Identification And Biological Characteristics Of Pestalotiopsis-like Fungi On Blueberry

The disease caused by Pestalotiopsis-like fungi is one of the most important diseases in blueberry production,resulting in production and economic losses.With the promotion of the blueberry planting to new area,the diversity of diseases was caused by different conditions.According to the difference of blueberry cultivar,the complexity of planting condition,and the diversity of Pestalotiopsis-like fungi,it was hypothesized that there were various Pestalotiopsis species infected blueberry in different cultivation conditions.In order to ascertain the main pathogenic species and provide the foundation of etiology for preventing disease caused by Pestalotiopsis-like fungi,we sought to identify Pestalotiopsis-like fungi isolated from diverse blueberry plantation by combining molecular biology and morphological characteristics.Meanwhile,their biological characteristics were alsoinvestigated.1.The morphological identification of Pestalotiopsis-like fungiFrom 2013 to 2016,the samples of leaf spot disease and twig dieback disease were collected.A total of 53 isolates were isolated and purified from diseased twigs and leaves.Seven isolates were no pathogenic,and 46 isolates have pathogenicity in different levels.The leaf spot disease showed symptoms of yellow-brown,circle or subcircle spots with the dark brown edge,and the acervuli emerged at the later period.The twig dieback disease showed symptoms characterized by brown necrotic spots on twigs,with leaves and young shoots later turning brown and dry.The main stem blighted and died at the later stage,and black acervuli were observed on symptomatic twigs.The morphological identification was carried out by observing the culture characters.Furthermore,the freehand section,frozen section,and temporary slide were made for observing characteristics of conidia and conidiogenous cells.The results showed that colonies were diversiform.The color of the colony was always white,some isolates have striations,and fruiting bodies were black,aggregated or solitary.Conidia presented three median versicoloured cells and two hyaline cells.Apical cells had two or more appendages,and baseal cells had one to two appendages.Conidial characters were different among the isolates.The isolates were divided into two groups according to the color of three median cells,conidiogenous cells,and conidiophores:(1)Pestalotiopsis sp.,the three median cells were concolorous,conidiogenous cells hyaline,thin-walled,smooth,conidiophores distinct(2)Neopestalotiopsis sp.,the three median cells were versicolorous,conidiogenous cells were hyaline,thin-walled,smooth,and conidiophores were indistinct.2.Molecular identification of Pestalotiopsis-like fungiThe LSU gene was used for distinguishing different genera by Maximum Parsimony(MP)analysis and Bayesian Inference(BI)analysis,The combination of rDNA-ITS,?-tubulin,and tefl gene was used for distinguishing different species by Maximum Likelihood(ML)analysis and MP analysis,and the morphological characteristics was also used for identification.The results showed that Neopestalotiopsis spp.and Pestalotiopsis spp.were the main pathogens causing diseases on blueberry in China.N.chrysea was the pathogen that triggered twig dieback on blueberry in Fujian.P.trachicarpicola was the casual agent for leaf spot disease on blueberry in Guangxi.Both Neopestalotiopsis spp.and Pestalotiopsis spp.could infect leaves and twigs of blueberry,leading to twig dieback and leaf spot disease.3.Biological characteristics of Pestalotiopsis-like fungiThe representative isolates of different groups were selected for biological characteristics study.The results showed that there were differences among different species.Glucose was the most appropriate carbon source for Pestalotiopsis-like fungi;cubic niter was the best nitrogen source for mycelia of N.chrysea,and glycine was the best nitrogen source for P.trachicarpicola;PDA is the best medium;the mycelia could grow between 10 ? to 30 ?,and the suitable temperature was 25 ? to 28 ?;light had no effects on mycelium growth;the mycelia could grow at a range of pH 4 to 9.The suitable pH for different species is as follows:8 for N.chrysea,5-6 for SCCDO1Cb(Neopestalotiopsis sp.),4 for P.trachicarpicola,and 6 for YN06F(Pestalotiopsis sp.).