Isolation And Identification Of Related Microorganisms On Leaf Disease Of Three Oak Species

The oak tree is widely distributed in China and has strong adaptability.Its timber is widely used and has high application value and ornamental value.The main purpose of this study is to investigate leaf diseases of oak trees.During the period of high incidence of diseases from June to September,the concentrated and relatively primitive areas of oak forests were selected as far as possible for investigation and sampling.Tissue block separation and dilution coating methods were used to isolate and identify the samples in the laboratory.In the investigation of Quercus variabilis leaf diseases,93 strains of fungi belonging to 48species in 30 genera and 32 strains of bacteria belonging to 27 species in 18 genera were collecteded from 8 locations in 4 provinces（Henan,Shaanxi,Anhui and Guizhou）.This study identified 6 common leaf diseases on Q.variabilis,including anthracnose disease,red spot disease,brown spot disease,shot-hole disease,maculopathy and powdery mildew.Colletotrichum gloeosporioide,Tubakia Dryina and Ampelomyces quisqualis were also identified as the common pathogenic of anthracnose,brown spot and powdery mildew disease,respectively.In the Quercus mongolica leaf disease survey,Q.mongolica disease were mainly investigated in Ningcheng County of Chifeng City,the Inner Mongolia Autonomous Region,China.27 fungus strains were isolated,belonging to 10 species in 10 genera.30 bacteria strains belonging to 17 species in 11 genera were isolated and identified.Four common diseases of the leaves of Q.mongolica were discovered,including macular disease,shot-hole disease,powdery mildew disease with its pathogen was Erysiphe euonymicola and brown spot disease.The isolated strain Bleaf8^T was a potential new species with 96.7%similarity with Mucilaginibacter daejeonensis Jip10^T,and was identified by polyphasic classification.The investigation of Cyclobalanopsis patelliformis leaf diseases was conducted in Bawangling National Forest Park,Hainan Province.And 4 strains of fungi were separated into4 species belonging to 4 genera.2 strains of bacteria were isolated and identified and strain TPQG1-4^T was regarded as a novel strain and was classified and identified by polyphasic taxonomy identification.This study identified three common diseases of C.patelliformis leaves,including black spot disease,brown spot disease and shot-hole disease.This study not only enriched the forest microbial resources,but provided knowledge reserve for the future diagnosis and prevention of more serious diseases.The main results are as follows,（1）Description of Mucilaginibacter quercina sp.nov.Strain Bleaf8^T which cannot grow in the absence of oxygen,is a pale pink rod-shaped Gram-stain-negative bacterium,0.3-0.8μm wide and 1.1-1.8μm long.Bleaf8^T is tolerant to temperature of 20-37℃,p H of 6.0-8.0 and Na Cl of 0-3%.Catalase reaction was positive and oxidase reaction was positive.The main fatty acids（>10.0%）were iso-C_15:0（36.9%）,and the summed feature 3(C_16:1ω7c and/or C_16:1ω6c;35.3%).The G+C content of the genomic DNA was 43.9%.The 16S r RNA sequence has the highest similarity with Mucilaginibacter daejeonensis Jip10^T（96.7%）.Bleaf8^T is a new species of Mucilaginibacter.The type strain is Bleaf8^T isolated from the leaf spot disease of Q.mongolica（CFCC 16165^T）.（2）Description of Stenotrophomonas cyclobalanopsidis sp.nov.TPQG1-4^T,a Gram-negative,facultatively anaerobic,motile bacterial strain with an average cell size of 0.5-1.0×2.5-3.0μm.The growth temperature range of the strain was 4-37℃（optimum 30℃）,the p H value suitable for growth was 4.0-10.0（optimum 7.0）,and the Na Cl tolerance range was 0-3%.Catalase reaction was positive and oxidase reaction was positive.It cannot reduce nitrogen.16S r RNA gene sequencing and analyzing revealed that the novel strain shares the highest sequence similarity with Stenotrophomonas lactitubi M15^T（99.6%）,and the（average nucleotide identity）ANI value between the two species was 87.2%.The DNA G+C%content of the strain was 67.1%.The main fatty acids are iso-C_15:0（25.4%）and anteiso-C_15:0（17.0%）.The polar lipid components include phosphatidylethanolamine,phosphatidylglycerol,diphosphatidylglycerol,amino phospholipid and phospholipid.TPQG1-4^T is a new species of Stenotrophomonas,and the type strain is TPQG1-4^T isolated from the leaf spot disease of Cyclobalanopsis patelliformis（CFCC 15341^T=LMG 31208^T）.