Antagonistic activity of Bacillus velezensis strain P2-1 against tan-brown leaf spot of strawberry and its growth-promoting effect

Abstract: 【Objective】Strawberry is a perennial herb crop with high nutritional and economic value. Strawberry is grown all over the world because of its highly adaptive capacity. With the development of modern urban agriculture, the planting area of strawberry in China is increasing in recent years. However, the frequent occurrence of plant diseases seriously restricts the healthy development of the strawberry industry. Among them, tan-brown leaf spot of strawberry caused by Pilidium concavum or P. lythri is a newly discovered disease, which has been reported in the world. The disease mainly affects leaves and fruits, which causes serious loss in the yield and quality of strawberries. Controlling plant disease with chemical agent is the most common and effective method. However, the long-term use of chemical fungicides could increase the selection pressure on the presence of fungicides resistant isolates and cause environmental pollution. Biocontrol of plant diseases with endophytes is the hot spot of plant disease control research and has the characteristics of safety, low toxicity and high efficiency. So far, there has no prior report on biocontrol of tan-brown leaf spot of strawberry. The aim of this study is to analyze the biocontrol effect of endophyte Bacillus velezensis strain P2-1 against strawberry brown leaf spot and its growth-promoting efficacy on strawberry. The result will lay a foundation for the application of this antagonistic agent in strawberry disease.【Methods】The antagonistic activity of endo-phyte B. velezensis strain P2-1 against P. concavum was tested by plate confrontation experiment culture method. Colony growth rate and mycelium morphology of P. concavum was analyzed. Protease, cellulase and β-1, 3-glucanase activities of strain P2-1 were determined by placing strain P2-1 cells on skim-milk agar, CMC, and aniline blue agar media, respectively. The numbers of strain P2-1 colonies in inoculated strawberry leaves were counted at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 days after inoculation, respectively. The antagonistic activity of strain P2-1 on strawberry brown leaf spot was determined on detached leaves of strawberry. After treatment with strain P2-1 cell suspension, the leaves of strawberry were inoculated with mycelia plugs of P. concavum. Disease incidence and lesion diameter were measured at 5 and 7 days after inoculation. Water treatment was used as negative control and tebuconazole treatment was used as positive control. Photographs of the disease were taken at 7 days after inoculation. To analyze the effect of strain P2-1 on growth-promoting traits, strawberry seedlings were treated with 5 mL P2-1 cell suspension (10⁸ ∙mL^-1 ) by root irrigation and plant height, root length, fresh weight and dry weight were measured at 30 days after inoculation.【Results】The endophyte B. velezensis strain P2-1 showed strong inhibition effect on mycelia growth of P. concavum. Statistical result indicated that the inhibition rate of strain P2-1 on P. concavum reached to 66.38%, with inhibition zone being about 0.76 cm. Microscope observations revealed that strain P2-1 resulted in abnormal mycelial morphology of P. concavum. After antagonistically treated with strain P2-1, the hypha of P. concavum became fractured, digested, transparent and slender. Statistical result showed that the treated hyphal diameter with stain P2-1 was about 4.12 μm, which was significantly reduced, comparing to CK (7.71 μm). Strain P2-1 could form clear and transparent circles on skim milk medium and CMC medium, but could not form clear circles on aniline blue medium, indicating that metabolites of strain P2-1 had protease and cellulase activities. The statistical results showed that after spraying strawberry leaves with stain P2- 1, the number of colonies of strain P2-1 increased rapidly, and reached the maximum at 7 days after inoculation, with the number of colonies being 1.15 × 10¹⁰ CFU. Subsequently, the number of colonies showed a downward trend, and at 10 days after treatment, the number of colonies tended to be stable and remained at about 4.07×10⁸ CFU. This result indicated that strain P2-1 was able to successfully colonize in strawberry leaves. The result of pathogenicity test showed that strain P2- 1 cell suspension could inhibit the development of tan-brown leaf spot of strawberry caused by P. concavum. The statistical results showed that strain P2-1 could reduce the disease incidence and lesion diameter at 5 and 7 days after inoculation, comparing to CK treatment. The control efficiency of tan- brown leaf spot of strawberry by strain P2-1 was comparable to the positive control tebuconazole. In addition, strain P2-1 could significantly improve plant height, root length, wet weight and dry weight of strawberry seedlings.【Conclusion】The endophyte B. velezensis strain P2-1 showed strong antagonistic activity against strawberry tan- brown leaf spot caused by P. concavum, which can be exploited as a bio- controlling strain of the disease for further study.