Identification and pathogenicity of Alternaria species causing black spot in pear producing regions in China

Abstract:【Objective】Pear trees have been widely cultivated in China and the total yield and cultiva- tion area of Chinese pear ranks first in the world. Pear belongs to a kind of perennial fruit tree and it is frequently infected by a large number of fungal diseases during growth and development. Black spot is one of the important diseases in pear production areas in China, which mainly damages leaves and fruits, resulting in serious precocious defoliation and fruit decay. Black spot has become a worldwide disease, especially in Japan, South Korea and the south of China. The disease has caused severe losses to the economic benefits and greatly affected the development of pear industry in major pear-cultivation areas in China. This study aimed to clarify the species of Alternaria spp. associated with black spot of pear in China based on multi-gene and morphological identification, as well as to determine the compo- sition, distribution, morphological and pathogenic characteristics of the pathogens. The results of this study are expected to provide a better understanding on the etiology of the disease and scientific basis for its prevention and control.【Methods】The leaves and fruits infected by black spot collected from 14 provinces, municipalities or autonomous regions (including Anhui, Chongqing, Fujian, Gansu, Gui- zhou, Hubei, Jiangxi, Jilin, Shanxi, Shandong, Sichuan, Xinjiang, Yunnan and Zhejiang) were used as disease samples. The fungus pathogen was isolated by using the routine plant tissue isolation method. Total genomic DNA was extracted from pure cultures with a modified cetyltrimethyl ammonium bromide (CTAB) protocol, and subjected to polymerase chain reaction (PCR) amplification of partial re- gions of six loci including partial rDNA-ITS (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF 1), endo polygalacturonase (endoPG), Alternariamajor allergen gene (Alt a1) and histone 3 (His 3). Phylogenetic trees were constructed by mrbayes- 3.1.2. The best- fit models of nucleotide substitution for each partition were determined by using MrModeltest v.2.3. The sporulation phenotype and conidia morphology of representative strains were recorded under a microscope. The pathogenicity was determined on detached leaves, fruits and branch- es (wounded or unwounded) of pear inoculated with mycelial plugs of representative strains and the host ranges were determined on detached leaves of peach (Prunus persica), kiwifruit (Actinidia chinen- sis) and citrus (Citrus reticulate) inoculated with mycelial plugs of representative strains.【Results】A total of 405 strains of Alternaria, including 355 strains from leaves and 50 strains from fruits, were ob- tained. The obtained strains were identified by multiple genes, combined with morphological observa- tion, and they were confirmed to be 6 species of Alternaria, including A. tenuissima (267 strains), A. al- ternata (115 strains), A. arborescens (14 strains), A. gaisen (6 strains), A. gossypina (2 strains) and A. longipes (1 strains). Phylogenetic analysis showed that specific species could not be identified only by ITS sequences. A. gaisen, A. arborescens and A. alternata could be identified by multiple genes (ITS,GAPDH, Alt a1, TEF 1 and endoPG). A. tenuissima and A. alternata could be identified by His 3 gene. Through PCR amplification, A. tenuissima produced a fragment of 550 bp, while A. alternata produced a fragment of 440 bp. However, SC-5, SC-10 and SC-16 strains were clustered together with A. lon- gipes and A. gossypina, so there was no clear identification between A. longipes and A. gossypina. Com- bined with morphological identification, the conidia morphology of A. longipes and A. gossypina were different. The conidia of A. gossypina were dark brown and pear-shaped, while the conidia of A. lon- gipes were gray and spindle-shaped. The results of pathogenicity test showed that the representative strains of A. alternata (SX-1), A. tenuissima (SX-2), A. arborescens (XJ-13), A. gaisen (CQ-35), A. lon- gipes (SC-10) and A. gossypina (SC-5) could induce lesions on leaves and fruits of Pyrus pyrifolia cv. Cuiguan, but there were significant differences in their pathogenicity. However, under the same inocula- tion conditions, these representative strains could not induce lesions on pear branches. The pathogens isolated from the infected sites after inoculation were identical with the inoculated strains. These results showed that they were all pathogenic and responsible for black spot by fulfilling the Koch’s postulates. The results of the determination of host range showed that these representative strains could infect the leaves of peach (Prunus persica) and kiwifruit (Actinidia chinensis), but there were differences in their pathogenicity. None of these representative strains could cause symptom on leaves of citrus (Citrus re- ticulate). Analysis of the prevalence of 6 species of Alternaria revealed that A. tenuissima and A. alter- nata were dominant species, which occurred in all Pyrus spp. cultivated in 14 provinces, municipalities or autonomous regions, accounting for 65.9% and 28.4% of the total isolates, respectively. A. longipesand A. gossypina were only separated from the samples of Sichuan, A. arborescens was isolated from the samples of Xinjiang, Sichuan and Gansu, and A. gaisen was isolated from the samples of Chongq- ing, Hubei, and Anhui.【Conclusion】Based on the phylogenetic analysis and morphological characteris- tics of the 405 strains obtained in this study, the pathogens causing black spot of pear in China were as- sociated with six species of Alternaria including A. tenuissima, A. alternata, A. arborescens, A. gaisen, A. gossypina and A. longipes. Among them, A. tenuissima and A. alternata were the dominant patho- gens of the disease. This research is the first report on the identification of pathogens associated with black spot in the main pear producing areas in China.