Pathogen identification of loquat stem blight disease and its pathogenicity

Abstract:【Objective】Loquat (Eriobotrya japonica) is a subtropical tree with sour and sweet fruit and itsleaf derivatives are often used to treat cough and asthma with phlegm. In 2011, a severe stem and leaf dis-ease, with a 30% to 60% incidence rate, was observed in a local loquat cultivar ‘Ninghaibai' in Ninghai,Zhejiang province, China. Infected plants exhibited brown spots on the stem and leaves, which becameblackish-brown to grayish white with some little sporadic black acervuli (137.9-189.3 μm in diameter). Leaf symptoms showed large necrotic lesions(more than 20 mm) that frequently, but not exclusively, appeared in diameter along the leaf margin or dorsal surface. Necrotic spots were water-soaked, irregularshape and bordered by a tan halo. The disease resulted in a wide reduction of output and raised the anxi-ety of the farmers and some of them also lost confidence for curing some of the diseased old trees and hadto fall them to restrain the spread of the disease. To effectively try to control the disease, some studieswere carried out to identify the pathogen of the disease.【Methods】Several different areas with large cultur-ing of loquat in Zhejiang and Fujian province, including Ninghai, Yuhang, Jiande, Zhangzhou, and Putian,were selected to collect samples. The 89 diseased samples, which were not only the typical loquat stemblight disease but also the leaf spot disease, were numbered and tissues were cut from the disease by thehealth organization and were examined by microscopy and cultured on different media, including PSA,OMA, CA, NA, starch, Richard, Czapek, juice of the loquat branch and leaves (loquat branch and leaves200 g, water 1 L, agar 20 g). Some isolates were obtained and separated by different phenotype and werepurified to allow for carrying out tests of the pathogenicity by Koch's postulates. And then the sclerotium (20 mm×5 mm) of isolates would be in vivo or in vitro inoculated on the healthy three year trees of‘Ning-haibai'with wound or without wound treatment. Control seedlings were only encircled by the PSA blocks.To identify the pathogen species, we extracted the DNA of isolates and compared them with the gene bankof NCBI, a type of fungi (NNH-B-4) was successfully separated from one hundred eighty seven isolatesand showed the strong pathogenicity to the host loquat and which proved to be the pathogen of the disease.【Results】The obtained isolates were divided into two types: bacteria and fungi. The bacteria showed nopathogenicity to the host, and the fungi were classified as Pestalotiopsis sp., Phyllosticta sp. and Alternaria sp. with separate rates of 84.3%, 2.6% and 5.7% respectively. The in vitro inoculation results indicatedthat most of the fungi were obtained without pathogenicity to the loquat stem, except three kinds of Pestalotiopsis sp.. One Pestalotiopsis sp. isolate named NNH-B-4 could infect the detached tender stem within 48 h in both wound and non-wound treatments and after 72 h the brown lesion length would reach to 4-5cm. The less pathogenicity of the other isolates and control fungi P. eriobotrifolia had been observed whencompared to the NNH-B-4's. After 72 h the lesion sizes of the stems which were inoculated by the other Pestalotiopsis sp. were almost the same as that of the sclerotium and their pathogenicity rate was about 40%-60%. And then the three isolates mentioned above were inoculated with the loquat seedling. Thesimilar result from NNH-B-4 had been presented as a rigescent blade and brown stem being inoculatedafter 4 days. Symptoms observed on artificially inoculated seedlings were similar to that observed in natu-rally infected plants. The control plants remained healthy. And the pathogen of the loquat leaf spot diseaseshowed no symptoms of the same condition. So it would be correct to assume that the isolation NNH-B-4was the pathogen of the disease. The colonies of pathogen were white (front) and honey gold(reverse) witha ring radius on the PSA,and produced some small black acervuli containing black, slimy spore masses at 25 ℃ for 7 d. The conidia typically had 5 cells, fusiform or fusoid, straight or slightly curved; three medi-an cells were heterochrome, the first and second cell of which was dark brown and the third was lightbrown, and the place of the septate was constricted; the apical cells were Hyaline and triangular with 2-3wide-angle apart apical appendages; basal cells were hyaline, triangular or coniform, with a middle han-dle. The apical cells were obviously bigger than the basal cells. The acervuli of the pathogen dispersed orassembled grew under the epidermis cell of the host with a diameter of 150 um to 300 um, and it wouldbreak the epidermis cell once matured. The character of the conidia from NNH-B-4 obviously differedwith the pathogen of the loquat leaf spot disease P. eriobotrifolia and P. eriobotryae-japonica. For the Pestalotiopsis genus, the taxonomy system was not a form of complete distinction based on the clear mor-phological characteristics and relatively complete types, which were mainly according to the shape of theconidia spore morphology, cell number, scale, intermediate cell color, chromatic aberration and the position, number, and length of the appendage of the fungus itself. It might be a Pestalotiopsis sydowiana refer-ence to the books and papers of Pestalotiopsis classification. So the sequence of pathogen rDNA ITS wasextracted and sequenced by the Sangon Biotech to do a molecular identification classification of the genus. The results of the sequence alignment indicated that the homology of the pathogen had reached 100%compared with the sequence of the ITS disclosed Pestalotiopsis sydowiana.【Conclusion】It was clear thatthe new loquat disease in the town of Ninghai was caused by the Pestalotiopsis sydowiana(Bres.)P. L.Zhu, Q. X. Ge & T. Xu. according to their molecular and morphology identification.Key wordsThe new loquat diseaseLoquat branch blast diseaseBres.P. L.