紫花苜蓿致病菌的分离及鉴定

Isolation and identification of pathogenic bacteria from alfalfa

为了明确紫花苜蓿叶部病害具体发病原因并分离致病菌,试验于2022年6月16日于黑龙江八一农垦大学试验田采集紫花苜蓿患病植株叶片,对其致病菌进行分离纯化;将分离得到的致病菌接种发芽期紫花苜蓿种子,根据菌株名称分组,同时设置接种等量无菌水的对照组,12 d后测量紫花苜蓿幼芽芽长,统计发病率和病情指数;对分离菌株进行形态学观察及分子生物学鉴定;以紫花苜蓿的芽长、发病率、病情指数为依据,通过隶属函数法评价分离的致病菌对紫花苜蓿的致病性。结果表明:从紫花苜蓿叶部病害组织中分离获得34株菌株,其中3株真菌菌株对紫花苜蓿幼芽具有致病性,分别命名为HB4、HB6、PB8。接种致病菌后紫花苜蓿幼芽均出现芽长变短、部分腐烂等症状,其中HB6致病性最强。HB4组、HB6组、PB8组间紫花苜蓿的发病率及病情指数均差异不显著(P>0.05),但均显著高于对照组(P<0.05);芽长均显著低于对照组(P<0.05),其中HB4组芽长最短,为1.92 cm。筛选出的HB4、HB6、PB83种紫花苜蓿致病菌经鉴定分别为麦斑点附球霉(Epicoccum tritici)、腐皮镰刀菌(Fusarium solani)、枝状枝孢菌(Cladosporium cladosporioides)。3种致病菌的致病性强弱顺序为HB4>HB6>PB8。说明3种菌株对紫花苜蓿幼芽均有致病性。

In order to clarify the specific causes of alfalfa leaf disease and isolate its pathogenic bacteria,the leaves of diseased alfalfa plants were collected from the experimental field of Heilongjiang Bayi Agricultural University on June 16,2022,and its pathogenic bacteria were isolated and purified.The isolated pathogenic bacteria were inoculated into alfalfa seeds at the germination stage and grouped according to the strain name,at the same time,the control group was inoculated with an equal amount of sterile water.After 12 d,the sprout length of alfalfa was measured,and the morbidity rate and disease condition index were calculated.Morphological observation and molecular biological identification of the isolated strains were performed.Based on the bud length,incidence,and disease index of alfalfa,the pathogenicity of isolated pathogenic bacteria to alfalfa was evaluated by the membership function method.The results showed that 34 strains were isolated from the leaf disease tissues of alfalfa,among which 3 fungal strains were pathogenic to the young buds of alfalfa,which were named HB4,HB6 and PB8,respectively.After inoculation with pathogenic bacteria,the young buds of alfalfa showed some symptoms such as shortening and partial rot,among which HB6 was the most pathogenic.The incidence and disease index of alfalfa between HB4,HB6 and PB8 were not significantly different(P>0.05),but they were significantly higher than those in the control group(P<0.05),and the bud length was significantly lower than that in the control group(P<0.05),and the bud length of HB4 group was the shortest(1.92 cm).The selected pathogen(HB4,HB6 and PB8)of alfalfa were identified as Epicoccum tritici,Fusarium solani and Cladosporium cladosporioides,respectively.The pathogenic order of the three pathogenic bacteria was HB4>HB6>PB8.The results indicated that the three strains were pathogenic to young buds of alfalfa.