三株球孢白僵菌对栗实象甲幼虫的感染和致病力研究
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摘要
为了明确球孢白僵菌Beauveria bassiana 3菌株TST05、FDB01、SYN01对栗实象甲老熟幼虫的感染、致死率、致死中时、以及胞外蛋白酶、几丁质酶、脂肪酶的活性与毒力的关系,本研究采用3个菌株的孢子悬浮液(1.0×108孢子/m L),浸虫法感染栗实象甲幼虫,统计连续8 d的校正死亡率;以栗实象甲幼虫的虫体作为菌株的唯一碳源制备液体培养基,测定培养过程中各菌株的脂肪酶、几丁质酶、类枯草杆菌蛋白酶(Pr1酶)连续8 d的酶活性变化。结果表明,球孢白僵菌3个菌株TST05、FDB01、SYN01感染栗实象甲幼虫后,1-4 d内表现出染病和死亡的症状,连续8 d累积校正死亡率分别为40%、44.4%、62.22%,菌株SYN01对栗实象甲的致死率最高,与其他两菌株的差异显著;菌株TST05、FDB01、SYN01的Pr1酶活性最大峰值分别为(175.56±0.7)U/m L、(172.74±2.32)U/m L、(195.71±5.41)U/m L。几丁质酶活性最大峰值分别为(12.58±0.58)U/m L、(13.06±1.16)U/m L、(15.12±0.32)U/m L。都是菌株SYN01的酶活性最大,而TST05和FDB01之间差异不显著;球孢白僵菌3个菌株的Pr1酶、几丁质酶、脂肪酶在8 d内的酶活性平均值与染菌幼虫8 d累积校正死亡率的线性回归方程分别为y=0.4641x+122.45(R2=0.8854)、y=0.034x+10.473(R2=0.9328)、y=0.0354x+2.4586(R2=0.1201),说明菌株的Pr1酶和几丁质酶活性与幼虫死亡率之间呈显著的线性相关,而菌株的脂肪酶活性与菌株对幼虫的致死率不具有线性关系。综合分析,菌株SYN01可以作为生物防治栗实象甲的病原菌种;Pr1酶与几丁质酶的活性可作为菌株的毒力因子。
This research is to explore the infection symptoms,lethallty rate,the median lethal time( LT50) of the larvae of Curculio davidi infected with the three strains of Beauveria bassiana( TST05,FDB01 and SYN01),and to explore the relationship between the activities of extracelluar enzymes and virulence of the three strains. The following methods were used. The C. davidi were infected by immersing the insects with conidial suspension( 1. 0 × 108 conidia/m L) of the three strains. The corrected lethallty rates of the larvae were recorded every day. The baked dry larvae were used as the sole carbon source in the medium for fungal culturing,and the activities of threes extracellular enzymes,including lipase,protease( Pr1) and chintinase produced by the three strains were determined. Theresults showed the diseased symptoms and death of larvae started in 1- 4 d after inoculation,and the cumulative mortality on 8 d of TST05, FDB01 and SYN01 were 40%, 44. 4% and 62. 22%,respectively. In which,the SYN01 strain caused the highest mortality rates of the larvae and showed significant difference compared to the strains TST05 and FDB01. The maximum peak values of Pr1 enzyme activities of the strains TST05,FDB01 and SYN01 were( 175. 56 ± 0. 7) U/m L,( 172. 74 ±2. 32) U/m L and( 195. 71 ± 5. 41) U/m L,respectively. The maximum peak values of chitinase activity of the three strains were( 12. 58 ± 0. 58) U/m L,( 13. 06 ± 1. 16) U/m L and( 15. 12 ± 0. 32)U/m L,respectively. The Pr1 activities and chitinase activity of strain SYN01 were the highest in the three strains. And no difference existed between TST05 and FDB01. The relationships between the average values of Pr1 activity,chitinase activity and lipase activity of three strains in the period of 8 d and to the cumulative mortality of the infected larvae were analyzed,and the linear equations were y =0. 4641 + 122. 45( R2= 0. 8854),y = 0. 034 x + 10. 473( R2= 0. 9328),y = 0. 0354 x + 2. 4586( R2=0. 1201),respectively. It is indicated a significant linear relationship between Pr1 activity and chitinase activity of the strains and the larval mortality. Based on comprehensive analysis of the results,the strain SYN01 can be considered as a better pathogenic fungus for biological control of Curculio davidi larvae.Pr1 and chitinase activity can be used as virulence index for strain choice.
This research is to explore the infection symptoms,lethallty rate,the median lethal time( LT50) of the larvae of Curculio davidi infected with the three strains of Beauveria bassiana( TST05,FDB01 and SYN01),and to explore the relationship between the activities of extracelluar enzymes and virulence of the three strains. The following methods were used. The C. davidi were infected by immersing the insects with conidial suspension( 1. 0 × 108 conidia/m L) of the three strains. The corrected lethallty rates of the larvae were recorded every day. The baked dry larvae were used as the sole carbon source in the medium for fungal culturing,and the activities of threes extracellular enzymes,including lipase,protease( Pr1) and chintinase produced by the three strains were determined. Theresults showed the diseased symptoms and death of larvae started in 1- 4 d after inoculation,and the cumulative mortality on 8 d of TST05, FDB01 and SYN01 were 40%, 44. 4% and 62. 22%,respectively. In which,the SYN01 strain caused the highest mortality rates of the larvae and showed significant difference compared to the strains TST05 and FDB01. The maximum peak values of Pr1 enzyme activities of the strains TST05,FDB01 and SYN01 were( 175. 56 ± 0. 7) U/m L,( 172. 74 ±2. 32) U/m L and( 195. 71 ± 5. 41) U/m L,respectively. The maximum peak values of chitinase activity of the three strains were( 12. 58 ± 0. 58) U/m L,( 13. 06 ± 1. 16) U/m L and( 15. 12 ± 0. 32)U/m L,respectively. The Pr1 activities and chitinase activity of strain SYN01 were the highest in the three strains. And no difference existed between TST05 and FDB01. The relationships between the average values of Pr1 activity,chitinase activity and lipase activity of three strains in the period of 8 d and to the cumulative mortality of the infected larvae were analyzed,and the linear equations were y =0. 4641 + 122. 45( R2= 0. 8854),y = 0. 034 x + 10. 473( R2= 0. 9328),y = 0. 0354 x + 2. 4586( R2=0. 1201),respectively. It is indicated a significant linear relationship between Pr1 activity and chitinase activity of the strains and the larval mortality. Based on comprehensive analysis of the results,the strain SYN01 can be considered as a better pathogenic fungus for biological control of Curculio davidi larvae.Pr1 and chitinase activity can be used as virulence index for strain choice.
引文
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Kepenekci I,Gokce A,Gaugler R.Virulence of three species of entomopathogenic nematodes to the chestnut weevil,Curculio elephas(Coleoptera:Curculionidae)[J].Nematropica,2004,34(2):199-204.
Li CY,Kong FT.The damage characteristics and control strategy of Curculio davidi Fairmaire[J].Fruit Growers'Friend,2012,2:43-43.[李春野,孔凡涛.栗实象鼻虫的危害特点及防治对策[J].果农之友,2012,2:43-43]
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Qu DZ,Huang YC,Zhang YR.The biological property and integrated control of Curculio davidi Fairmaire[J].Shaanxi Forest Science and Technology,2009,1:71-73.[屈顶柱,黄应成,张宜仁.栗实象甲生物学特性及综合防治技术研究[J].陕西林业科技,2009,1:71-73]
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Sun SF,uo YG,Tang YJ,et al.Observation on the life history of Curculio bimaculatus Faust and its control with Paecilomyces farinosus[J].Forest Pest and Disease,2004,23(5):21-24.[孙绍芳,郭亚钢,唐永军,等.二斑栗实象甲生活史观察及粉拟青霉菌防治试验[J].中国森林病虫,2004,23(5):21-24]
St Leger RJ,Cooper RM,Charnley AK.Production of cuticledegrading enzymes by the entomopathogen Metarhizium anisopliae during infection of cuticles form Calliphora vomitoria and Manduca sexta[J].Journal of General Microbiology,1987,133(5):1371-1382.
Silva WOB,Mitidieri S,Schrank A,et al.Production and extracellular lipase from the entomopathogenic fungus Metarhizium anisopliae[J].Process Biochemistry,2005,40(1):321-326.
Svedese VM,Tiago PV,Bezerra JDP,et al.Pathogenicity of Beauveria bassiana and production of cuticle-degrading enzymes in the presence of Diatraea saccharalis cuticle[J].African Journal of Biotechnology,2013,12:6491-6497.
Wei MX,Liu RJ.The occurrence and control of the main pest of Jingdong Chestnut[J].Beijing Agriculture,2014,30:158-159.[魏敏宣,刘瑞江.京东板栗主要病虫害的发生与防治[J].北京农业,2014,30:158-159]
Yuan SJ,Sun Y,Wang XD,et al.The biological property and control of Curculio davidi Fairmaire[J].Liaoning Agricultural Sciences,2010,5:9-60.[袁少杰,孙羽,王晓东,等.栗实象鼻虫生物学特性与防治[J].辽宁农业科学,2010,5:9-60]
Yu WJ,Song XS,Sun Y.Progress on the control of Curculio davidi Fairmaire[J].Jilin Agriculture,2015,9:94-95.[遇文婧,宋小双,孙妍.栗实象甲的防治研究进展[J].吉林农业,2015,9:94-95]
Cao GC.Studies on Resistance of Diamondback Moth Plutella xylostella(L.)to Tebufenozide and the Mechanisms[D].Nanjing:Nanjing Agricultural University,2007.[曹广春.小菜蛾Plutella xylostella(L.)对虫酰肼的抗性及其机理研究[D].南京:南京农业大学,2007]
Fang WG.Cloning and Characterization of Cuticle Degrading Enzyme Genes from Entomopathogenic Fungi and Improvement of Beauveria bassiana by Genetic Modification[D].Chongqing:Southwest Agricultural University,2003.[方卫国.昆虫病原真菌降解寄主体壁酶基因的克隆及球孢白僵菌高毒力重组菌株的获得[D].重庆:西南农业大学,2003]
Feng MG.Reliability of extracellular protease and lipase activity of Beauveria bassiana isolates used as their virulence indices[J].Acta Microbiologica Sinica,1998,38(6):461-467.[冯明光.胞外蛋白酶和脂酶活性作为球孢白僵菌毒力指标的可靠性分析[J].微生物学报,1998,38(6):461-467]
Huang LN.The occurrence rule damage characteristics and integrated control of Curculio davidi Fairmaire[J].Friends of Farmers to Get Rich,2014,22:81-81.[黄莉宁.栗实象的发生规律、危害特点及综合防治[J].农民致富之友,2014,22:81-81]
Ihara F,Toyama M,Higaki M,et al.Comparison of pathogenicities of Beauveria bassiana and Metarhizium anisopliae to chestnut pests[J].Applied Entomology&Zoology,2009,44(1):127-132.
Kepenekci I,Gokce A,Gaugler R.Virulence of three species of entomopathogenic nematodes to the chestnut weevil,Curculio elephas(Coleoptera:Curculionidae)[J].Nematropica,2004,34(2):199-204.
Li CY,Kong FT.The damage characteristics and control strategy of Curculio davidi Fairmaire[J].Fruit Growers'Friend,2012,2:43-43.[李春野,孔凡涛.栗实象鼻虫的危害特点及防治对策[J].果农之友,2012,2:43-43]
Lin HP,Wei JY,Mao SF,et al.Correlation between protease,chitinase and lipase activities and virulence of Beauveria bassiana against Monochamus alternates[J].Chinese Journal of Biological Control,2008,24(3):290-292.[林海萍,魏锦瑜,毛胜凤,等.球孢白僵菌蛋白酶、几丁质酶、脂肪酶活性与其毒力相关性[J].中国生物防治,2008,24(3):290-292]
Mohanty SS,Raghavendra K,Dash AP.Induction of chymoelastase(Pr1)of Metarhizium anisopliae and its role in causing mortality to mosquito larvae[J].World Journal of Microbiology&Riotechnology,2008,24(10):2283-2288.
Qu DZ,Huang YC,Zhang YR.The biological property and integrated control of Curculio davidi Fairmaire[J].Shaanxi Forest Science and Technology,2009,1:71-73.[屈顶柱,黄应成,张宜仁.栗实象甲生物学特性及综合防治技术研究[J].陕西林业科技,2009,1:71-73]
Speranza S,Paparatti B,Bounous G,et al.Chemical control of chestnut weevils in central Italy[J].Acta Horticulturae,2010,866(3):411-415.
Sun SF,uo YG,Tang YJ,et al.Observation on the life history of Curculio bimaculatus Faust and its control with Paecilomyces farinosus[J].Forest Pest and Disease,2004,23(5):21-24.[孙绍芳,郭亚钢,唐永军,等.二斑栗实象甲生活史观察及粉拟青霉菌防治试验[J].中国森林病虫,2004,23(5):21-24]
St Leger RJ,Cooper RM,Charnley AK.Production of cuticledegrading enzymes by the entomopathogen Metarhizium anisopliae during infection of cuticles form Calliphora vomitoria and Manduca sexta[J].Journal of General Microbiology,1987,133(5):1371-1382.
Silva WOB,Mitidieri S,Schrank A,et al.Production and extracellular lipase from the entomopathogenic fungus Metarhizium anisopliae[J].Process Biochemistry,2005,40(1):321-326.
Svedese VM,Tiago PV,Bezerra JDP,et al.Pathogenicity of Beauveria bassiana and production of cuticle-degrading enzymes in the presence of Diatraea saccharalis cuticle[J].African Journal of Biotechnology,2013,12:6491-6497.
Wei MX,Liu RJ.The occurrence and control of the main pest of Jingdong Chestnut[J].Beijing Agriculture,2014,30:158-159.[魏敏宣,刘瑞江.京东板栗主要病虫害的发生与防治[J].北京农业,2014,30:158-159]
Yuan SJ,Sun Y,Wang XD,et al.The biological property and control of Curculio davidi Fairmaire[J].Liaoning Agricultural Sciences,2010,5:9-60.[袁少杰,孙羽,王晓东,等.栗实象鼻虫生物学特性与防治[J].辽宁农业科学,2010,5:9-60]
Yu WJ,Song XS,Sun Y.Progress on the control of Curculio davidi Fairmaire[J].Jilin Agriculture,2015,9:94-95.[遇文婧,宋小双,孙妍.栗实象甲的防治研究进展[J].吉林农业,2015,9:94-95]