isolates 21040 and B4 were identified as Bacillus subtilis, and ASV3 – as Bacillus vallismortis. The identified isolates of lactic acid bacteria were Lactobacillus pentosus F1 and Lactobacillus sakei 8/1. Conclusions. In addition to phytopathogenic bacteria, from soryz plants we isolated the strains of spore-forming bacteria Bacillus subtilis 21040, B4, Bacillus vallismortis AVS3 and such lactic acid bacteria as Lactiplantibacillus pentosus and Lactobacillus sakei 8/1 (Latilactobacillus sakei 8/1), promising for the elaboration of methods for the biocontrol of the agents of bacterial diseases.
Bacteria-antagonists of the agents of soryz bacterial diseases
Aim. To isolate and identify bacteria with antagonist properties for biocontrol of the agents of bacterial diseases of soryz (Sorghum oryzoidum) and sorghum crops. Methods. The studies were conducted in 2021-2023. Spore-forming bacteria were isolated from the soryz samples, collected in the fields of the experimental farm of the Uman National Horticulture University (Cherkasy region, Uman). Lactic acid bacteria were isolated from soryz plants, collected in the private land plot, located between the villages of Teolyn, Vladyslavchyk, Kniazhyky in Monastyryshche com- munity, Uman district, where Pershotravneve hamlet used to be situated. A total of 1,250 samples were analyzed. The experiment had three repeats. Spore-forming and lactic acid bacteria were isolated from the surface of soryz plants on the firm ripe stage in summer while isolating phytopathogenic bacteria. The isolates of lactic acid bacteria- antagonists were also isolated from the inner part of winter stubble stalk of soryz, collected from the tilled field. The antagonistic activity of the strains of lactic acid bacteria and spore-forming bacteria, isolated from different ecological niches, to phytopathogens of soryz and sorghum crops was determined in vitro. The strains of Pseudomonas syringae, the agents of soryz bacterial spots, were used as test-cultures: 211141a, 211141, 210341, 21034, and 210521, along with the collection strains of phytopathogens: Pseudomonas syringae 8299, Pseudomonas syringae subsp. syringae UKM B-1021, X. oryzae 8375, Dickeya chrysanthemi 8683, Diskeya chryzanthemi 8683. The antagonistic activity of the extracted isolates of spore-forming and lactic acid bacteria was studied using the method of radial strokes (joint cultivation of the antagonist and the strains under investigation). The bacterial isolates were deemed inactive if the growth delay zone was 0–5 mm (–), from 5 to 10 mm (+) – low activity, 11–20 mm (++) – moderate activity, over 20 mm (+++) – high activity regarding the test-cultures. To check the effect of the isolate-antagonist of phytopathogenic bacteria, artificial infecting was conducted in the field conditions. For this purpose, a diurnal culture of the antagonist was introduced into the stalk of plants in the concentration of 1×108 colony-forming units, and 24 h later, a culture of test-strain of the phytopathogen was administered above the previous puncture. The results were evaluated 7–14 days after the artificial infection. The experiment had three repeats. The isolates of bacteria which demonstrated their an- tagonistic properties regarding the phytopathogenic bacteria were identified by their morphological properties, Gram staining, catalase test, profile of carbohydrate fermentation and mass-spectrometry (MALDI-TOF – Matrix Assisted Laser Desorption/Ionization) using VITEK MS mass-spectrometer. Results. Thirty-eight spore-forming bacterial iso- lates were extracted from soryz; among these, 21030, 21095, 21040, ASV1, ASV3, B4 demonstrated their antagonistic activity towards the investigated phytopathogenic bacteria. Isolate 21040 showed high antagonistic activity to most test-strains of P. syringae from soryz (the zone of negative culture – 23–30 mm) and lower activity regarding the collection cultures. Isolates B4 and AVS3 demonstrated their selective activity regarding the investigated phytopatho- gens. Twenty isolates of lactic acid bacteria were extracted. Higher antagonistic activity was noted for the isolates of lactic acid bacteria 8/1 and F1 to the strains of P. syringae, isolated from soryz and collection cultures. The highest antagonistic activity of isolate 8/1 was noted regarding test-strains of P. syringae 210521 and X. oryzae 8375 (the zone of negative culture – 40–35 mm). In the field conditions, the treatment of sorghum plants with F1 affected the pathological process that developed due to the impact of the phytopathogenic bacteria P. syringae, which led to the reduction in disease symptoms. The taxonomic position of the isolates of bacteria, which seem to be promising for the control of disease agents, was determined. In terms of morphology of cells and colonies, the biochemical profile, and mass-spectrometry MALDI-TOF, the spore-forming isolates 21040 and B4 were identified as Bacillus subtilis, and ASV3 – as Bacillus vallismortis. The identified isolates of lactic acid bacteria were Lactobacillus pentosus F1 and Lactobacillus sakei 8/1. Conclusions. In addition to phytopathogenic bacteria, from soryz plants we isolated the strains of spore-forming bacteria Bacillus subtilis 21040, B4, Bacillus vallismortis AVS3 and such lactic acid bacteria as Lactiplantibacillus pentosus and Lactobacillus sakei 8/1 (Latilactobacillus sakei 8/1), promising for the elaboration of methods for the biocontrol of the agents of bacterial diseases.
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