Isolation and Identification of Three <i>Beauveria bassiana</i> Isolates in Chongqing Area

Yan-yan WU; Yun-lin TANG; Ruo-cheng GU; Tian-tian LI; Jia-ling BAO; Tian LI; Chun-feng LI; Jun-hong WEI; Guo-qing PAN; Ze-yang ZHOU

doi:10.13718/j.cnki.xdzk.2019.08.003

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China is one of the biggest agricultural and forestry countries in the world, and agricultural and forestry pests cause serious economic losses annually. At the same time, chemical control measures have caused many problems such as environmental pollution. Therefore, the development of bioinsecticides based on entomogenous fungi is of great importance. In order to enrich the resources of entomopathogenic fungi, we obtained entomogenous fungal isolates via isolating and cultivating from wild stinkbug and silkworm infected by Beauveria sp. and identified their biological characteristics. In this experiment, three isolates (NB-1, PW-1 and JCY) were identified. Morphological observation showed that the cultural characteristics of all the three strains were consistent with those of Beauveria bassiana. ITS of each isolate shared 99% homology to many isolates of B. bassiana in GenBank. Thus, isolates NB-1, PW-1 and JCY were all identified as B. bassiana. Significant differences in conidia production were detected between different isolates via biological characteristics observation. The conidial yield of PW-1 and NB-1 isolated from the infected stinkbug was (7.55±0.15)×10⁷ and (6.30±0.40)×10⁷ spores/mL, respectively, which was higher than that of JCY isolated from the infected silkworm, (3.02±0.22)×10⁷ spores/mL.

Corresponding author: Jun-hong WEI ;

Received Date: 13/07/2017

Available Online: 20/08/2019

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Abstract: China is one of the biggest agricultural and forestry countries in the world, and agricultural and forestry pests cause serious economic losses annually. At the same time, chemical control measures have caused many problems such as environmental pollution. Therefore, the development of bioinsecticides based on entomogenous fungi is of great importance. In order to enrich the resources of entomopathogenic fungi, we obtained entomogenous fungal isolates via isolating and cultivating from wild stinkbug and silkworm infected by Beauveria sp. and identified their biological characteristics. In this experiment, three isolates (NB-1, PW-1 and JCY) were identified. Morphological observation showed that the cultural characteristics of all the three strains were consistent with those of Beauveria bassiana. ITS of each isolate shared 99% homology to many isolates of B. bassiana in GenBank. Thus, isolates NB-1, PW-1 and JCY were all identified as B. bassiana. Significant differences in conidia production were detected between different isolates via biological characteristics observation. The conidial yield of PW-1 and NB-1 isolated from the infected stinkbug was (7.55±0.15)×10⁷ and (6.30±0.40)×10⁷ spores/mL, respectively, which was higher than that of JCY isolated from the infected silkworm, (3.02±0.22)×10⁷ spores/mL.

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虫生真菌作为昆虫病原微生物中的最大类群，是自然界中控制害虫种群数量的一个重要影响因素.自然界中全部病死昆虫约60%是因真菌感染致死^[1].因此虫生真菌在害虫防治中的地位举足轻重，已受到研究者们的广泛关注.中国是世界上较早记载虫生真菌的国家之一，早在2000多年前的《神农本草经》中就记载了受白僵菌属感染而死亡的家蚕幼虫；古代史书中也多次记载蝗虫感染蝗噬虫霉后“抱草而死”的现象^[2].

20世纪50年代，我国开始利用虫生真菌资源来防治害虫，经过几十年的发展，虫生真菌资源的开发利用取得了很大的进步.宋晓兵等学者分离得到1株对柑橘木虱具有强致病性的球孢白僵菌，以浓度为1.0×10⁸个/mL的分生孢子悬浮液处理柑橘木虱成虫7 d后，其校正死亡率达到95.7%^[3]；孙艺昕等学者分离筛选得到6株对亚洲玉米螟具有高致病力的球孢白僵菌菌株，丰富了玉米地下害虫及土传病害的生物防治资源^[4]；蔡守平通过测定不同球孢白僵菌菌株的生长速度、产孢量及对星天牛幼虫的致病力，筛选到两株具强致病力且生长速度快、产孢量高的球孢白僵菌菌株，以期作为防治星天牛的优良菌株^[5].

白僵菌(Beauveria)因寄主范围广、致病性强等特点，逐渐成为农林害虫防治中广泛使用的昆虫生防菌之一^[6]，在分类地位上属于半知菌亚门，丝孢纲，丛梗孢目，丛梗孢科，白僵菌属.本研究从重庆地区野外感染白僵菌的蝽蟓及室内感染的家蚕僵虫上分离获得3株球孢白僵菌菌株，并通过形态学观察及分子生物学方法进行鉴定，以期丰富重庆本地的虫生真菌资源库，为进一步研发农林害虫生物农药奠定基础.

1. 材料及方法

1.1. 供试虫源

分离虫生真菌所用的蝽蟓僵虫于2018年9月采集自重庆市北碚区缙云山，僵蚕样品为实验室饲养家蚕时所得病蚕.

1.2. 供试培养基

试验采用的培养基为马铃薯葡萄糖琼脂培养基(PDA)、营养琼脂培养基(NA)和酵母浸出粉胨葡萄糖培养基(YPD).

1.3. 虫生真菌的分离纯化

将蝽蟓僵虫及家蚕僵虫样品在超净工作台上进行分离操作.在僵虫体表挑取孢子粉，涂布于PDA等平板上分离培养；将僵虫虫体浸入75%的酒精中，消毒5 s，再用灭菌水漂洗3次，用灭菌滤纸吸净水分，将虫体剪开，放置于PDA等平板上.将平板放置于27 ℃恒温培养箱中培养，待虫体周围长出菌丝后，挑取菌丝转至新的平板上，重复3次后，挑取单菌落进行分离纯化.

1.4. 菌株形态学鉴定

将分离纯化的菌株接种到PDA平板中央，置于27 ℃恒温条件下培养，定期观察菌落生长情况，记录其颜色及形态，并在光学显微镜下观察菌丝及孢子形态，对分离到的菌株进行形态学鉴定.

1.5. ITS序列鉴定

使用CTAB法^[7]提取菌株的基因组DNA.采用真菌通用引物ITS1(5′-TCCGTAGGTGAACCTGCGG-3′)和ITS4(5′-TCCTCCGCTTATTGATATGC-3′)进行ITS序列扩增，将扩增产物送往生工生物工程(上海)股份有限公司测序.将所得基因序列与GenBank数据库中保存的基因序列进行比对，得到与目的菌株具有同源性的多株菌株相应序列.从中选择下载同源性较高的序列，使用MEGA5.2软件，运用NJ法进行1 000次步长计算，构建系统发育进化树.

1.6. 菌株生物学特性观察

取各菌株少量孢子，在PDA平板上划线，然后置于26 ℃的恒温培养箱中培养2 d(略长出菌丝)，在线上切取菌落块，倒置于PDA平板培养基中央，采用十字交叉法测量菌落生长直径，共测量7 d.每个菌株设3次重复.

培养7 d后，在距平皿边缘等距离处随机切取3块直径为8 mm的菌块，浸入0.5%吐温80溶液中，振荡获取孢子，用血球计数板测定并计算菌株产孢量^[8].

1.7. 数据分析

采用Excel 2016对原始数据初步分析，用SPSS 21软件对实验数据进行统计分析，以Duncan氏新复极差法进行显著差异性检验(p=0.05；p=0.01).

3. 讨论

球孢白僵菌是世界范围内害虫生物防治中推广和应用最为成功的虫生真菌之一，具有杀虫谱广、分布广泛、致病性强等优点，被用于防治草地贪夜蛾、柑橘木虱和玉米螟等多种农林害虫^[9-11]，同时还能定殖于宿主植物进行垂直传播，从而达到持续有效的防治目标^[12].白僵菌被认为是一种对环境无害的生物防治剂，对人类健康几乎没有威胁，对非目标生物通常也是无害的，因此有效利用白僵菌进行生物防治，有利于环境保护和可持续发展战略.白僵菌通常以有丝分裂孢子进行增殖，其分生孢子通过风、雨水飞溅或甚至通过节肢动物载体分散传播，在易感宿主中建立感染，在定植期间能产生多种毒性代谢物，伴随宿主内部组织的破坏和营养物耗尽，并最终导致宿主死亡^[13-14].

本研究共分离纯化获得NB-1，PW-1和JCY3株菌株，形态学观察和基于ITS rDNA序列分析相结合，将菌株NB-1，PW-1和JCY均鉴定为球孢白僵菌(Beauveria bassiana).生物学特性观察结果表明，不同菌株间的菌落生长情况和分生孢子产生量存在显著差异，这可能是菌株来源寄主物种的不同以及生理环境各异所致.球孢白僵菌PW-1，NB-1菌株分生孢子产量分别为(7.55±0.15)×10⁷个/mL，(6.30±0.40)×10⁷个/mL，与陈方新等从玉米根际土中分离得到的39株球孢白僵菌菌株产孢量相比，均达到了较高水平^[15].

从生物防治的发展趋势来看，白僵菌作为一种重要的虫生真菌，其制剂的应用开发潜力巨大，但目前从菌株分离、菌株毒力保持到利用白僵菌菌剂在自然环境中进行大规模病虫害防治，仍然面临着诸多问题.未来如何有效利用已有白僵菌菌种资源以及筛选新型高致病力菌株、生产致病性强的广谱性生物制剂，都需要进一步的研究.研究发现，LI Huiping等利用肿腿蜂携带白僵菌对桑天牛进行防治试验，发现这种联合寄生的方法较单一方法能够得到更好的防治效果^[16]；Borja等人通过将化学杀虫剂(毒死蜱乙基、多杀菌素)和虫生真菌(球孢白僵菌、金龟子绿僵菌)相结合，可以增加真菌虫生孢子形成，提高草地贪夜蛾的死亡率^[17].这表明将白僵菌制剂结合多种防治措施，可以对病虫害进行更为有效的防治，为未来白僵菌用于生物防治提供了新思路.

虫生真菌的宿主种属特异性较强，不同菌株的宿主谱和毒力的差别也比较大，因此分离鉴定更多的菌株有助于丰富虫生真菌资源，为虫生真菌生防农药的开发提供更多的材料.这3株分离于重庆地区的球孢白僵菌，对重庆地区常见的农林害虫的杀虫效果如何，特别是对近期入侵的重大迁飞害虫草地贪夜蛾具有怎样的杀虫效果，还需开展进一步研究.

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Isolation and Identification of Three Beauveria bassiana Isolates in Chongqing Area

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Isolation and Identification of Three Beauveria bassiana Isolates in Chongqing Area

Corresponding author: Jun-hong WEI ;