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1 Chinese Journal of Applied Entomology 2015, 52(3): DOI: /j.issn 几种杀虫真菌对二化螟幼虫的毒力测定与 * 高毒力菌株的分子鉴定洪勇 1** 林华峰 1*** 岳霄霄 1 刘苏 1 叶恭银 ; 摘要目的 Chilo suppressalis (Walker) 方法 10 Beauveria sp. 18S rdna ITS PCR 结果 YQ147 QING Bbr14 Bbr /ml YQ % LT d Beauveria bassiana 结论关键词 2 Virulence of several insecticidal fungi against Chilo suppressalis larvae and molecular identification of highly virulent strains HONG Yong 1** LIN Hua-Feng 1*** YUE Xiao-Xiao 1 LIU Su 1 YE Gong-Yin 2 (1. School of Plant Protection, Anhui Agricultural University, Hefei , China; 2. Institute of Insect Sciences, Zhejiang University, Hangzhou , China) Abstract [Objectives] To identify fungal strains with high virulence to Chilo suppressalis larvae and provide a theoretical foundation for the biological control of C. suppressalis. [Methods] The virulence of 10 insecticidal fungi to C. suppressalis larvae were measured under the same conditions (spore concentration, temperature and humidity) in a laboratory and the infestation process of insecticidal fungal spores observed. A pair of 18S rdna universal primers of Beauveria sp. were used to identify the most virulent strains. [Result] At a concentration of spores/ml, the fungal strains YQ147, QING, Bbr14, and Bbr84 displayed strong virulence against 2 nd instar larvae of C. suppressalis, and the YQ147 strain achieved the highest cumulative corrected mortality of 78.4% with a LT 50 of 5.8 d after 10 d. Molecular identification confirmed that this fungus was Beauveria bassiana. [Conclusion] Beauveria bassiana is highly virulent to C. suppressalis and may be an effective agent for the biological control of this pest. Key words Beauveria bassiana, Chilo suppressalis, molecular identification, electron microscope, phylogenetic tree Chilo suppressalis Walker Han and Wang * Supported projects ( ) (2011AA10A204) ** First author @qq.com *** Corresponding author hf.lin@163.com Received Accepted

2 3 : 703 Cao et al He et al Coppling and Duke Clarkson et al Beauveria sp Myzus persicae 1999 Dendrolimus punctatus 2000 Ostrinia nubilalis 2005 Lymantria xylina 2003 Plutella xyllostella 材料与方法 1.1 试验材料供试菌株及来源 Bbr14 Bbr84 Qing Pf10 Nr07 PC31 YQ147 FY324 WL324 ANJ327 SDAY 4 g 1 g 1 g 2 g 100 ml 25 1 RZH 供试幼虫真菌对二化螟幼虫的毒力测定真菌孢子悬浮液制备 10 SDAY d 20 ml 0.05% /ml 室内毒力测定 % 80% 14L.. 10D % d 扫描电镜样品制备 YQ147 3% 3 10 min/ 50% 70% 80% 90% 100% 10 min 100% min CO 2 2 h S-4800 EVACSEQ ERROR CODES 数据统计与分析 = / DPS Tang and Zhang 2013 LSD d X Y

3 704 Chinese Journal of Applied Entomology 52 LT 真菌基因组 DNA 的提取与分子测序鉴定真菌基因组 DNA 提取 DNA DNA DNA ITS ITS4 5 -TCCTCCGCTTATTG ATATGC-3 ITS5 5 -GGAAGTAAAAGTCGTAA CAAGG PCR 反应体系与 PCR 方案 25 µl : 4 µl 4 µl 10 PCR Buffer10 µl Mg mmol/l 6 µl dntp 10 mmol /L 2 µl Taq DNA 2 µl ddh 2 O 70 µl PCR 94 4 min min 56 1 min 72 2 min min min 1 PCR % PCR 分子测序与菌种鉴定 PCR GenBank 99% Rehner et al MEGA 2 结果与分析 2.1 真菌对二化螟幼虫的毒力真菌孢子液对二化螟 3 龄幼虫的致死效果 /ml 2 3 YQ147 QING Bbr14 Bbr84 20% YQ YQ147 QING Bbr14 Bbr84 35% WL324 PC31 Nr07 ANJ327 Pf10 FY324 6 YQ147 QING Bbr14 Bbr84 7 YQ % 8 Bbr YQ % 10 QING 74.5% 0.05 YQ YQ LT 50 2 YQ147 QING LT d 6.3 d YQ147 LT 50 YQ147 YQ 杀虫真菌侵染二化螟后致病症状 1 YQ147 1 A Bbr14 1 B

4 3 : 705 Table 1 Strains 表 1 二化螟幼虫感染 10 种杀虫真菌后的累计校正死亡率 Chilo suppressalis larvae cumulative corrected mortality after infected with 10 strains of insecticidal fungus (%) C. suppressalis larvae infected with different time cumulative corrected mortality ( /ml) Concentration 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d YQ ±5.8a 35.1±6.3a 43.9±3.5a 51.9±3.7a 63.0±3.7a 72.6±3.9a 78.4±2.0a 78.4±2.0a QING ±7.6a 28.1±1.8a 36.8±3.0ab 44.4±0.0b 57.4±1.9ab 66.7±2.0ab 72.6±3.9ab 74.5±3.9ab Bbr ±1.7ab 29.8±1.8a 42.1±3.0ab 46.3±1.9ab 50.0±3.2b 52.9±3.4c 66.7±2.0bc 66.7±2.0bc Bbr ±5.8a 31.6±5.3a 35.1±3.5b 46.3±3.2ab 53.7±6.7ab 58.8±3.4bc 58.8±3.4c 60.8±5.2cd WL ±1.7abc 12.3±1.8b 17.5±1.8cd 20.4±1.9d 35.2±1.9c 41.2±3.4d 49.0±1.9d 52.9±2.0de PC ±3.3c 5.3±3.0bc 19.3±3.5c 27.8±3.2c 33.3±3.2cd 41.2±3.4d 47.1±3.4de 51.0±3.9ef Nr ±1.7c 3.5±1.8bc 14.0±1.8cd 18.5±1.9de 29.6±1.9cd 33.3±2.0de 45.1±1.9de 49.0±2.0ef ANJ ±1.7c 1.8±1.8c 10.5±3.0d 13.0±1.9e 24.1±1.9de 25.5±1.9e 43.1±3.9de 47.1±0.0ef Pf ±2.9bc 3.5±1.8bc 12.3±1.8cd 14.8±1.9de 22.2±3.2e 27.5±1.9e 37.3±3.9e 45.1±3.9f FY ±2.9bc 10.5±3.0bc 12.3±3.5cd 16.7±3.2de 24.1±1.9de 33.3±2.0de 37.3±2.0e 43.1±3.9f ± LSD (P 0.05) The data are mean±se, and followed by different letters in the same column indicate significant difference (P 0.05) by LSD test. 表 2 不同真菌对二化螟幼虫的致死中时间 Table 2 LT 50 of different fungi on Chilo suppressalis larvae Treatment Regression equation (r) Correlation coefficient LT 50 (d) YQ147 Y = X QING Y = X Bbr14 Y = X Bbr84 Y = X WL324 Y = X PC31 Y = X Nr07 Y = X ANJ327 Y = X FY324 Y = X Pf10 Y = X d 1 A~F QING YQ147 Bbr14 Bbr d YQ 分生孢子对幼虫体表的侵染过程 YQ147 2 A 2 B, 2 C D 2 E ( 2 F)

706 2.2 应用昆虫学报 Chinese Journal of Applied Entomology 高毒力菌株的分子鉴定与系统树分析以菌株 YQ147 的基因组 DNA 为模板用特 Fig.

5 应用昆虫学报 Chinese Journal of Applied Entomology 高毒力菌株的分子鉴定与系统树分析以菌株 YQ147 的基因组 DNA 为模板用特 Fig 卷异性引物 ITS4 和 ITS5 进行 PCR 扩增获得 500 bp 左右的扩增产物 PCR 产物经上海立菲生物图 1 杀虫真菌侵染二化螟后致病症状的观察 Pathogenic symptoms observed after insecticidal fungal infect Chilo suppressalis A, F 为菌株 YQ147 B 为菌株 Bbr14 C, D 为菌株 Bbr84 E 为菌株 QING A, F is the strain YQ147 B is the strain Bbr14 C, D is the strain Bbr84 E is the strain QING.

3期洪勇等: 几种杀虫真菌对二化螟幼虫的毒力测定与高毒力菌株的分子鉴定 707 技术有限公司进行序列测定将该序列与 AY531981 AY532045 AY531972 AY531992 GenBank NCBI 中的球孢白僵菌菌株 AY532015 和 AY531995

6 3期洪勇等: 几种杀虫真菌对二化螟幼虫的毒力测定与高毒力菌株的分子鉴定 707 技术有限公司进行序列测定将该序列与 AY AY AY AY GenBank NCBI 中的球孢白僵菌菌株 AY 和 AY 序列进行同源性比较 Fig. 2 图 2 分生孢子在二化螟体表上的萌发及入侵部位 Germination and invasion position of Beauveria bassiana conidia on the cuticle of Chilo suppressalis

7 708 Chinese Journal of Applied Entomology 52 NCBI Beauveria bassiana 99% 40 3 Fig. 3 图 3 基于 18S rdna 基因序列构建的真菌的进化树 ( 邻接法 ) The neighbor joining tree resulting from analysis of the 18S rdna gene sequences of fungi GenBank The strains and their GenBank accession numbers: ARSEF1969: AY31998; ARSEF2641: AY532008; ARSEF4149: HQ880804; ARSEF7542: HQ880805; ARSEF4384: AY532026; ARSEF4474: AY532027; ARSEF4850: AY532028; ARSEF4580: HQ880788; ARSEF4598: HQ880789; ARSEF4622: HQ880790; ARSEF300: AY532015; ARSEF751: AY532045,;ARSEF1040: AY531972; ARSEF1478: AY531981; ARSEF1811: AY531992; ARSEF1848: AY531995; ARSEF979: HQ880783; ARSEF2271: HQ880779; ARSEF4362: AY532025; ARSEF6214: HQ880774; ARSEF7058: HQ880773; ARSEF1567: AY531986; ARSEF2251: AY532003; ARSEF2567: AY532006; ARSEF7032: HQ880803; ARSEF7760: DQ376247; ARSEF1855: HQ880796; ARSEF3220: AY532020; ARSEF6229: HQ880799; ARSEF4933: AY532029; ARSEF1685: AY531990; ARSEF5689: AY532030; ARSEF7043: AY532039; ARSEF7044: AY532040, ARSEF2694: HQ880802; ARSEF8257: HQ880800; ARSEF2922: AY532012; ARSEF442: AF516300; ARSEF703: AF516301; ARSEF7487: HQ331446; ARSEF2038: FJ617319; ARSEF1184: AF138267; ARSEF1768: AF

8 3 : 709 YQ147 3 讨论 10 YQ147 rdna ITS GenBank AY AY AY AY AY AY S rdna ITS 99% B. bassiana (Bals.) Vuill /ml 10 YQ % LT d QING 74.5% LT d 2 YQ147 Bbr14 Bbr84 WL324 PC31 Nr07 ANJ327 FY324 Pf10 10 YQ147 QING Bbr14 Bbr84 LT /ml LT 50 8 d 18 d 98.87% 2011 Ostrinia furnacalis % LT d 2010 Frankliniella occidentalis LT d LC /ml Solenopsis invicta 参考文献 (References) Cao MZ, Shen J, Liu XY, Li M, The insecticide resistance in stripped stem borer, Chilo suppressalis (Walker). CRRN, 9(1): 6 7. Clarkson JM, Charnley AK, New insights into mechanisms of fungal pathogenesis in insects. Trends Microbiol. Lett., 133(4): Coppling CP, Duke SO, Review: natural products that have been used commercially as crop protection agents. Pest Manag. Sci., 63(1): Han ZJ, Wang YC, Biochemical features of a resistant population of the ricestem borer, Chilo suppressalis (Walker). Acta Entomologica Sinica, 46(2): He YR, Lu LH, Kuang ZB, Feng X, Chen HY, Wu YJ, Effect of temperature and humidity on the virulence of beetle-derived Beauveria bassiana (Balsamo) Vuillemin (Deuteromycetes: Moniliales) against the daikon leaf beetle, Phaedon brassicae Baly (Coleoptera: Chrysomelidae). Acta Entomologica Sinica, 48(5):

9 710 Chinese Journal of Applied Entomology 52 Rehner SA, Minnis AM, Sung GH, Luangsa-ard JJ, Devotto L, Humber RA, Phylogeny and systematics of the anamorphic, entomopathogenic genus Beauveria. Mycologia, 103(5): Tang QY, Zhang CX, Data processing system (DPS) software with experimental design, statistical analysis and data mining developed for use in entomological research. Insect Science, 20(2): Deng CS, Zhang YR, Zhang MM, Geng B, Wu W, Fu WJ, Zhang DM, Effect of Beauveria bassiana wettable powder against Leptinotarsa decemlineata (Say). Chinese Journal of Biological Control, 28(1): [,,,,,,, , 28(1): ] Fang ZG, Zhang LQ, Zhao RY, Pathogenicity of 9 strains of Beauveria Bassiana to masson-pine caterpillar. Journal of Zhejiang Forestry College, 16 (4): [,,, , 16(4): ] Fu ZJ, Chen JX, Fu LJ, A review of studies on the pathogenesis of Beauveria bassiana to insects. Wuyi Science Journal, 16(1): [,,, , 16(1): ] Huang FR, Studies on mixing Beauveria bassiana, the nuclear polyhedrosis virus, Bacillus thuringiensis and deetamethrin to control Lymantria xylina. Journal of Fujian Forestry Sci. and Tech., 27(3): [, , 27(3): ] Huang SJ, Qin WJ, Liu H, Using 20% chlorantraniliprole sc to controlrice stem borer (Chilo suppressalis Walker). Acta Agriculturae Jiangxi, 21(5): [,,, % SC., 21( 5): ] Lei YY, Lv LH, Chen DH, Correlation between biological characteristics of Beauveria bassiana and its virulence to Plutella xylostella. Chinese Journal of Biological Control, 26(2): [,,, , 26(2): ] Li CX, Gu LQ, Zhang SH, Xia YM, Studies on the efficacy of Beauveria bassiana against on the Plutella Xylostella. Journal of Tangshan Teachers College, 29(2): [,,,, , 29(2): ] Li MY, Lin HF, Jin L, Zhang SY, Observations on infection of Nilaparvata lugens by Metarhizium flavoviride using a scanning electron microscope. Chinese Journal of Applied Entomology, 48(5): [,,,, , 48(5): ] Lin HF, Li MY, Zhang SY, Li SG, Zhang CQ, Molecular biological identification of entomogenous fungi isolated from the brown planthopper. Chinese Journal of Applied Entomology, 48(5): [,,,,, , 48(5): ] Lin HF, Huang B, Li ZZ, Hu C, Electron microscopic scanning observation of the survival and infection of Beauveria bassiana on dendrolimus punctatus. Mycosytema, 17(4): [,,,, , 17(4): ] Liu J, Chen HZ, Li ZH, Process development and future for entomopathogenic fungus Beauveria bassiana production. Chinese Journal of Biological Control, 19(2): [,,, , 19(2): ] Liu YQ, Feng MG, Liu SS, Effect of temperature on virulence of Beauveria bassiana against myzus persicae. Chinese Journal of Biological Control, 16(2): [,,, , 16(2): ] Luo HD, Yan JL, Yu Y, Tan WZ, 2012, Isolation, screening and identification of bacterial agents for biological control of colorado potato beetle (Leptinotarsa decemlineata). Scientia Agricultura Sinica, 45(18): [,,,, , 45(18): ] Wang LJ, Lv LH, He YR, Xie MQ, Observation on infection process of Beauveria bassiana on cuticle of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), using

10 3 : 711 scanning electron microscope. Acta Entomologica Sinica, 53(1): [,,,, , 53(1): ] Wei YB, Shi SH, Liu MX, Interactions between infestation regularity of Chilo suppressalis and cultivation factor. Chinese Bulletin of Entomology, 42(6): [,,, , 42(6): ] Xue J, Ge F, Li JW, The interrelation between Chilo suppressalis and crops and its influencing factors. Chinese Bulletin of Entomology, 42(3): [,,,, , 42(3): ] Yang MZ, Li XH, Tan YF, Tian ZL, Different material packaging three temperature storage of Beauveria bassiana powder on the research of the corn borer control effect. Journal of Jilin Agricultural Sciences, 30(2): [,,,, , 30(2): ] Yao YJ, Xu XL, Zeng SG, Xu RZ, Xiong QH, Shu PP, Susceptibility of Chilo suppressalis to insecticides. Plant Protection, 39(6): [,,,,,, , 39(6): ] Yuan SY, Kong Q, Chen B, Xue CL, Tian XJ, Xiao C, Li ZY, Study on virulence of Beauveria bassiana MZ to larvae of Ostrinia furnacalis. Southwest China Journal of Agricultural Sciences, 24(2): [,,,,,,, , 24(2): ] Yuan SY, Zhang HR, Kong Q, Li ZY, Luo DQ, Wang XL, Dong LL, Detect ion of pathogenicity of Beauveryia bassiana MZ against Frankliniella occidentalis. Journal of Northwest A&F University(Nat. Sci. Ed.), 38(3): [,,,,,,, MZ , 38(3): ]

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<D2BDC1C6BDA1BFB5CDB6C8DAD7CAB8DFB7E5C2DBCCB3B2CEBBE1C3FBB5A52E786C7378> 参会人员名单 Last Name 姓名公司 Tel Fax Bai 柏煜康复之家 8610 8761 4189 8610 8761 4189 Bai 白威久禧道和股权投资管理 ( 天津 ) 有限公司 8610 6506 7108 8610 6506 7108 Bao 包景明通用技术集团投资管理有限公司 8610