杧果露水斑病菌和细菌性黑斑病菌双重PCR体系的建立
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摘要
杧果露水斑病和细菌性黑斑病在国内杧果产区普遍发生,严重威胁杧果安全生产,俗称杧果"两病"。建立准确高效的检测方法,提早检出杧果两病并及时采取防治措施,可有效减轻为害。本实验通过筛选反应体系和电泳条件,建立了可同时检测杧果中的露水斑病菌和细菌性黑斑病菌的双重PCR检测体系。结果表明:25μL反应体系,2×Taq PCR MasterMix 12.5μL,引物ML-SF9/SR5 (10μmol/L)各1.5μL、XcmHF/HR(10μmol/L)各1μL,模板各1μL,加ddH2O补足至25μL。反应条件为94℃预变性4 min;94℃变性45 s,65℃退火45 s,72℃延伸1 min,35个循环;72℃延伸5 min。电泳检测条件为1.5%琼脂糖凝胶浓度、120 V电压电泳25 min。该体系检测下限为杧果露水斑病原菌g DNA 0.224 ng/μL、细菌性黑斑菌液2.6×1~04cfu/m L。该技术可应用于田间杧果"两病"的早期检测与鉴定工作中。
Cladosporium cladosporioides(Fr.) de Vries and Xanthomonas campestris pv. mangiferaeindicae of mango called the "two mango diseases" occur in most mango producing regions of China, which seriously affects mango production. In order to effectively reduce the loss, it is necessary to establish an accurate and efficient detection method to detect two mango diseases and prevent them in time. By screening the reaction system and electrophoresis conditions in this study, a double PCR detection system for detecting Cladosporium cladosporioides(Fr.) de Vries and Xanthomonas campestris pv. mangiferaeindicae of mango was established. The results showed that 25 μL reaction system consisted of 12.5 μL 2×Taq PCR MasterMix, 1.5 μL 10 μmol/L ML-SF9, 1.5 μL 10 μmol/L ML-SR5,1 μL 10 μmol/L XcmHF, 1 μL 10 μmol/L Xcm HR, and 1 μL of each template, made up to 25 μL with ddH2 O.The reaction conditions were as follows: one cycle for pre-degeneration at 94℃ for 4 min, 35 cycles at 94℃ for 45 s for degeneration, 65℃ for 45 s for annealing, and 72℃ for 1 min for extension, followed by one cycle at 72℃ for 5 min for extension. The electrophoresis detection conditions were 1.5% agarose gel and electrophoresis under 120 V for25 minutes. The results showed that the lower limit was 0.224 ng/μL for Cl. cladosporioides g DNA and 2.6 ×10~4cfu/mL for Xcm using this double PCR detection system. This method could be applied to the early detection and identification of "two diseases" on mangoes in field.
Cladosporium cladosporioides(Fr.) de Vries and Xanthomonas campestris pv. mangiferaeindicae of mango called the "two mango diseases" occur in most mango producing regions of China, which seriously affects mango production. In order to effectively reduce the loss, it is necessary to establish an accurate and efficient detection method to detect two mango diseases and prevent them in time. By screening the reaction system and electrophoresis conditions in this study, a double PCR detection system for detecting Cladosporium cladosporioides(Fr.) de Vries and Xanthomonas campestris pv. mangiferaeindicae of mango was established. The results showed that 25 μL reaction system consisted of 12.5 μL 2×Taq PCR MasterMix, 1.5 μL 10 μmol/L ML-SF9, 1.5 μL 10 μmol/L ML-SR5,1 μL 10 μmol/L XcmHF, 1 μL 10 μmol/L Xcm HR, and 1 μL of each template, made up to 25 μL with ddH2 O.The reaction conditions were as follows: one cycle for pre-degeneration at 94℃ for 4 min, 35 cycles at 94℃ for 45 s for degeneration, 65℃ for 45 s for annealing, and 72℃ for 1 min for extension, followed by one cycle at 72℃ for 5 min for extension. The electrophoresis detection conditions were 1.5% agarose gel and electrophoresis under 120 V for25 minutes. The results showed that the lower limit was 0.224 ng/μL for Cl. cladosporioides g DNA and 2.6 ×10~4cfu/mL for Xcm using this double PCR detection system. This method could be applied to the early detection and identification of "two diseases" on mangoes in field.
引文
Jia J.,Pu J.J.,Lu Y.C.,Zhan W.,Wang F.,and Xie Y.X.,2012,Detection of m ango malformation disease by nested-PCR,Guangdong Nongye Kexue(Guang dong Agriculture Sciences),39(22):162-164(贾静,蒲金基,吕延超,占魏,王芳,谢艺贤,2012,杧果畸形病的巢式PCR检测,广东农业科学,39(22):162-164)
Jing M.M.,Tian Y.Q.,Wang R.H.,and Shao Y.Z.,2017,Isolation and identifi cation of pathogen causing mango dew blotch disease and screening of its antagonistic microorganisms,Shipin Gongye Keji(Science and Technology of Food Industry),38(5):163-167(井敏敏,田亚琴,王润菡,邵远志,2017,杧果露水斑病病原菌分离鉴定及其拮抗菌筛选,食品工业科技,38(5):163-167)
Mills P.R.,Sreeivasaprasad S.,and Brown A.E.,1992,Detection and differentiation of Colletotrichum gloeosporioides isolates using PCR,Fems Microbiol.Lett.,77(1-3):137-143
Pu J.J.,Qi Y.X.,and Xie Y.Y.,2012,NY/T2257 2012 Molecular detection for pathogen of mango bacterial black spot dis ease,China Agriculture Press,Beijing,China,pp.1-8(蒲金基,漆艳香,谢艺贤,2012,NYT 2257-2012杧果细菌性黑斑病原菌分子检测技术规范,中国农业出版社,中国,北京,pp.1-8)
Qi Y.X.,Zhng H.,Yu Q.F.,Xie Y.X.,Zhang X.,Lu Y.,Zhang H.Q.,and Pu J.J.,2015,Survival period of Xanthomonas campestris pv.mangiferaeindicae in different places,Zhiwu Baohu(Plant Protection),41(5):140-144(漆艳香,张贺,喻群芳,谢艺贤,张欣,陆英,张辉强,蒲金基,2015,杧果细菌性黑斑病菌在不同场所的存活期,植物保护,41(5):140-144)
Sreenivasaprasad S.,Sharada K.,Brown A.E.,and Mills P.R.,1996,PCR-based detection of Colletotrichum acutatum on strawberry,Plant Pathol.,45(4):650-655
Wen Y.T.,and Huang S.M.,1994,Identification of symptoms and pathogen of mango bacterial black spot,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),(1):79-85(文衍堂,黄圣明,1994,杧果细菌性黑斑病症状与病原菌鉴定,热带作物学报,(1):79-85)
Wu J.B.,Zhan R.L.,Liu F.,Zhao Y.L.,He Y.B.,and Chang J.M.,2016,Rapid molecular detection of Fusarium mangiferae,the pathogen of mango malformation disease,Junwu Xuebao(Mycosystema),35(3):298-308(吴婧波,詹儒林,柳凤,赵艳龙,何衍彪,常金梅,2016,杧果畸形病病原菌Fusarium mangiferae的快速分子检测,菌物学报,35(3):298-308)
Zhang H.,Liu X.M.,Yu Q.F.,Qi Y.X.,Xie Y.Y.,and Pu J.J.,2013,Preliminary studies of mango sooty blotch of Cladosporium cladosporioides in Hainan Province,Guangdong Nongye Kexue(Guangdong Agriculture Sciences),40(7):75-77(张贺,刘晓妹,喻群芳,漆艳香,谢艺贤,蒲金基,2013,海南杧果露水斑病的初步鉴定,广东农业科学,40(7):75-77)
Jing M.M.,Tian Y.Q.,Wang R.H.,and Shao Y.Z.,2017,Isolation and identifi cation of pathogen causing mango dew blotch disease and screening of its antagonistic microorganisms,Shipin Gongye Keji(Science and Technology of Food Industry),38(5):163-167(井敏敏,田亚琴,王润菡,邵远志,2017,杧果露水斑病病原菌分离鉴定及其拮抗菌筛选,食品工业科技,38(5):163-167)
Mills P.R.,Sreeivasaprasad S.,and Brown A.E.,1992,Detection and differentiation of Colletotrichum gloeosporioides isolates using PCR,Fems Microbiol.Lett.,77(1-3):137-143
Pu J.J.,Qi Y.X.,and Xie Y.Y.,2012,NY/T2257 2012 Molecular detection for pathogen of mango bacterial black spot dis ease,China Agriculture Press,Beijing,China,pp.1-8(蒲金基,漆艳香,谢艺贤,2012,NYT 2257-2012杧果细菌性黑斑病原菌分子检测技术规范,中国农业出版社,中国,北京,pp.1-8)
Qi Y.X.,Zhng H.,Yu Q.F.,Xie Y.X.,Zhang X.,Lu Y.,Zhang H.Q.,and Pu J.J.,2015,Survival period of Xanthomonas campestris pv.mangiferaeindicae in different places,Zhiwu Baohu(Plant Protection),41(5):140-144(漆艳香,张贺,喻群芳,谢艺贤,张欣,陆英,张辉强,蒲金基,2015,杧果细菌性黑斑病菌在不同场所的存活期,植物保护,41(5):140-144)
Sreenivasaprasad S.,Sharada K.,Brown A.E.,and Mills P.R.,1996,PCR-based detection of Colletotrichum acutatum on strawberry,Plant Pathol.,45(4):650-655
Wen Y.T.,and Huang S.M.,1994,Identification of symptoms and pathogen of mango bacterial black spot,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),(1):79-85(文衍堂,黄圣明,1994,杧果细菌性黑斑病症状与病原菌鉴定,热带作物学报,(1):79-85)
Wu J.B.,Zhan R.L.,Liu F.,Zhao Y.L.,He Y.B.,and Chang J.M.,2016,Rapid molecular detection of Fusarium mangiferae,the pathogen of mango malformation disease,Junwu Xuebao(Mycosystema),35(3):298-308(吴婧波,詹儒林,柳凤,赵艳龙,何衍彪,常金梅,2016,杧果畸形病病原菌Fusarium mangiferae的快速分子检测,菌物学报,35(3):298-308)
Zhang H.,Liu X.M.,Yu Q.F.,Qi Y.X.,Xie Y.Y.,and Pu J.J.,2013,Preliminary studies of mango sooty blotch of Cladosporium cladosporioides in Hainan Province,Guangdong Nongye Kexue(Guangdong Agriculture Sciences),40(7):75-77(张贺,刘晓妹,喻群芳,漆艳香,谢艺贤,蒲金基,2013,海南杧果露水斑病的初步鉴定,广东农业科学,40(7):75-77)