山东桃褐腐病病原菌种群鉴定及致病性分析

Population identification and pathogenicity analysis of peach brown rot pathogens in Shandong province

【目的】明确山东省桃褐腐病病原菌种群结构及其致病力差异,为山东桃褐腐病病原菌的多样性研究及有效防控提供理论依据。【方法】采集烟台、威海、临沂等地桃褐腐病样本,利用形态学鉴定、rDNA-ITS序列分析、欧氏距离非加权组平均法(UPGMA)等技术手段,对桃褐腐病病原菌种类、致病力等进行分析。【结果】采集桃树叶片、果实、枝条褐腐病样品,通过组织分离获得41株桃褐腐病病原菌,这些菌株在菌落形态上存在较大差异,结合rDNA-ITS序列分析,分别鉴定为Monilinia fructicola、Monilia yunnanensis及Monilia polystroma,三者占比分别为80.48%、9.76%、9.76%。桃褐腐病病原菌菌丝生长速率为0.47~1.09 cm·d-1,UPGMA聚类分析证实,其生长速率可被划分为慢、中、快三大类。采用桃叶片有伤接种菌饼方法,确定桃褐腐病病原菌引起的病斑大小范围为0~2.32 cm,UPGMA聚类分析证实,其致病力可被划分为强、中、弱三类。桃褐腐病病原菌菌丝生长速率及产孢量与致病力相关性分析发现,相关系数r分别为0.297 5、0.030 0,表明菌丝生长速率及产孢量均与致病力无相关性。【结论】山东省桃褐腐病病原菌主要为Monilinia fructicola,Monilia yunnanensis及Monilia polystroma,其中Monilinia fructicola为优势菌种,Monilia yunnanensis是首次在山东省被鉴定,证实了山东省桃褐腐病病原菌趋于多样化,不同菌株间菌丝生长速率、产孢量及致病力存在较大差异,菌丝生长速率及产孢量均与致病力无相关性。

【Objective】China has the biggest peach planting area and output in the world. There are 20 provinces with a peach planting area exceeding 10 000 hm~2, and Shandong province ranks first. Peach brown rot is an important peach disease caused by Monilinia spp. Peach brown rot mainly damages fruits, but also flowers, leaves and shoots. Fruit can be damaged from the young fruit stage to the mature stage. If it rains in the later stage of growth, disasters are common, and the incidence rate is more than 80% or even there is no harvest. It can also occur during transportation and storage, causing the fruit to lose its commercial value. This study aimed to clarify the species of Monilinia spp. associated with peach brown rot in Shandong province based on ITS sequencing and morphological identification,as well as to determine the distribution, morphological and pathogenic characteristics of the pathogens.The results are expected to provide a better acknowledge of the disease and scientific basis for its prevention and control.【Methods】The leaves, branches and fruits infected by peach brown rot were collected from main producing areas in Shandong province and were used as disease samples. The pathogens were isolated by the routine plant tissue isolation method. The pathogens were identified through microscopic observation of the morphological characteristics of hyphae, conidia and sporulation. To further identify the pathogens, total genomic DNA was extracted using a fungal genomic DNA extraction kit, and subjected to polymerase chain reaction(PCR) amplification of partial region of rDNA-ITS(ITS). PCR products were sequenced for phylogenetic analysis by the blast comparison and the neighboring method(NJ) by MEGA 6.0 to identify the taxonomic status of the pathogens on peach brown rot. The average growth rate of the mycelial was measured by the criss-cross method after 5 days culture on PDA plate. The spore production was recorded under a microscope using hemocytometer. The pathogenicity was determined on leaves inoculated with mycelial plugs of isolates with the postulates of Koch’s. Correlation analysis of mycelial growth rate and pathogenicity was determined by Pearson method.【Results】A total of 41 strains were isolated from the collected samples infected by the peach brown rot disease. There were significant differences in the colony morphology of 41 isolates. The strains represented by THF-01 had neat colony edges, grayish-yellow color, abundant spore production and short sporophytes. Spore piles could form concentric ring structures, and the size of conidia was(15.3-18.9) μm×(13.4-14.8) μm. The strains represented by THF-06 had neat colony edges, dark gray color and sparse spore production. Aerial mycelium grew close to petri dish. Sporogenous stems varied in length. The size of conidia was(14.9-18.0) μm ×(13.6-14.9) μm. The strains represented by THF-14had gray-white and irregular colony edges, which had obvious cracks. They had abundant spore production and long sporophytes. The size of conidia was(15.6-18.2) μm ×(14.4-15.8) μm. To further classify the taxonomic status of the pathogens, the specific primers of rDNA-ITS were used to amplify the genomic DNA of 41 isolates. The results indicated that the similarity between THF-01 and M. fructicola(accession number: MZ047241.1, EF207419.1) was as high as 99%. The similarity between THF-06and Monilia yunnanensis(accession number: MW355895.1) was as high as 100%. The similarity between THF-14 and M. polystroma(accession number: LT615178.1, LT615192.1) was as high as 99%.Those pathogens were identified as M. Fructicola, M. Yunnanensis and M. polystroma, accounting for80.48%, 9.76% and 9.76%, respectively. The mycelial growth rates of those strains ranged from 0.47 to 1.09 cm·d-1, and the UPGMA clustering analysis confirmed that the mycelial growth rates could be divided into three categories: slow, medium and fast. The lesion length of different peach brown rot pathogens ranged from 0 to 2.32 cm by using the method of injury-inoculated cake on peach leaves. UPGMA clustering analysis confirmed that the pathogenicity of those strains could be divided into three categories: strong, medium and weak. By analyzing the correlation between mycelium growth rate, spore output and pathogenicity, the correlation coefficient r=0.226 7 and 0.030, so it was clear there were no correlation between mycelial growth rate, spore output and pathogenicity.【Conclusion】The peach brown rot pathogens in Shandong province were mainly M. fructicola, M. yunnanensis and M. polystroma,among which M. fructicola was the dominant strain. M. yunnanensis was the first time to be identified in Shandong province, which confirmed that the peach brown rot pathogens in Shandong province tended to be diversified and different. There were great differences in the growth rate, spore output and pathogenicity of mycelium among different strains, and there was no correlation between mycelial growth rate, spore output and pathogenicity. This study can provide a scientific basis for the diversity research and effective prevention and control of peach brown rot in Shandong.