梨阿太菌果腐病菌(Athelia bombacina)单核化菌丝制备及其致病力评价  

Preparation and pathogenicity evaluation of mononuclear hyphae of Athelia bombacina

作　　者：贾晓辉[1,2] 张鑫楠[1] 杜艳民 周如军[2] 王文辉[1] 傅俊范[2] JIA Xiaohui;ZHANG Xinnan;DU Yanmin;ZHOU Rujun;WANG Wenhui;FU Junfan(Research Institute of Pomology,Chinese Academy of Agricultural Sciences/Key Laboratory of Fruit Storage and Processing of Liaoning Province,Xingcheng 125100,Liaoning,China;College of Plant Protection,Shenyang Agricultural University,Shenyang 110161,Liaoning,China)

机构地区：[1]中国农业科学院果树研究所·辽宁省果品贮藏与加工重点实验室,辽宁兴城125100 [2]沈阳农业大学植物保护学院,沈阳110161

出　　处：《果树学报》2021年第8期1340-1348,共9页Journal of Fruit Science

基　　金：国家现代农业产业技术体系建设专项资金项目(CARS-28-21);中央级公益性科研院所基本科研业务费专项(1610182019027)。

摘　　要：【目的】建立梨阿太菌果腐病菌(Athelia bombacina)单核化菌丝制备方法,并对其致病性进行评价,为后续该病原菌的分子生物学研究奠定基础。【方法】以阿太菌果腐病菌HG-AB20170418为供试菌株,研究了菌龄、酶解时间、裂解酶等对阿太菌果腐病菌原生质体制备的影响,并比较分析了单核体菌丝与原始菌丝之间形态、生长速率以及致病力差异,最后对单核化处理前后菌丝杂合度进行了评估。【结果】以溶壁酶为裂解酶制备的原生质体产量最高,崩溃酶和纤维素酶对菌丝裂解作用极弱。溶壁酶和蜗牛酶裂解时间较短,最佳裂解时间为2.0 h,而崩溃酶和纤维素酶裂解时间较长。培养3 d菌丝经酶解后在显微镜下可观察到大量的原生质体,且在较短时间内菌丝全部裂解。单核化处理后菌丝在PDA上培养呈螺旋状生长,且生长速率显著低于原始菌株。经单核化处理的菌株接种至黄冠梨后,所有果实接种部位均在第3天产生病斑,但不同菌株间致病力差异显著。经单核化处理后菌丝杂合率为0.00%,属于简单基因组。【结论】单核化菌丝最佳制备条件为PDB液体培养3 d的A.bombacina菌丝经1.5%(w,后同)溶壁酶和1.5%蜗牛酶裂解1.5 h,该制备方法的建立为该病原菌的分子致病机制研究提供重要的方法基础。【Objective】This study aimed to establish a method for the preparation of mononucleated mycelium of Athelia bombacina and evaluate its pathogenicity,so as to lay a foundation for the following-up study on molecular biology.【Methods】The mycelia were inoculated on PDB medium and cultured in shaking table at 25℃and 120 r·min-1 for 1,2,3 and 4 d.Fresh mycelia with a mass of 0.5 g were collected and washed twice with sterile water and twice with 0.6 mol·L^(-1) mannitol osmotic stabilizer.5 mL of 1.5% snailase,1.5% driselase,1.5% cellulase and 1.5% lyase were added respectively.The mycelia were hydrolyzed at 30℃ and 120 r·min^(-1),for 1.5,2.0,2.5,3.0,3.5,4.0 and 4.5 h,the number of protoplasts was observed and calculated with the blood cell counting plate,and the type of cell wall lyase with high protoplast yield and the enzymolysis time were determined.The effects of age,lyase and enzymolysis time on the protoplast preparation of fruit rot fungus HG-AB20170418 were studied,and the differences of morphology,growth rate and pathogenicity between the monokaryon hyphae and the original hyphae were compared and analyzed.Finally,the heterozygosity of hyphae before and after the treatment was evaluated.【Results】The highest yield of protoplasts was obtained with lysozyme as lysozyme,and the enzymolysis effect of cellulase was very weak.The enzymolysis time of the lyase and the snail enzyme was shorter,and the optimal enzymolysis time was 2.0 h,while the enzymolysis time of the driselase and the cellulase was longer.After 3 days of culture,a large number of protoplasts could be observed under the microscope,and all the hyphae were lysed in a short time.After 4 days of culture,a large number of hyphae could still be seen in 2.5 hours under the action of the optimal lyase.The original strain grew radially on the PDA,but the mycelial growth was spiral.On the 3rd and 5th day of culture,the colony diameter of the original strain was 37.54 mm and 65.00 mm,respectively.The growth rate was significantly lower than th

关 键 词：梨阿太菌果腐病菌 原生质体 单核化 杂合率 致病力 

分 类 号：S661.2[农业科学—果树学] S436.612[农业科学—园艺学]