Identification of 29 strains of entomogenous fungi and their toxicity to Bemisia tabaci
-
摘要:目的
对29株虫生真菌菌株进行分类学鉴定并筛选出对烟粉虱Bemisia tabaci具有高毒力的菌株。
方法采用形态学及ITS区、TEF区、Bloc区基因序列相似性分析鉴定菌株，并采用浸渍法测定真菌对烟粉虱的毒力。
结果29株虫生真菌有26株符合白僵菌Beauveria的特征，菌株SB003、SP665和SP670被鉴定属于棒束孢属Isaria；利用TEF区序列、Bloc区序列、TEF-Bloc联合序列构建的系统发育树，结果显示29株菌株共包括24株球孢白僵菌B. bassiana、2株假性球孢白僵菌B. pseudobassiana和3株环链棒束孢I. cateinannulata。烟粉虱2龄若虫的死亡率随孢子浓度的增加而增加，不同菌株致病力不同；用1×108 mL−1的孢子悬浮液处理烟粉虱2龄若虫7 d，致死率最高的菌株为SP433，其次为SB009，分别为87.37%和82.93%。
结论本研究可为虫生真菌的分类提供理论基础，筛选出的高致病力菌株SP433和SB009可为烟粉虱的生物防治提供参考。
Abstract:ObjectiveTo identify 29 strains of entomogenous fungi and select high virulent strains against Bemisia tabaci.
MethodIdentification of fungi were based on morphological analysis and genetic homology analysis of sequences in ITS, TEF and Bloc regions. The fungal toxicity to B. tabaci was evaluated by the immersion method.
ResultTwenty-six of 29 entomogenous fungal strains had the characteristics of Beauveria and the other three strains of SB003, SP665 and SP670 were identified as Isaria. Phylogenetic tree was constructed using TEF region sequence, Bloc region sequence and TEF-Bloc combined sequence, which showed that 29 strains included 24 strains of Beauveria bassiana, 2 strains of B. pseudobassiana, and 3 strains of Isaria cateinannulata. The mortality of the 2nd instar nymph of B. tabaci increased with the increase of spore concentration, and the pathogenicity of different strain was different. The 2nd instar nymphs of B. tabaci were treated with 1×108 mL−1 spore suspension for 7 days, and the SP433 strain had the highest lethality rate of 87.37%, followed by SB009 with a lethality rate of 82.93%.
ConclusionThis study can provide a theoretical basis for the classification of entomophytic fungi. The highly pathogenic strains of SP433 and SB009 were screened out, which can provide a reference for the biological control of B. tabaci.
-
-
![]()
图 1 菌株的菌落形态(奇数为正面，偶数为反面)
1、2：SB003；3、4：SB004；5、6：SB006；7、8: SB009；；9、10：SB010；11、12：SB015；13、14: SB026；15、16：SB032；17、18：SB035；19、20：SB036；21、22：SB037；23、24：SB038；25、26：SB039；27、28：SB041；29、30：SB043；31、32：SB050；33、34：SB051；35、36：SB057；37、38：SB062；39、40：SB063；41、42：SB671；43、44：SB672；45、46：SB673；47、48：SB674；49、50：SP016；51、52：SP031；53、54：SP433；55、56：SP665；57、58：SP670
Figure 1. Colony morphology of strain(the odd number showed the front and the even number showed the back )
![]()
图 4 基于不同序列以最大似然法构建的真菌系统发育树
▲为供试菌株
Figure 4. Phylogenetic tree of fungi constructed using the maximum likelihood method and based on different sequences
▲ indicates test strains
表 1 供试菌株信息
Table 1 Information of test strains
菌株
Strain采样地
Sampling place采样时间
Sampling timeSB003 湖北省神农架林区 Shennongjia forest area, Hubei Province 2007−07 SB004 甘肃省天水市麦草沟自然保护区
Maicaogou nature reserve, Tianshui, Gansu Province2007−08 SB006 甘肃省临夏回族自治州莲花山自然保护区
Lianhuashan nature reserve, Linxia Hui Autonomous Region, Gansu Province2007−08 SB009 四川省成都市青羊区文殊院 Wenshuyuan, Qingyang district, Chengdu, Sichuan Province 2007−10 SB010 云南省昆明市 Kunming, Yunnan Province 2007−09 SB015 河南省鹤壁市 Hebi, Henan Province 2007−10 SB026 四川省雅安市宝兴县蜂桶寨
Fengtong village, Baoxing county, Ya’an, Sichuan Province2007−07 SB032 韩国济州岛天地渊瀑布 Tiandiyuan waterfall, Jeju Island, South Korea 2008−07 SB035 云南省红河州石屏县 Shiping county, Honghe Prefecture, Yunnan Province 2009−05 SB036 内蒙古自治区呼伦贝尔市新巴尔虎左旗
Xinbalhuzuoqi, Hulunbuir, Inner Mongolia Autonomous Region2009−08 SB037 内蒙古自治区兴安盟扎赉特旗
Zhalaitqi, Xing’an League, Inner Mongolia Autonomous Region2009−08 SB038 内蒙古自治区锡林郭勒盟正镶白旗
Zhengxiangbaiqi, Xilinguole League, Inner Mongolia Autonomous Region2009−08 SB039 内蒙古自治区阿拉善盟贺兰山国家级自然保护区
Helan Mountain National Nature Reserve, Alxa League, Inner Mongolia Autonomous Region2009−08 SB041 内蒙古自治区锡林郭勒盟正镶白旗
Zhengxiangbaiqi, Xilinguole League, Inner Mongolia Autonomous Region2009−08 SB043 西藏自治区林芝市波密县古乡
Gu village, Bomi county, Linzhi, Tibet Autonomous Region2009−09 SB050 广东省广州市白云山 Baiyun Mountain, Guangzhou, Guangdong Province 2009−10 SB051 广东省河源市龙川县 Longchuan county, Heyuan, Guangdong Province 2010−05 SB057 贵州省雷山县方祥乡 Fangxiang village, Leishan county, Guizhou Province 2010−07 SB062 内蒙古自治区阿拉善盟古拉本敖包镇
Gulaben Aobao Town, Alxa League, Inner Mongolia Autonomous Region2010−08 SB063 内蒙古自治区阿拉善盟贺兰山脉中部哈拉乌
Harau, central Helan Mountain, Inner Mongolia Autonomous Region2010−08 SB671 云南省镇沅彝族哈尼族拉祜族自治县
Zhenyuan Yi Hani Lahu Autonomous county, Yunnan Province2009−10 SB672 福建省厦门市鼓浪屿 Gulangyu, Xiamen, Fujian Province 2010−09 SB673 云南省迪庆州维西县保和镇 Baohe town, Weixi county, Diqing Prefecture, Yunnan Province 2010−09 SB674 广东省广州市华南农业大学林学与风景园林学院
College of forestry and landscape architecture, South China Agricultural University, Guangzhou,
Guangdong Province2010−10 SP016 湖北省神农架林区老君山 Laojun Mountain, Shennongjia forest area, Hubei Province 2007−08 SP031 甘肃省张掖市肃南县东柳沟 Dongliugou, Sunan county, Zhangye, Gansu Province 2007−08 SP433 内蒙古自治区呼伦贝尔市牙克石市
Yakeshi, Hulunbuir, Inner Mongolia Autonomous Region2009−08 SP665 云南省楚雄彝族自治州峨绿公园西灵宫
Xiling Palace, Erlu Park, Chuxiong Yi Autonomous Prefecture, Yunnan Province2009−09 SP670 湖北省宜昌市蛤蟆泉 Toad Spring, Yichang, Hubei Province 2009−10 
下载: 导出CSV
表 2 虫生真菌DNA的PCR扩增引物
Table 2 PCR amplification primers for entomogenous fungal DNA
基因序列 Gene sequence 引物 Primer 引物序列(5′→3′) Primer sequence ITS ITS4F TCCTCCGCTTATTGATATGC ITS5R GGAAGTAAAAGTCGTAACAAGG Bloc B5.1F CGACCCGGCCAACTACTTTGA B3.1R GTCTTCCAGTACCACTACGCC TEF 983F GCYCCYGGHCAYCGTGAYTTYAT 2218R ATGACACCRACRGCRACRGTYTG
下载: 导出CSV
表 3 虫生真菌的产孢量1)
Table 3 Spore productions of entomogenous fungi
菌株
Strain产孢量/(×107 mL−1)
Spore production菌株
Strain产孢量/(×107 mL−1)
Spore production菌株
Strain产孢量/(×107 mL−1)
Spore productionSB003 3.25±0.59f SB037 9.86±0.69abcdef SB671 4.64±0.71ef SB004 14.27±2.63abc SB038 14.78±2.06ab SB672 8.35±1.72abcdef SB006 13.65±1.59abcd SB039 16.38±2.83a SB673 5.15±1.10def SB009 9.42±1.19abcdef SB041 3.48±0.47f SB674 6.61±1.75bcdef SB010 6.72±1.92bcdef SB043 11.16±1.48abcdef SP016 4.04±0.49ef SB015 8.29±0.74abcdef SB050 12.64±3.07abcde SP031 9.32±1.73abcdef SB026 9.19±1.73abcdef SB051 6.54±0.47bcdef SP433 12.54±2.36abcde SB032 16.56±1.59a SB057 9.30±0.37abcdef SP665 5.57±0.89cdef SB035 3.84±0.56ef SB062 6.02±1.76bcdef SP670 7.26±0.49bcdef SB036 11.23±2.66abcdef SB063 7.88±1.59abcdef 1)数据后不同小写字母者表示不同菌株间菌落产孢量差异显著(P<0.05，Duncan’s法)
1) Different lowercase letters indicate significant differences in spore production among different strains (P<0.05, Duncan’s test)
下载: 导出CSV
表 4 供试菌株ITS区、TEF区和Bloc区的序列比对结果
Table 4 Results of comparing the ITS regions, TEF regions and Bloc regions of test strains
真菌
Fungus菌株登录号 Accession number of strain ITS区
ITS regionTEF区
TEF regionBloc区
Bloc region球孢白僵菌 Beauveria bassiana KX901309 JQ867178 JQ867116 球孢白僵菌 B. bassiana AY531976 JQ867172 JQ867106 球孢白僵菌 B. bassiana KX901310 JQ867171 JQ867109 球孢白僵菌 B. bassiana GU233705 JQ867157 JQ867118 球孢白僵菌 B. bassiana GU233701 JQ867151 JQ867125 球孢白僵菌 B. bassiana AY883691 AY883788 球孢白僵菌 B. bassiana KU725693 KU725706 假球孢白僵菌 Beauveria pseudobassiana MH259852 HQ880999 HQ880727 假球孢白僵菌 B. pseudobassiana LC163567 LC163595 环链棒束孢 Isaria cateniannulata GU734760 GU734758
下载: 导出CSV
表 5 不同浓度的孢子悬浮液处理烟粉虱2龄若虫7 d的死亡率1）
Table 5 Mortality of the 2nd instar nymphs of Bemisia tabaci infected with spore suspensions of different concentrations for 7 days
% 菌株 Strain 1×107 mL−1 1×108 mL−1 菌株 Strain 1×107 mL−1 1×108 mL−1 CK 9.43±0.58d 11.73±2.00d SB043 47.67±4.00abc 80.33±3.93ab SB003 56.43±3.45abc 60.06±2.68bc SB050 49.47±1.47abc 81.30±4.86ab SB004 55.23±5.85abc 75.23±5.49abc SB051 59.67±4.17abc 70.87±5.90abc SB006 51.03±0.54abc 72.33±6.90abc SB057 51.67±3.55abc 76.00±2.34abc SB009 67.93±1.68ab 82.93±2.88ab SB062 35.81±3.54c 63.34±3.93abc SB010 47.90±4.42abc 70.20±4.94abc SB063 68.03±5.46ab 71.40±2.03abc SB015 64.30±7.27ab 77.07±4.95abc SB671 59.80±1.12abc 59.50±9.22bc SB026 51.77±4.15abc 67.80±3.0abc SB672 44.67±6.17bc 62.43±7.28bc SB032 56.57±2.95abc 77.69±3.88ab SB673 53.23±5.62abc 70.53±2.57abc SB035 53.07±2.37abc 59.99±2.77bc SB674 67.20±7.04ab 77.20±2.70abc SB036 55.33±6.36abc 80.67±4.41ab SP016 54.47±8.25abc 72.07±4.82abc SB037 50.17±7.69abc 74.00±3.21abc SP031 49.43±2.13abc 52.85±1.00c SB038 48.03±6.08abc 70.87±5.90abc SP433 69.03±3.76ab 87.37±0.43a SB039 72.77±2.14a 73.13±5.20abc SP665 49.70±4.65abc 64.90±3.26abc SB041 50.33±4.36abc 70.33±5.42abc SP670 65.77±0.50ab 72.40±1.56abc 1)相同浓度数据后不同小写字母者表示不同菌株间致病力差异显著(P<0.05，Duncan’s法)
1)Different lowercase letters after data at the same concentration indicate significant differences in pathogenicity among different strains (P<0.05, Duncan’s test)
下载: 导出CSV
-
[1] 查岭生, 文庭池, KEVIN D H, et al. 中国虫霉目真菌及其寄主昆虫名录更新[J]. 菌物学报, 2016, 35(6): 666-683. [2] 阙生全, 喻爱林, 刘亚军, 等. 白僵菌应用研究进展[J]. 中国森林病虫, 2019, 38(2): 29-35. doi: 10.3969/j.issn.1671-0886.2019.02.007 [3] 蒲蛰龙, 李增智. 昆虫真菌学[M]. 合肥: 安徽科学技术出版社, 1996: 402-715. [4] WRIGHT M S, LAX A R. Improved mortality of the Formosan subterranean termite by fungi, when amended with cuticle-degrading enzymes or eicosanoid biosynthesis inhibitors[J]. Folia Microbiol, 2016, 61(1): 73-83. doi: 10.1007/s12223-015-0412-0
[5] HEINIG R L, PAAIJMANS K P, HANCOCK P A, et al. The potential for fungal biopesticides to reduce malaria transmission under diverse environmental conditions[J]. J Appl Ecol, 2015, 52(6): 1558-1566. doi: 10.1111/1365-2664.12522
[6] 刘晓晓, 王星冉, 王义勋, 等. 马尾松毛虫高毒力球孢白僵菌菌株的生物学特性[J]. 南方农业学报, 2017, 48(6): 1019-1023. doi: 10.3969/j.issn.2095-1191.2017.06.13 [7] 辛正, 张晓, 刘慧媛, 等. 白僵菌在蟑螂防治中的研究进展[J]. 中华卫生杀虫药械, 2017, 23(5): 401-406. [8] SUN M, REN Q, GUAN G, et al. Eectiveness of Beauveria bassiana sensu lato strains for biological control against Rhipicephalus (Boophilus)microplus (Acari: Ixodidae) in China[J]. Parasitol Int, 2013, 62(5): 412-415. doi: 10.1016/j.parint.2013.04.008
[9] CHEN W, HASEGAWA D D, KAUR N, et al. The draft genome of whitefly Bemisia tabaci MEAM1, a global crop pest, provides novel insights into virus transmission, host adaptation, and insecticide resistance[J]. BMC Biol, 2016, 14(1): 110. doi: 10.1186/s12915-016-0321-y
[10] CHEN M J, HUANG B, LI Z Z, et al. Morphological and genetic characterisation of Beauveria sinensissp. nov. from China[J]. Mycotaxon, 2013, 124(1): 301-308. doi: 10.5248/124.301
[11] KESSIA F C, PANTOJA, KELLY C G, et al. Identification of Capsicum accessions to lerantto Tomato severe rugose virus and resistant to Bemisia tabaci Middle East-Asia Minor1(MEAM1)[J]. Trop Plant Pathol, 2018, 43(2): 138-145. doi: 10.1007/s40858-018-0212-6
[12] 李英梅, 杨苗苗, 刘晨, 等. 杀虫剂对烟粉虱传番茄黄化曲叶病毒病的防控效果[J]. 西北农业学报, 2019, 28(3): 466-474. [13] JURAJ M, JAROSLAV M, JANKA M, et al. Phylogenetic structure and habitat associations of Beauveria species isolated from soils in Slovakia[J]. J Invertebr Pathol, 2016, 140: 46-50. doi: 10.1016/j.jip.2016.08.009
[14] 潘李隆, 迟瑶, 范运运, 等. 烟粉虱对菜豆金黄花叶病毒属病毒的传播:病毒对媒介昆虫的适应[J]. 昆虫学报, 2019, 62(7): 885-894. [15] GILBERTSON R L, BATUMAN O, WEBSTER C G, et al. Role of the insect supervectors Bemisia tabaci and Frankliniella occidentalisin the emergence and global spread of plant viruses[J]. Annu Rev Virol, 2015, 2(1): 67-93. doi: 10.1146/annurev-virology-031413-085410
[16] ABDESSAMAD I, MUZAMMIL H, PAUL M, et al. Entomopathogenic fungus Beauveria: Host specificity, ecology and significance of morpho-molecular characterization in accurate taxonomic classification[J]. J Asia-Pac Entomol, 2017, 20(4): 1204-1212. doi: 10.1016/j.aspen.2017.08.015
[17] 褚栋, 张友军. 近10年我国烟粉虱发生为害及防治研究进展[J]. 植物保护, 2018, 44(5): 51-55. [18] 郭东升, 翟颖妍, 任广伟, 等. 白僵菌属分类研究进展[J]. 西北农业学报, 2019, 28(4): 497-509. doi: 10.7606/j.issn.1004-1389.2019.04.001 [19] 余垚颖, 郭应菊, 杨雪, 等. 我国虫生真菌的研究现状[J]. 四川农业科技, 2018(12): 27-29. doi: 10.3969/j.issn.1004-1028.2018.12.015 [20] REHNER S A, BUCKLEY E P. A Beauveria phylogeny inferred from nuclear ITS and EF1-α sequences: Evidence for cryptic diversification and links to Cordyceps teleomorphs[J]. Mycologia, 2005, 97: 84-98.
[21] REHNER S A, MINNIS A M, SUNG G H, et al. Phylogeny and systematics of the anamorphic, entomopathogenic genus Beauveria[J]. Mycologia, 2011, 103: 1055-1073. doi: 10.3852/10-302
[22] MEYLING N V, PILZ C, KELLER S, et al. Diversity of Beauveria spp. isolates from pollen beetles Meligethes aeneusin Switzerland[J]. J Invertebr Pathol, 2012, 109: 76-82. doi: 10.1016/j.jip.2011.10.001
[23] 张云月, 张军, 李丽莉, 等. 球孢白僵菌有性世代培养及子代毒力稳定性鉴定[J]. 中国生物防治学报, 2017, 33(3): 364-370. [24] 芦俊佳, 夏举飞, 庄飞, 等. 楚雄腮扁叶蜂虫生真菌粉红粘帚霉的鉴定及其生物学特性[J]. 植物保护学报, 2018, 45(3): 622-631. [25] 杨帆, 曹涵铭, 陈园园, 等. AM真菌结构及鉴定方法的研究进展[J]. 现代农业科技, 2019(16): 152-154. doi: 10.3969/j.issn.1007-5739.2019.16.095 [26] 张伟铮, 关文苑, 李松, 等. ITS序列分析与MALDI-TOF MS质谱技术在丝状真菌鉴定中的应用[J]. 菌物学报, 2019, 38(8): 1298-1305. [27] 甘森宁, 陆梅颖, 肖志邦, 等. 植物内生真菌的分类鉴定方法[J]. 安徽农学通报, 2019, 25(10): 36-37. doi: 10.3969/j.issn.1007-7731.2019.10.014 [28] 李美君, 白庆荣, 臧连生, 等. 水稻二化螟病原真菌鉴定及其致病力[J]. 中国生物防治学报, 2019, 35(1): 63-69. [29] 方志刚, 张立钦, 林新春. 不同球孢白僵菌菌株DNA的RAPD分析[J]. 南京林业大学学报(自然科学版), 2001, 25(4): 65-68. doi: 10.3969/j.issn.1000-2006.2001.04.015 [30] 王慧. 绿僵菌和白僵菌不同菌株对烟粉虱若虫的致病力及应用研究[D]. 合肥: 安徽农业大学, 2013. [31] 吴乾兴, 刘勇, 张友军, 等. 烟粉虱常用药剂抗性监测研究[J]. 现代农业科技, 2018(2): 119-120. [32] 王登杰, 王海鸿, 张桃, 等. 三株球孢白僵菌侵染烟粉虱的比较生长动力学及其毒力[J]. 菌物学报, 2016, 35(5): 577-585. [33] 银航, 窦雪绒, 张云霞, 等. 球孢白僵菌、保幼激素联用防治花椒蚜虫[J]. 陕西农业科学, 2019, 65(11): 57-61. doi: 10.3969/j.issn.0488-5368.2019.11.018 [34] 张爽, 杨佳利, 马军, 等. 苏云金杆菌(Bacillus thuringiensis)与球孢白僵菌(Beauveria bassiana)复配对小菜蛾幼虫(Plutella xylostella)的影响研究[J]. 生物灾害科学, 2017, 40(4): 288-291. doi: 10.3969/j.issn.2095-3704.2017.04.63 [35] 杨芷, 路杨, 毛刚, 等. 赤眼蜂携带球孢白僵菌防治亚洲玉米螟技术研究与应用[J/OL]. 中国生物防治学报, 2019. doi: 10.16409/j.cnki.2095-039x.2019.05.011.
