Isolation and identification of the pathogen causing cherry black spot in Gansu province

Abstract:【Objective】Cherry black spot disease caused by Alternaria spp., induces rot and shrinkage fruit and perforated leaves. It tends to be serious over the years and affects the economic benefits of the cherry industry. Alternaria spp. are common pathogens, and they have cultural diversity, strong adaptability and broad hosts. It is important to clarify the pathogen species causing cherry spot for the diagnosis and comprehensive control of the disease.【Methods】Based on the tissue isolation method, the cherry leaves with black spot collected from Qinzhou district, Tianshui city, Gansu province. The pieces of the leaves (5 mm×5 mm) were disinfected with 10% sodium hypochlorite and cultured on PDA plate. After 3 days, the edge of colony were picked to purify and then stored at 4 °C. In order to determine the pathogenicity of the isolates with the postulates of Koch’s, the isolates cultured for 7 days were diluted to suspensions (108 spore per mL), and then were sprayed on the healthy cherry leaves with wound stabbed with insect needle, respectively. The cherry leaves inoculated with sterile water were used as a control. All treatments were moisturized with sterile cotton wool and placed in incubator at 25 °C. The assay was repeated three times. The pathogens were identified through micro- scopic observation of the morphological characteristics of hyphae, conidia and sporulation. To further identify the pathogens, the DNAs of the them were extracted with the OMEGA fungal extraction kit (D3195-01), and the sequence of them were amplified with the Alternaria major allergen gene (Alta1), calmodulin gene (CAL) and plasma membrane adenosine triphosphatase gene (ATPase), respectively, and the products detected by 1% agarose gel electrophoresis were sequenced by Xi'an TSINGKE Zexi Biotechnology Co., Ltd. The phylogenetic tree was constructed by the Blast comparison and the neigh- boring method (NJ) by MEGA 7.0 to identify the taxonomic status of the pathogens on cherry black spot.【Results】9 isolates, named TYY1-9, were isolated from the collected leaves of cherry infected by the black spot disease. After 5 days of inoculation, the cherry leaves inoculated with the isolates TTY2, TYY6 and TYY9 exhibited infected symptoms. Among them, the isolate TYY2 produced dark brown irregular lesions on the leaves and gray black mold layer on the back of the leaves. The isolate TTY6 induced browned leaves and black mold layer on the lesions. In addition, the leaves inoculated with isolate TYY9 showed brown spots and inconspicuous concentric stripes. The symptoms appeared on the inoculated leaves were similar to that collected from field, however the control was asymptom- atic. The diseased leaves were isolated again and the isolates TTY2, TYY6 and TYY9 were obtained. According to Koch's postulates, it was concluded that all three isolates were pathogens causing cherry black spot. After culturing on PDA plate for 7 days at 25 °C, the colony of TTY2 was concentric with white and dark green, and aerial hyphae was hyaline, and the size of conidia was 7.68-32.31 μm×3.50- 8.32 μm. The strain TYY6 was dark green with irregular edges. Conidia were single or skewered, mostly stick-shaped, a few were oval or round, light brown or brown, with 1-4 septa, 0-5 longitudinal membranes, and spore size was 51.51-7.44 μm × 3.85-8.65 μm. The strain TYY9 was gray to dark gray round colony on the PDA plate, and the back of the colony was black. The conidia size was 7.46- 34.85 μm×3.58-10.67 μm, with 1-6 septa and 0-4 mediastinum. To further classify the taxonomic sta- tus of the pathogens, the specific primers of Alternaria major allergen gene (Alta1), calmodulin gene (CAL) and plasma membrane adenosine triphosphatase gene (ATPase) were used to amplify the ge- nomic DNA of the 3 isolates. The results indicated that the TYY2 were 99.15% (accession number: MG199093), 98.75% (accession number: MG925123) and 99.23% (accession number: MH492681) identical to the three sequences of A. alternate, respectively. The identities of three genes for the strain TYY6 were 99.73% (accession number: KY561842), 99.72% (accession number: MG925159) and 99.92% (accession number: MF073265) in comparison to the sequence of A. tenuissima. In addition, the identities of genes for the strain TYY9 were 100.00% (accession number: MG199093), 100.00% (accession number: KJ920975) and 99.49% (accession number: MG736305) in comparison to the se- quence of A. alternate.【Conclusion】9 isolates were isolated from the cherry leaves with black spot, and the isolates TTY2, TYY6 and TYY9 were confirmed as pathogens causing cherry black spot in Tianshui city. Combined with morphological characteristics and phylogenetic analysis, strain TYY6 was identified as A. tenuissima, which was firstly reported on cherry in China, and the strains TYY2 and TYY9 were identified as A. alternate. Therefore, the quantity and species of pathogens causing cherry black spot disease should be comprehensively considered in the diagnosis and the control of the disease.