Identification of the pathogens of loquat leaf spot caused by Neopestalotiopsis sp. in Medog and the effect of antagonistic bacteria on disease

【Objective】The Medog areas of Xizang is located in the southwest of China, surrounded by mountains. The unique ecological and climatic characteristics are suitable for the development of the loquat industry. However, with the expansion of the loquat cultivation area, loquat leaf spot disease frequently occurs in this region, and has become a major disease in local loquat production. Loquat leaf spot disease often causes leaves to deform, scorch and wither, directly threatening plant health and affecting the yield and quality. However, the pathogen causing loquat leaf spot disease in Medog areas of Xizang is unclear, and there is a lack of research on the occurrence regularity of the disease and effective biological control techniques. Therefore, this study aimed to isolate and identify the pathogen species that cause loquat leaf spot disease in the Medog areas of Xizang, and clarify the control effect of an-tagonistic bacteria on the disease. The study was expected to provide a better acknowledge of the disease and scientific basis for its prevention and control.【Methods】The fresh leaves with the symptoms were collected from 7 plantations (including Dexing village, Madi village, Xirang village, Disha village, Beibeng village, Hezha village and Miri village) in 2023 and 2024. Leaf spot tissues (approximately 5 mm2 in size) were excised from the margins of lesions with typical symptoms, washed with sterile water, the surface was sterilized in 75% ethanol for 45 seconds, and then washed three times with sterile water again, dried on sterilized filter papers, and finally placed on PDA medium containing rifampin. The isolates were incubated at 25 ℃ in dark for 12 days, and then purified with single spore isolation method. The pathogens were identified through morphological observation, multi-site sequence analysis and pathogenicity determination (Koch's postulates). The morphology of pathogen was preliminarily identified through microscopic observation of the colony, hyphae and spores. To further identify the pathogens, the sequences of genes [rDNA internal transcribed spacer (rDNA- ITS), mtSSU, β- tubulin (TUB) and translation elongation factor 1-α (TEF-1α)] were amplified with primers TIS1/TIS4, NMS1/ NMS2, T1/T22 and EF1/EF2, respectively. PCR products were sequenced for phylogenetic analysis by the blast comparison in NCBI and constructed a molecular phylogenetic tree with the neigh- boring method by MEGA 7.0. The pathogenicity detection was carried out using the method of needle puncture inoculation. The surface of loquat leaves was washed with sterilized water, wounded by sterilized needles, and then inoculated with pathogen spore suspension at the concentration of 1×105 spores·mL-1 . After inoculation, the treated leaves were incubated at 25 ℃ with 85% relative humidity for 7 days. The pathogen was reisolated from the inoculated sites. In addition, the inhibition width was measured by the plate confrontation to determine the activity of antagonistic bacteria to pathogens, and the control effect of antagonistic bacteria on loquat leaf spot and fruit disease was measured with the spray and immersion method, respectively.【Results】A total of 22 strains were isolated from the leaf spot tissue. The result of pathogenicity test showed that all the isolated strains could induce spot on the leaves, and the symptoms were similar to those of the disease in the field. The pathogens isolated from the infected lesions after inoculation were identical with the inoculated strain. According to the morphological observation (colony, mycelium and conidium), multiple gene analyses and molecular phylogenetic tree constructed by multi- gene association analysis, the strains were identified as 4 species, which were Neopestalotiopsis clavispora (18 strains), N. eucalypticola (1 strain), N. formicarum (1 strain) and N. cubana (2 strains). The occurrence frequencies of the strains were 81.82%, 4.55%, 4.55% and 9.10%, respectively. It was found that the 5 tested strains of antagonistic bacteria had good antagonistic activity against N. clavispora with plate confrontation. The inhibition widths of the tested strains were 7.33- 14.33 mm, among which the strain NJ-4 displayed the strongest effect. In vitro study showed that the control efficiency of 20-fold dilution of 3 antagonistic bacteria fermentation broths (S8, T122F and NJ- 1) on the disease was 81.25%, 66.52% and 63.84%, respectively, and the control efficiency on fruit spot were 48.24%, 39.68% and 49.31%, respectively.【Conclusion】Loquat leaf spot disease occurs seriously in the Medog areas of Xizang. In this study, there were 4 species of pathogens (including N. clavispora, N. eucalypticola, N. formicarum and N. cubana) isolated from the leaf spot tissue. This is the first report on the identification of pathogens associated with loquat leaf spot disease in Xizang. Among them, N. clavispora was the dominant species, accounting for 81.82% of Neopestalotiopsis strains. All the isolated strains showed pathogenicity. Our study also indicated that the antagonistic bacteria (S8, T122F and NJ-1) had good biocontrol potential against loquat leaf spot disease, which showed a certain preser-vative effect on fruit during storage. The results could provide important references for the green prevention and control on loquat leaf spot disease in Medog areas of Xizang.