Identification and pathogenicity of pathogenic species of plum leaf spot disease in Fujian

Abstract：【Objective】In recent years, a new leaf disease has occurred in plum producing areas in Fujian Province, causing yellow to brown spots on the leaves in preliminary stage, irregular gray to dark brown stripes in later period and leaf fall in severe cases. According to the symptoms, it is named plum leaf spot disease. The disease has a trend of spreading and increasing every year, which has aroused high local attention. This study investigated the occurrence of the disease and identified the pathogen species.【Methods】A survey of leaf spot disease was conducted in two plum orchards from 2020 to 2021 in Liancheng county, Fujian province. The leaves with the symptoms were collected from the plum trees of Furong, Younai and Huanai (Prunus sallcina) in the surveyed orchards and 4-5 mm² diseased tissues (neighboring the asymptomatic regions) were surface-sterilized with 75% ethanol for 45 s, washed two times in sterile water and dried on sterilized filter paper, and placed onto potato dextrose agar (PDA) plates and incubated at a temperature of 28 ℃ in the dark. The single mycelium was used for purifying strains, and pure cultures were stored in 25% glycerol at -80 ℃. The pathogen genomic DNA was extracted with cetyltrimethylammonium bromide (CTAB) buffer, which was identified through partial actin (ACT), beta-tubulin (TUB2), chitin synthase (CHS-1), a 200 bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and partial regions of six loci including partial rDNA-ITS (ITS) region sequence analysis. The multiple sequences were aligned using MAFFT v.7 with default settings, manually adjusted in MEGA v.5.2.2 if necessary and phylogenetic tree was constructed using maximum-likelihood (ML) by IQ-TREE. The morphological characteristics, sporulation phenotype, conidia morphology and ascospore morphology for representative strains of the identified Colletotrichum spp. were recorded. The pathogenicity was determined on the detached leaves and fruits of plum inoculated with mycelial plugs and conidia suspension of representative strains of the identified Colletotrichum spp., the host ranges were determined on the detached leaves of peach (Prunus persica), pear (Pyrus pyrifolia), citrus (Citrus reticulate) and kiwifruit (Actinidia chinensis) inoculated with representative strains of the identified Colletotrichum spp..【Results】Through tissue isolation and purification, 66 strains with the similar morphology to Colletotrichum spp. were isolated in total. The obtained strains were identified by multiple genes (ACT, TUB2, CHS-1, GAPDH, and ITS) and were clustered in six different branches, including 59 strains clustered in the branch of C. fruticola, 2 strains in C. karstii, 2 strains in C. pluriforum, 1 strain in C. siamense, 1 strain in C. wuxiensis and 1 strain (2FRL-3-1) close to C. tropicicola. Morphological observation showed that 2FRL- 3- 1 was significantly different from C. tropicicola in colony color and conidia size, which indicated that 2FRL-3-1 was a novel species of Colletotrichum spp. identified in this study, named as C. pruni-salicinae. The results of pathogenicity test showed that the representative strains of the above 6 species of Colletotrichum spp. induced lesions on the leaves and fruits of P. salicina Lindl. The strains isolated from infected sites were identical to the strains inoculated. The results showed that they were all pathogenic and responsible for plum leaf spot and fulfilled the Koch’s postulates. But there were significant differences of pathogenicity in the fruits. The pathogenicity of C. siamense on the fruits was significantly stronger than the other 5 species. The pathogenicity of HN-6-1 was significantly stronger than that of 2FRL-2-3, although they all belonged to the species C. karstii. The results indicated that different species and different strains of the same species of Colletotrichum spp. had significant pathogenicity differentiation against the same host. The results of determination for the host range showed that the above six species of Colletotrichum spp. infected the leaves of the plants other than plum, C. fruticola and C. wuxiense infected the leaves of peach (P. persica), pear (P. pyrifolia), citrus (Citrus reticulata) and kiwifruit (A. chinensis), C. siamense and C. pluriforum infected the leaves of peach, pear and kiwifruit, while C. karstii and C. prunisalicinae also infected the leaves of peach and pear.【Conclusion】Based on morphological characteristics and phylogenetic analysis of the multiple genes (ACT, TUB2, CHS-1, GAPDH, and ITS), the pathogens of plum leaf spot disease in Fujian were identified as 6 species, including C. fruticola, C. karstii, C. pluriforum, C. siamense, C. wuxiense and C. pruni-salicinae. Among them, C. fruticola was the dominant species, accounting for 89.4% of Colletotrichum strains. There were significant differences in pathogenicity between different species and strains of the same species on the same host. C. siamense had the strongest pathogenicity on the fruits of plum. This study is the first report on the identification of pathogens associated with plum leaf spot disease in Fujian.