球孢白僵菌有性世代培养及子代毒力稳定性鉴定
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摘要
在利用球孢白僵菌Beauveria bassiana有性态改良球孢白僵菌菌株的研究中,菌株的选择非常重要,其中利用交配型基因选择菌株是必要手段。而且虫生真菌的有性态能够使菌株间产生基因交流,促进其进化的过程。为更好地研究球孢白僵菌的遗传规律,以获得避免菌种衰退的方法,本研究建立了分别以柞蚕蛹为活体寄主和糙米培养基为基质的2种人工培养有性世代繁殖体系。结果表明,交配型为MAT1-1-1型和MAT1-2-1型混合型菌株BbDPSD5,经过自交在2种基质上均能够产生有性态。而具有2种单一交配型的菌株BbOFDH1-5和BbDPSD2进行配伍试验,BbOFDH1-5×BbDPSD2、BbOFDH1-5×BbDPSD5和BbDPSD2×BbDPSD5在2种基质上均没有有性态产生。对获得的有性态子囊孢子经无性繁殖产生的菌株进行r DNA-ITS片段以及交配型基因检测,并对测序结果进行比对分析。结果显示,扩增得到的r DNA-ITS序列和MAT1-2-1交配型基因与亲本序列同源性为100%,而没有检测到MAT1-1-1型。2种培养方式获得的结果完全一致。对有性态子代子囊孢子产生的株系进行毒力测试结果表明,所有子代菌株与原始菌株BbDPSD5的毒力差异不显著,子代交配型基因虽然缺失了MAT1-1-1,但是毒力没有衰退,未出现菌种退化现象。
Strains selected by improved and typical mating practice are very important to enhance sexual reproduction of Beauveria bassiana.Gene flow and evolution can occur during the stage of teleomorph.To study the inheritance of B.bassiana and solve the recession-related problem,we developed two novel methods for cultivating sexual generation,Cordyceps bassiana,using two kinds of media,pupae of Antheraea pernyi and brown rice.The results showed that BbDPSD5 inbred strain of B.bassiana which possessed two mating type genes,MAT1-1-1 and MAT1-2-1,could develop into C.bassiana through these two media.No fruit bodies,however,were developed in other combinations,i.e.BbOFDH1-5×BbDPSD2,BbOFDH1-5×BbDPSD5,and BbDPSD2×BbDPSD5,on the two media.Mating type genes and partial segments of rDNA-ITS gene were amplified and sequenced from the cloning strains of ascospores.The results indicated that the r DNA-ITS gene and MAT1-2-1 gene from strains of ascospores had 100% homologous with their parental type,and no MAT1-1-1 gene was detected.The results were consistent between the two media treatments.There was no significant difference on mortality between the ascospore strains and Bb DPSD5 strain in toxicity tests.The results also indicated that there was no decrease in toxicity and no recession on parental strain.
Strains selected by improved and typical mating practice are very important to enhance sexual reproduction of Beauveria bassiana.Gene flow and evolution can occur during the stage of teleomorph.To study the inheritance of B.bassiana and solve the recession-related problem,we developed two novel methods for cultivating sexual generation,Cordyceps bassiana,using two kinds of media,pupae of Antheraea pernyi and brown rice.The results showed that BbDPSD5 inbred strain of B.bassiana which possessed two mating type genes,MAT1-1-1 and MAT1-2-1,could develop into C.bassiana through these two media.No fruit bodies,however,were developed in other combinations,i.e.BbOFDH1-5×BbDPSD2,BbOFDH1-5×BbDPSD5,and BbDPSD2×BbDPSD5,on the two media.Mating type genes and partial segments of rDNA-ITS gene were amplified and sequenced from the cloning strains of ascospores.The results indicated that the r DNA-ITS gene and MAT1-2-1 gene from strains of ascospores had 100% homologous with their parental type,and no MAT1-1-1 gene was detected.The results were consistent between the two media treatments.There was no significant difference on mortality between the ascospore strains and Bb DPSD5 strain in toxicity tests.The results also indicated that there was no decrease in toxicity and no recession on parental strain.
引文
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[14]夏玉先,罗义辉,金凯.高毒力杀虫真菌基因工程菌株选育的进展与方向[J].中国生物防治学报,2011,27(1):1-5.
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[19]李增智,李春如,黄勃,等.重要虫生真菌球孢白僵菌有性型的发现和证实[J].科学通报,2001,46(6):470-473.
[20]Sung J M,Lee J O,Humber R A,et al.Cordyceps bassiana and production of stromata in vitro showing Beauveria anamorph in Korea[J].Mycobiology,2006,34(1):1-6.
[21]Lee J O,Shrestha B,Kim T W,et al.Stable formation of fruiting body in Cordyceps bassiana[J].Mycobiology,2007,35(4):230-234.
[22]Lee J O,Shrestha B,Sung G H,et al.Cultural characteristics and fruiting body production in Cordyceps bassiana[J].Mycobiology,2010,38(2):118-121.
[23]Lee J O,Shrestha B,Sung G H,et al.Successful development of Cordyceps bassiana stromata from Beauveria bassiana[J].Mycobiology,2010,38(1):13-16.
[24]宋彦英,周大荣,何康来.亚洲玉米螟无琼脂半人工饲料的研究与应用[J].植物保护学报,1999,26(4):324-328.
[25]朱衡,翟峰,朱立煌.利用氯化苄提取适于分子生物学分析的真菌DNA[J].真菌学报,1994,13(1):34-40.
[26]White T J,Bruns T,Lee S J W T,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[J].PCR Protocols:A Guide to Methods and Applications,1990,18(1):315-322.
[27]李晓慧,汪洋洲,张军,等.不同交配型球孢白僵菌在玉米植株中的定殖及其交配型基因变异比较研究[J].中国生物防治学报,2014,30(6):743-749.
[28]努尔买买提,焦子伟,杨晓绒,等.不同人工栽培培养基对蛹虫草子实体发育的影响及效益分析[J].新疆农业科学,2016,53(2):359-366.
[29]马文石,李凤芹,金英.柞蚕蛹虫草栽培技术[J].食用菌,2008,30(4):45-46.
[30]Dijksterhuis J,Samson R A.Food Mycology:A Multifaceted Approach to Fungi and Food[M].Boca Raton:CRC Press,2007,101-117.
[31]Whittle C A,Nygren K,Johannesson H.Consequences of reproductive mode on genome evolution in fungi[J].Fungal Genetics Boilogy,2011,48(7):661-667.
[32]Xiong C,Xia Y,Zheng P,et al.Increasing oxidative stress tolerance and subculturing stability of Cordyceps militaris by overexpression of a glutathione peroxidase gene[J].Applied Microbiology and Biotechnology,2013,97(5):2009-2015.
[33]Luo Z,Qin Y,Pei Y,et al.Ablation of the cre A regulator results in amino acid toxicity,temperature sensitivity,pleiotropic effects on cellular development and loss of virulence in the filamentous fungus Beauveria bassiana[J].Environmental Microbiology,2014,16(4):1122-1136.
[34]Turgeon B G.Application of mating type gene technology to problems in fungal biology[J].Annual Review of Phytopathology,1998,36(1):115-137.
[2]Smith S M,Moor E D,Oduor G I,et al.Effect of wood ash and conidia of Beauveria bassiana(Balsamo)Vuillemin on mortality of Prostephanus truncatus(Horn)[J].Journal of Stored Products Research,2006,42(3):357-366.
[3]Thompson S R,Brandenburg R L,Arends J J.Impact of moisture and UV degradation on Beauveria bassiana(Balsamo)Vuillemin conidial viability in turfgrass[J].Biological Control,2006,39(3):401-407.
[4]Moorthi P V,Balasubramanian C.Aulacophora foveicollis,a natural diet to entomopathogenic fungus,Beauveria bassiana[J].The Journal of Basic and Applied Zoology,2016,73(1):28-31.
[5]Tamayo-Mejía F,Tamez-Guerra P,Guzmán-Franco A W,et al.Developmental stage affects survival of the ectoparasitoid Tamarixia triozae exposed to the fungus Beauveria bassiana[J].Biological Control,2016,93(2):30-36.
[6]唐晓庆,樊美珍,李增智.球孢白僵菌继代培养中菌落局变现象及环境影响因素的研究[J].真菌学报,1996,15(3):188-196.
[7]Butt T M,Wang C,Shah F A,et al.Degeneration of entomogenous fungi[M]//Eilenberg J.An Encological and Societal Approach to Biological Control.New York:Springer,2006,213-226.
[8]徐金柱,黄勃,李增智.球孢白僵菌单孢子分离株在继代培养过程中菌落局变的遗传分析[J].菌物学报,2010,29(2):279-284.
[9]Nelson T L,Mc Neill M R,van Koten C,et al.Comparative virulence of Beauveria bassiana isolates against clover root weevil(Sitona obsoletus)[J].New Zealand Plant Protection,2015,68:139-145.
[10]李银平,雷仲仁,王海鸿.对西花蓟马高效的球孢白僵菌菌株筛选及产孢特性研究[J].中国生物防治学报,2013,29(2):219-226.
[11]王曦茁,汪来发,马建伟,等.松褐天牛球孢白僵菌高毒力航天诱变菌株的筛选[J].昆虫学报,2014,57(11):1299-1305.
[12]Shang Y,Duan Z,Huang W,et al.Improving UV resistance and virulence of Beauveria bassiana by genetic engineering with an exogenous tyrosinase gene[J].Journal of Invertebrate Pathology,2012,109(1):105-109.
[13]FernandeséK,Rangel D E,Braga G U,et al.Tolerance of entomopathogenic fungi to ultraviolet radiation:a review on screening of strains and their formulation[J].Current Genetics,2015,61(3):427-440.
[14]夏玉先,罗义辉,金凯.高毒力杀虫真菌基因工程菌株选育的进展与方向[J].中国生物防治学报,2011,27(1):1-5.
[15]O’Gorman C M,Fuller H T,Dyer P S.Discovery of a sexual cycle in the opportunistic fungal pathogen Aspergillus fumigatus[J].Nature,2009,457(7228):471-474.
[16]Yokoyama E,Yamagishi K,Hara A.Development of a PCR-based mating-type assay for Clavicipitaceae[J].FEMS Microbiology Letters,2004,237(2):205-212.
[17]Simon H M,Emma T S,Michael J W,et al.Mate-recognition and species boundaries in the ascomycetes[J].Fungal Diversity,2013,58(1):1-12.
[18]Shiu P K T,Glass N L.Cell and nuclear recognition mechanisms mediated by mating type in filamentous ascomycetes[J].Current Opinion in Microbiology,2000,3(2):183-188.
[19]李增智,李春如,黄勃,等.重要虫生真菌球孢白僵菌有性型的发现和证实[J].科学通报,2001,46(6):470-473.
[20]Sung J M,Lee J O,Humber R A,et al.Cordyceps bassiana and production of stromata in vitro showing Beauveria anamorph in Korea[J].Mycobiology,2006,34(1):1-6.
[21]Lee J O,Shrestha B,Kim T W,et al.Stable formation of fruiting body in Cordyceps bassiana[J].Mycobiology,2007,35(4):230-234.
[22]Lee J O,Shrestha B,Sung G H,et al.Cultural characteristics and fruiting body production in Cordyceps bassiana[J].Mycobiology,2010,38(2):118-121.
[23]Lee J O,Shrestha B,Sung G H,et al.Successful development of Cordyceps bassiana stromata from Beauveria bassiana[J].Mycobiology,2010,38(1):13-16.
[24]宋彦英,周大荣,何康来.亚洲玉米螟无琼脂半人工饲料的研究与应用[J].植物保护学报,1999,26(4):324-328.
[25]朱衡,翟峰,朱立煌.利用氯化苄提取适于分子生物学分析的真菌DNA[J].真菌学报,1994,13(1):34-40.
[26]White T J,Bruns T,Lee S J W T,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[J].PCR Protocols:A Guide to Methods and Applications,1990,18(1):315-322.
[27]李晓慧,汪洋洲,张军,等.不同交配型球孢白僵菌在玉米植株中的定殖及其交配型基因变异比较研究[J].中国生物防治学报,2014,30(6):743-749.
[28]努尔买买提,焦子伟,杨晓绒,等.不同人工栽培培养基对蛹虫草子实体发育的影响及效益分析[J].新疆农业科学,2016,53(2):359-366.
[29]马文石,李凤芹,金英.柞蚕蛹虫草栽培技术[J].食用菌,2008,30(4):45-46.
[30]Dijksterhuis J,Samson R A.Food Mycology:A Multifaceted Approach to Fungi and Food[M].Boca Raton:CRC Press,2007,101-117.
[31]Whittle C A,Nygren K,Johannesson H.Consequences of reproductive mode on genome evolution in fungi[J].Fungal Genetics Boilogy,2011,48(7):661-667.
[32]Xiong C,Xia Y,Zheng P,et al.Increasing oxidative stress tolerance and subculturing stability of Cordyceps militaris by overexpression of a glutathione peroxidase gene[J].Applied Microbiology and Biotechnology,2013,97(5):2009-2015.
[33]Luo Z,Qin Y,Pei Y,et al.Ablation of the cre A regulator results in amino acid toxicity,temperature sensitivity,pleiotropic effects on cellular development and loss of virulence in the filamentous fungus Beauveria bassiana[J].Environmental Microbiology,2014,16(4):1122-1136.
[34]Turgeon B G.Application of mating type gene technology to problems in fungal biology[J].Annual Review of Phytopathology,1998,36(1):115-137.