广西家蚕僵病流行趋势及其主要病原遗传规律研究

【目的】摸清广西家蚕僵病发生流行规律，研究病原特性及遗传规律，为精准防控家蚕僵病提供科学依据。【方法】以2008—2021年广西家蚕僵病发病原始数据为基础，分析广西家蚕僵病的发病规律。收集广西五大蚕区（桂中、桂南、桂西、桂西北和桂东南）的病蚕、病虫及生物防控白僵菌株，经显微形态鉴定和产孢测定后采用滤纸接触法测试其对家蚕的致病力；基于ITS4/ITS5引物进行PCR扩增，并将双向测序结果输入GenBank进行BLAST比对分析；根据ISSR-PCR扩增结果以Quantity One绘制电泳模式图，使用NTsys计算遗传相似系数并进行UPGMA聚类分析，同时以PopGene32对供试材料的遗传多样性指数进行分析。【结果】 2008—2021年广西家蚕僵病发病率整体上呈波动下降趋势，连续14年的平均发病率为5.19%。5月是广西家蚕僵病的发病率高峰，平均发病率为13.37%；其次是4月和10月，对应的发病率分别为7.91%和6.87%。山区地形的家蚕僵病发生率普遍高于平缓丘陵地区，其中，丘陵蚕区的家蚕僵病发病率排序为桂中（13.55%） >桂西北（12.66%） >桂南（11.56%） >桂西（11.05%） >桂东南（9.53%），山地蚕区的家蚕僵病发病率排序为桂中（18.91%） >桂西北（17.96%） >桂西（16.01%） >桂西（14.90%） >桂东南（11.61%）。广西家蚕僵病病原包括白僵菌属、曲霉属、拟青霉属和虫草菌属，其中球孢白僵菌（Beauveria bassiana）为主要病原，占比达86.67%，且白僵菌ITS序列高度保守，序列相似性在97.83%~99.83%。不同白僵菌株对家蚕的致病力表现为崇左白僵菌株（农药）的72 h累计死亡率达100.00%，明显高于蚕区僵蚕菌株（8.89%~93.33%）。筛选出的10条ISSR引物从50株菌株样本中扩增出152条清晰条带，其中多态位点147个，多态百分数为87.5%~100.0%； 50株菌株样本的Nei's基因多样性指数（H）为0.1740~0.3257，Shannon信息多样性指数（I）为0.2840~0.4880，均表现出极高的遗传多样性；基于遗传距离构建的UPGMA系统发育进化树显示，桂南、桂西北、桂东南、桂中等蚕区的病蚕白僵菌株与农药源白僵菌株存在非常近的亲缘关系。【结论】广西家蚕僵病危害高峰期为5月，球孢白僵菌是最主要的病原菌，且ITS序列高度保守。不同来源白僵菌对家蚕致病力的排序为农药源白僵菌株>松林白僵菌株>蚕区僵蚕菌株，但三者相互间存在非常高的基因关联性，提示广西家蚕僵病的病原遗传背景复杂，且蚕桑主产区应慎用白僵菌农药。

Prevalence tendency and genetic laws of main pathogens of silkworm muscardine diseases in Guangxi

【Objective】To find out the incidence and prevalence laws of silkworm muscardine diseases in Guangxi, and to study the pathogenic characteristics and genetic law, so as to provide a scientific basis for precise control of silkworm muscardine disease.【Method】Based on the survey data of silkworm disease in Guangxi in the past 14 years, the incidence law was analyzed. Diseased silkworms, pests and biological control Beauveria bassiana strains from 5 sericulture areas(central Guangxi, south Guangxi, west Guangxi, northwest Guangxi and southeast Guangxi) were collected, and their pathogenicity on silkworm were tested through microscopic morphology identification and filter paper contact method after sporulation determination. PCR amplification was conducted based on ITS4/ITS5 primer, and Bidirectional sequencing results were imported into GenBank for BLAST analysis. According to the results of ISSR-PCR amplification, the electrophoresis pattern map was drawn by Quantity One, the genetic similarity coefficient was calculated by NTsys and the UPGMA cluster analysis was carried out, and the genetic diversity index of the tested materials was analyzed by PopGene32.【Result】From 2008 to 2021, the incidence of silkworm muscardine disease in Guangxi showed a fluctuating downward trend, with an average incidence of 5.19% for 14 consecutive years. May was the peak of the incidence of silkworm muscardine disease in Guangxi, with an average incidence of 13.37%, followed by April and October, with the morbidity as 7.91% and 6.87%, respectively. The incidence of silkworm stiff disease in mountainous area was generally higher than that in gentle hilly area, and the incidence of silkworm muscardine disease in gentle hilly area was ranked as follows:central Guangxi(13.55%) > northwest Guangxi(12.66%) > south Guangxi(11.56%) > west Guangxi(11.05%) > southeast Guangxi(9.53%). The incidence rate of silkworm stiff disease in mountainous silkworm area is ranked as:central Guangxi(18.91%) > northwest Guangxi(17.96%) > west Guangxi(16.01%) > south Guangxi(14.90%) > southeast Guangxi(11.61%). The pathogens of muscardine diseases in Guangxi belonged to the genera Beauveria, Aspergillus, Paecilomyces and Cordyceps, in which B. bassiana was the main pathogen, which accounted for 86.67%, and its ITS sequence was highly conserved with similarity ranging from 97.83% to 99.83%. The 72-hour pathogenicity test on silkworm showed that the cumulative mortality rate of B. bassiana(pesticide) from Chongzuo in Guangxi was 100.00%, which was significantly higher than that of the silkworm muscardine strains in the sericulture area(8.89%-93.33%). A total of 152 clear bands were amplified from 50 strains by 10 ISSR primers, of which 147 were polymorphic, and the polymorphic percentage was 87.5%-100.0%. The Nei's gene diversity index(H) of the 50 strains was 0.1740-0.3257, and the Shannon's information diversity index(I) was 0.2840-0.4880, showing extremely high genetic diversity. The UPGMA phylogenetic tree based on genetic distance showed that the isolates from southern, northwestern, southeastern and central sericulture areas of Guangxi had close genetic relationship with the isolates from pesticide.【Conclusion】May is the peak month of silkworm muscardine diseases in Guangxi and B. bassiana is the main pathogen, whose ITS sequence is highly conserved. The order of pathogenicity of B. bassiana from different sources to silkworm was pesticide source B. bassiana strain > pine forest B. bassiana strain > silkworm strain in silkworm area, and there is a very high genetic correlation among the 3 sources, suggesting that the pathogenic genetic background of silkworm muscardine disease in Guangxi is complex, and B. bassiana pesticides should be used carefully in the main sericulture areas.