蔬菜土传病原菌三重PCR检测体系的建立与应用
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摘要
【目的】针对生产中危害严重的3种蔬菜土传病原菌瓜果腐霉(Pythium aphanidermatum)、尖镰孢(Fusarium oxysporum)、大丽轮枝菌(Verticillium dahliae),建立三重PCR(triplex PCR)检测体系,为蔬菜土传真菌病害的早期诊断和鉴定提供技术与方法。【方法】筛选3种病原菌特异性引物组合,通过设定不同的PCR退火温度、引物浓度、循环次数以及延伸时间,探索影响三重PCR扩增的因素,建立蔬菜土传病原菌三重PCR检测体系,并对体系灵敏度进行检测。为了验证建立体系的稳定性,选择2×Taq Master PCR Mix(北京博迈德生物技术有限公司)和TaKaRa Taq酶(大连宝生物工程有限公司)2个不同公司的试剂,分别利用C1000 Touch~(TM)型(赛默飞世尔科技有限公司)与Aeris~(TM)型(新加坡艺思高科技有限公司)热循环仪进行三重PCR反应,比较检测结果的可重复性。对田间采集的35份病害样本和149份土壤样本,分别进行三重PCR和病原菌分离检测,以确定所建立得三重PCR检测体系的适用性。【结果】三重PCR反应体系中引物对AsAPH2B/AsPyF、FOF1/FOR1、VActF/VActR可分别扩增出瓜果腐霉、尖镰孢、大丽轮枝菌长度为163、328和530 bp的特异性目的片段。反应体系(25μL):0.12μmol·L~(-1) AsAPH2B/AsPyF,0.16μmol·L~(-1) FOF1/FOR1,0.24μmol·L~(-1) VActF/VActR,2×Taq Master PCR Mix 12.5μL,退火温度为60.8℃,35个循环。该体系对病原菌纯培养物的检测灵敏度为10~(-1) ng·μL~(-1),对土壤中大丽轮枝菌、尖镰孢和瓜果腐霉的检测灵敏度分别为10~5、10~6个孢子/g土壤和10~(-2) mg菌丝/g土壤。分别采用2×Taq Master PCR Mix和TaKaRa Taq酶,利用C1000 Touch~(TM)型和Aeris~(TM)型热循环仪进行三重PCR,扩增结果一致,说明三重PCR体系检测稳定性好。对田间采集的病害和土壤样本进行检测,25份病害样本和71份土壤样本中检测出带菌,三重PCR检测与病原菌分离培养结果一致。【结论】本研究建立的瓜果腐霉、尖镰孢、大丽轮枝菌三重PCR检测体系具有灵敏度高、稳定性和重复性好的特点,能够快速、准确地检测田间病株及其根围土壤中的瓜果腐霉、尖镰孢、大丽轮枝菌,为蔬菜土传病害的早期预防和流行监测提供了有效的技术手段。
【Objective】The objective of this study is to establish a triplex PCR system for the detection of Pythium aphanidermatum, Fusarium oxysporum and Verticillium dahliae, and to provide technology and method for early diagnosis and identification of soil-borne diseases of vegetables.【Method】Three sets of specific primers were selected to analyze the influencing factors of triplex PCR and the primer concentration, annealing temperature, amplification cycles and extension time of PCR reaction were optimized. The best triplex PCR detection system for vegetable soil-borne pathogenic fungi was established and the sensitivity of the system was detected. In order to test the stability of the system, 2×Taq Master PCR Mix(Beijing Biomed Co. Ltd) and TaKaRa Taq enzyme(TaKaRa Biotechnology Dalian Co. Ltd) were used by C1000 Touch~(TM)(Thermo Fisher Scientific Co. Ltd) and Aeris~(TM) type(Esco Micro Pte Ltd) thermal cycle meter for amplification. The triplex PCR and pathogen isolation and detection of 35 disease samples and 149 soil samples collected in the field were carried out to determine the applicability of the triplex PCR detection system.【Result】In the triplex PCR detection system, the specific target fragments of P. aphanidermatum, F. oxysporum and V. dahliae with the length of 163, 328 and 530 bp could be amplified by AsAPH2B/AsPyF, FOF1/FOR1, VActF/VActR, respectively. The 25 μL reaction system contained 0.12 μmol·L~(-1) AsAPH2B/AsPyF, 0.16 μmol·L~(-1) FOF1/FOR1, 0.24 μmol·L~(-1) VActF/VActR, 2×Taq Master PCR Mix 12.5 μL. The annealing temperature was 60.8℃ and the number of cycles was 35. The detection limitation was 10~(-1) ng·μL~(-1) for pure culture of pathogens. For artificially infected substrate, the detection limitations were 10~5, 10~6 spores/g and 10~(-2) mg hyphae/g for V. dahliae, F. oxysporum and P. aphanidermatum, respectively. The results were consistent of 2×Taq Master PCR Mix and TaKaRa Taq enzyme, which were amplified by C1000 Touch~(TM) and Aeris~(TM) type thermal cycle meter. It meant that the triplex PCR system was stable. The infected tissues and soil samples collected in the field were detected, and 25 tissues and 71 soil samples were detected for carrying pathogen, which were consistent with the results of isolation and culture.【Conclusion】The triplex PCR detection system established in this study has the characteristics of high sensitivity, stability and reproducibility, and can be used for rapid and accurate detection of P. aphanidermatum, F. oxysporum and V. dahliae in the infected plant and the surrounding soil. It provides an effective technical means for early prevention and epidemic monitoring of vegetable soil-borne diseases.
【Objective】The objective of this study is to establish a triplex PCR system for the detection of Pythium aphanidermatum, Fusarium oxysporum and Verticillium dahliae, and to provide technology and method for early diagnosis and identification of soil-borne diseases of vegetables.【Method】Three sets of specific primers were selected to analyze the influencing factors of triplex PCR and the primer concentration, annealing temperature, amplification cycles and extension time of PCR reaction were optimized. The best triplex PCR detection system for vegetable soil-borne pathogenic fungi was established and the sensitivity of the system was detected. In order to test the stability of the system, 2×Taq Master PCR Mix(Beijing Biomed Co. Ltd) and TaKaRa Taq enzyme(TaKaRa Biotechnology Dalian Co. Ltd) were used by C1000 Touch~(TM)(Thermo Fisher Scientific Co. Ltd) and Aeris~(TM) type(Esco Micro Pte Ltd) thermal cycle meter for amplification. The triplex PCR and pathogen isolation and detection of 35 disease samples and 149 soil samples collected in the field were carried out to determine the applicability of the triplex PCR detection system.【Result】In the triplex PCR detection system, the specific target fragments of P. aphanidermatum, F. oxysporum and V. dahliae with the length of 163, 328 and 530 bp could be amplified by AsAPH2B/AsPyF, FOF1/FOR1, VActF/VActR, respectively. The 25 μL reaction system contained 0.12 μmol·L~(-1) AsAPH2B/AsPyF, 0.16 μmol·L~(-1) FOF1/FOR1, 0.24 μmol·L~(-1) VActF/VActR, 2×Taq Master PCR Mix 12.5 μL. The annealing temperature was 60.8℃ and the number of cycles was 35. The detection limitation was 10~(-1) ng·μL~(-1) for pure culture of pathogens. For artificially infected substrate, the detection limitations were 10~5, 10~6 spores/g and 10~(-2) mg hyphae/g for V. dahliae, F. oxysporum and P. aphanidermatum, respectively. The results were consistent of 2×Taq Master PCR Mix and TaKaRa Taq enzyme, which were amplified by C1000 Touch~(TM) and Aeris~(TM) type thermal cycle meter. It meant that the triplex PCR system was stable. The infected tissues and soil samples collected in the field were detected, and 25 tissues and 71 soil samples were detected for carrying pathogen, which were consistent with the results of isolation and culture.【Conclusion】The triplex PCR detection system established in this study has the characteristics of high sensitivity, stability and reproducibility, and can be used for rapid and accurate detection of P. aphanidermatum, F. oxysporum and V. dahliae in the infected plant and the surrounding soil. It provides an effective technical means for early prevention and epidemic monitoring of vegetable soil-borne diseases.
引文
[1]KAGEYAMA K,SUZUKI M,PRIYATMOJO A,OTO Y,ISHIGUROK,SUGA H,AOYAGI T,FUKUI H.Characterization and identification of asexual strains of Pythium associated with root rot of rose in Japan.Journal of Phytopathology,2003,151(9):485-491.
[2]刘会清.植病生防菌剂对连作设施蔬菜的防病促生效果研究[D].北京:中国农业科学院,2010.LIU H Q.Studies on the growth promoting and disease prevention effect of biological control fungus to continuous cropping vegetables in greenhouse[D].Beijing:Chinese Academy of Agricultural Sciences,2010.(in Chinese)
[3]乔世佳.生物熏蒸对土传病害及黄瓜生长的影响[D].哈尔滨:东北农业大学,2011.QIAO S J.Effect of biofumigation on soilborne diseases and cucumber growth[D].Harbin:Northeast Agricultural University,2011.(in Chinese)
[4]王丽丽,蔡超,罗明,顾爱星,王慧,李克梅.马铃薯黄萎病研究现状.生物安全学报,2017,26(1):30-38.WANG L L,CAI C,LUO M,GU A X,WANG H,LI K M.Research advances of Verticillium wilt of potato.Journal of Biosafety,2017,26(1):30-38.(in Chinese)
[5]张旭,尚楠,张宝,张志刚,尚庆茂.抗瓜果腐霉芽孢杆菌优良菌株的筛选及生物学特性.食品科学,2012,33(5):138-143.ZHANG X,SHANG N,ZHANG B,ZHANG Z G,SHANG Q M.Screening and biological characteristics of Bacillus sp.with high anti-fungal activity against Pythium aphanidermatum.Food Science,2012,33(5):138-143.(in Chinese)
[6]TAHMATSIDOU V,O’SULLIVAN J,CASSELLS A C,VOYIATZISD,PAROUSSI G.Comparison of AMF and PGPR inoculants for the suppression of Verticillium wilt of strawberry(Fragaria×ananassa cv.Selva).Applied Soil Ecology,2006,32:316-324.
[7]SASU M A,SEIDL-ADAMS I,WALL K,WINSOR J A,STEPHENSON A G.Floral transmission of Erwinia tracheiphila by cucumber beetles in a wild Cucurbita pepo.Environmental Entomology,2010,39(1):140-148.
[8]SCHROEDER K L,OKUBARA P A,TAMBONG J T,LéVESQUE CA,PAULITZ T C.Identification and quantification of pathogenic Pythium spp.from soils in eastern Washington using real-time polymerase chain reaction.Phytopathology,2006,96(6):637-647.
[9]王楠,王剑,尹丹韩,高观朋,王伟.三重PCR检测草莓灰霉病菌、炭疽病菌和黄萎病菌.中国农业科学,2010,43(21):4392-4400.WANG N,WANG J,YIN D H,GAO G P,WANG W.Triplex PCRdetection of Botrytis cinerea,Colletotrichum gloeosporioides and Verticillium dahlia in infected strawberry plant tissues.Scientia Agricultura Sinica,2010,43(21):4392-4400.(in Chinese)
[10]高永洋,王楠,高观朋,王伟.瓜黑星病菌、枯萎病菌和蔓枯病菌的三重PCR检测.植物病理学报,2010,40(4):343-350.GAO Y Y,WANG N,GAO G P,WANG W.Triplex PCR detection of Cladosporium cucumerinum,Fusarium oxysporum f.sp.niveum and Mycosphaerella melonis in infected plant tissues.Acta Phytopathologica Sinica,2010,40(4):343-350.(in Chinese)
[11]严蕾艳,张宴瑜,马凯慧,宋慧,王毓洪.两种瓜类镰刀菌的多重PCR检测.华北农学报,2014,29(增刊):44-47.YAN L Y,ZHANG Y Y,MA K H,SONG H,WANG Y H.Multiple PCR detection of Fusarium oxysporum and Fusarium solani in cucurbitaceous plants.Acta Agriculturae Boreali-Sinica,2014,29(Suppl.):44-47.(in Chinese)
[12]王楠,王伟.三重PCR检测黄瓜炭疽病菌、菌核病菌和细菌性萎蔫病菌.植物病理学报,2014,44(2):129-138.WANG N,WANG W.Triplex PCR detection of Colletotrichum orbiculare,Sclerotinia sclerotiorum and Erwinia tracheiphila in infected cucumber tissues.Acta Phytopathologica Sinica,2014,44(2):129-138.(in Chinese)
[13]THOMAS S H,HOUSLEY J M,REYNOLDS A N,PENCZYKOWSKI R M,KENLINE K H,HARDEGREE N,SCHMIDT S,DUFFY M A.The ecology and phylogeny of oomycete infections in Asplanchna rotifers.Freshwater Biology,2011,56(2):384-394.
[14]MARCHAND G,CHEN Y,BERHANE N A,WEI L,ANDRéLéVESQUE C,XUE A G.Identification of Pythium spp.from the rhizosphere of soybeans in Ontario,Canada.Canadian Journal of Plant Pathology,2014,36(2):246-251.
[15]GEISER D M,JIMéNEZ-GASCO M M,KANG S,MAKALOWSKAI,VEERARAGHAVAN N,WARD T J,ZHANG N,KULDAU G A,O’DONNELL K.FUSARIUM-ID v.1.0:A DNA sequence database for identifying Fusarium.European Journal of Plant Pathology,2004,110:473-479.
[16]RAHJOO V,ZAD J,JAVAN-NIKKHAH M,MIRZADI GOHARI A,OKHOVVAT S M,BIHAMTA M R,RAZZAGHIAN J,KLEMSDALS S.Morphological and molecular identification of Fusarium isolated from maize ears in Iran.Journal of Plant Pathology,2008,90(3):463-468.
[17]LI K N,ROUSE D I,GERMAN T L.PCR primers that allow intergeneric differentiation of ascomycetes and their application to Verticillium spp.Applied and Environmental Microbiology,1994,60(12):4324-4331.
[18]BRESSAN M,BLUM A,CASTEL L,TRINSOUTROT-GATTIN I,LAVAL K,GANGNEUX C.Assessment of Verticillium flax inoculum in agroecosystem soils using real-time PCR assay.Applied Soil Ecology,2016,108:176-186.
[19]ASANO T,SENDA M,SUGA H,KAGEYAMA K.Development of multiplex PCR to detect five Pythium species related to turfgrass diseases.Journal of Phytopathology,2010,158(9):609-615.
[20]NEL B,STEINBERG C,N.LABUSCHAGNE N,VILJOEN A.Isolation and characterization of nonpathogenic Fusarium oxysporum isolates from the rhizosphere of healthy banana plants.Plant Pathology,2006,55(2):207-216.
[21]GHARBI Y,TRIKI M A,TRABELSI R,FENDRI I,DAAYF F,GDOURA R.Genetic structure of Verticillium dahliae isolates infecting olive trees in Tunisia using AFLP,pathogenicity and PCRmarkers.Plant Pathology,2015,64(4):871-879.
[22]康华军,柴阿丽,石延霞,谢学文,袁军海,李宝聚.番茄细菌性斑点病菌、溃疡病菌、青枯病菌和疮痂病菌的四重PCR检测方法.园艺学报,2018,45(11):2254-2264.KANG H J,CHAI A L,SHI Y X,XIE X W,YUAN J H,LI B J.Quadruple PCR detection of Pseudomonas syringae pv.tomato,Clavibacter michiganensis subsp.michiganensis,Ralstonia solanacearum and Xanthomonas campestris pv.vesicatoria in infected tomato tissues.Acta Horticulturae Sinica,2018,45(11):2254-2264.(in Chinese)
[23]李淑芬,莫照兰,凡娜,朱明,茅云翔.一株紫菜腐霉的鉴定及其对条斑紫菜的致病性.中国海洋大学学报,2016,46(7):27-34.LI S F,MO Z L,FAN N,ZHU M,MAO Y X.Identification of a Pythrium porphyrae strain causing the red rot disease of Porphyra yezoensis.Periodical of Ocean University of China,2016,46(7):27-34.(in Chinese)
[24]高苇,李宝聚,王万立,郝永娟,石延霞.土壤中黄瓜棒孢叶斑病病原菌实时荧光定量PCR检测技术研究.华北农学报,2014,29(2):71-74.GAO W,LI B J,WANG W L,HAO Y J,SHI Y X.Detection of Corynespora cassiicola in soil with real-time quantitative PCR.Acta Agriculturae Boreali-Sinica,2014,29(2):71-74.(in Chinese)
[25]张真和,马兆红.我国设施蔬菜产业概况与“十三五”发展重点--中国蔬菜协会副会长张真和访谈录.中国蔬菜,2017(5):1-5.ZHANG Z H,MA Z H.Overview of China’s facility vegetable industry and key points of the 13th five-year plan--Interview with Zhang Zhenhe,vice president of China Vegetable Association.China Vegetables,2017(5):1-5.(in Chinese)
[26]何玮玲,张驰,杨静,黄明,杨军.食品中4种肉类成分多重PCR的快速鉴别方法.中国农业科学,2012,45(9):1873-1880.HE W L,ZHANG C,YANG J,HUANG M,YANG J.A quick multiplex PCR method for the identification of four meat ingredients in food products.Scientia Agricultura Sinica,2012,45(9):1873-1880.(in Chinese)
[27]HE Y,ZHAO Y,ZHOU G,HUANG M.Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from aged refuse for microbial community analysis.World Journal of Microbiology and Biotechnology,2009,25(11):2043-2051.
[28]何宛芹,付瑶,鲁雯璐,常小丽,杨文钰.大豆根腐病致病镰孢菌的多重PCR检测技术.植物保护学报,2017,44(4):609-616.HE W Q,FU Y,LU W L,CHANG X L,YANG W Y.A multiplex PCR detection technique for the pathogenic Fusarium species causing soybean root rot.Journal of Plant Protection,2017,44(4):609-616.(in Chinese)
[29]孙娟,陈舜胜,胡培龙,于子翔,于翠,杨翠云.同步检测棉黄萎病菌、枯萎病菌和炭疽病菌三重PCR方法的建立.上海农业学报,2014,30(4):10-15.SUN J,CHEN S S,HU P L,YU Z X,YU C,YANG C Y.A triplex PCR method to simultaneously detect Verticillium dahlia,Fusarium oxysporum f.sp.vasinfectum and Colletotrichum gloeosporioiles.Acta Agriculturae Shanghai,2014,30(4):10-15.(in Chinese)
[2]刘会清.植病生防菌剂对连作设施蔬菜的防病促生效果研究[D].北京:中国农业科学院,2010.LIU H Q.Studies on the growth promoting and disease prevention effect of biological control fungus to continuous cropping vegetables in greenhouse[D].Beijing:Chinese Academy of Agricultural Sciences,2010.(in Chinese)
[3]乔世佳.生物熏蒸对土传病害及黄瓜生长的影响[D].哈尔滨:东北农业大学,2011.QIAO S J.Effect of biofumigation on soilborne diseases and cucumber growth[D].Harbin:Northeast Agricultural University,2011.(in Chinese)
[4]王丽丽,蔡超,罗明,顾爱星,王慧,李克梅.马铃薯黄萎病研究现状.生物安全学报,2017,26(1):30-38.WANG L L,CAI C,LUO M,GU A X,WANG H,LI K M.Research advances of Verticillium wilt of potato.Journal of Biosafety,2017,26(1):30-38.(in Chinese)
[5]张旭,尚楠,张宝,张志刚,尚庆茂.抗瓜果腐霉芽孢杆菌优良菌株的筛选及生物学特性.食品科学,2012,33(5):138-143.ZHANG X,SHANG N,ZHANG B,ZHANG Z G,SHANG Q M.Screening and biological characteristics of Bacillus sp.with high anti-fungal activity against Pythium aphanidermatum.Food Science,2012,33(5):138-143.(in Chinese)
[6]TAHMATSIDOU V,O’SULLIVAN J,CASSELLS A C,VOYIATZISD,PAROUSSI G.Comparison of AMF and PGPR inoculants for the suppression of Verticillium wilt of strawberry(Fragaria×ananassa cv.Selva).Applied Soil Ecology,2006,32:316-324.
[7]SASU M A,SEIDL-ADAMS I,WALL K,WINSOR J A,STEPHENSON A G.Floral transmission of Erwinia tracheiphila by cucumber beetles in a wild Cucurbita pepo.Environmental Entomology,2010,39(1):140-148.
[8]SCHROEDER K L,OKUBARA P A,TAMBONG J T,LéVESQUE CA,PAULITZ T C.Identification and quantification of pathogenic Pythium spp.from soils in eastern Washington using real-time polymerase chain reaction.Phytopathology,2006,96(6):637-647.
[9]王楠,王剑,尹丹韩,高观朋,王伟.三重PCR检测草莓灰霉病菌、炭疽病菌和黄萎病菌.中国农业科学,2010,43(21):4392-4400.WANG N,WANG J,YIN D H,GAO G P,WANG W.Triplex PCRdetection of Botrytis cinerea,Colletotrichum gloeosporioides and Verticillium dahlia in infected strawberry plant tissues.Scientia Agricultura Sinica,2010,43(21):4392-4400.(in Chinese)
[10]高永洋,王楠,高观朋,王伟.瓜黑星病菌、枯萎病菌和蔓枯病菌的三重PCR检测.植物病理学报,2010,40(4):343-350.GAO Y Y,WANG N,GAO G P,WANG W.Triplex PCR detection of Cladosporium cucumerinum,Fusarium oxysporum f.sp.niveum and Mycosphaerella melonis in infected plant tissues.Acta Phytopathologica Sinica,2010,40(4):343-350.(in Chinese)
[11]严蕾艳,张宴瑜,马凯慧,宋慧,王毓洪.两种瓜类镰刀菌的多重PCR检测.华北农学报,2014,29(增刊):44-47.YAN L Y,ZHANG Y Y,MA K H,SONG H,WANG Y H.Multiple PCR detection of Fusarium oxysporum and Fusarium solani in cucurbitaceous plants.Acta Agriculturae Boreali-Sinica,2014,29(Suppl.):44-47.(in Chinese)
[12]王楠,王伟.三重PCR检测黄瓜炭疽病菌、菌核病菌和细菌性萎蔫病菌.植物病理学报,2014,44(2):129-138.WANG N,WANG W.Triplex PCR detection of Colletotrichum orbiculare,Sclerotinia sclerotiorum and Erwinia tracheiphila in infected cucumber tissues.Acta Phytopathologica Sinica,2014,44(2):129-138.(in Chinese)
[13]THOMAS S H,HOUSLEY J M,REYNOLDS A N,PENCZYKOWSKI R M,KENLINE K H,HARDEGREE N,SCHMIDT S,DUFFY M A.The ecology and phylogeny of oomycete infections in Asplanchna rotifers.Freshwater Biology,2011,56(2):384-394.
[14]MARCHAND G,CHEN Y,BERHANE N A,WEI L,ANDRéLéVESQUE C,XUE A G.Identification of Pythium spp.from the rhizosphere of soybeans in Ontario,Canada.Canadian Journal of Plant Pathology,2014,36(2):246-251.
[15]GEISER D M,JIMéNEZ-GASCO M M,KANG S,MAKALOWSKAI,VEERARAGHAVAN N,WARD T J,ZHANG N,KULDAU G A,O’DONNELL K.FUSARIUM-ID v.1.0:A DNA sequence database for identifying Fusarium.European Journal of Plant Pathology,2004,110:473-479.
[16]RAHJOO V,ZAD J,JAVAN-NIKKHAH M,MIRZADI GOHARI A,OKHOVVAT S M,BIHAMTA M R,RAZZAGHIAN J,KLEMSDALS S.Morphological and molecular identification of Fusarium isolated from maize ears in Iran.Journal of Plant Pathology,2008,90(3):463-468.
[17]LI K N,ROUSE D I,GERMAN T L.PCR primers that allow intergeneric differentiation of ascomycetes and their application to Verticillium spp.Applied and Environmental Microbiology,1994,60(12):4324-4331.
[18]BRESSAN M,BLUM A,CASTEL L,TRINSOUTROT-GATTIN I,LAVAL K,GANGNEUX C.Assessment of Verticillium flax inoculum in agroecosystem soils using real-time PCR assay.Applied Soil Ecology,2016,108:176-186.
[19]ASANO T,SENDA M,SUGA H,KAGEYAMA K.Development of multiplex PCR to detect five Pythium species related to turfgrass diseases.Journal of Phytopathology,2010,158(9):609-615.
[20]NEL B,STEINBERG C,N.LABUSCHAGNE N,VILJOEN A.Isolation and characterization of nonpathogenic Fusarium oxysporum isolates from the rhizosphere of healthy banana plants.Plant Pathology,2006,55(2):207-216.
[21]GHARBI Y,TRIKI M A,TRABELSI R,FENDRI I,DAAYF F,GDOURA R.Genetic structure of Verticillium dahliae isolates infecting olive trees in Tunisia using AFLP,pathogenicity and PCRmarkers.Plant Pathology,2015,64(4):871-879.
[22]康华军,柴阿丽,石延霞,谢学文,袁军海,李宝聚.番茄细菌性斑点病菌、溃疡病菌、青枯病菌和疮痂病菌的四重PCR检测方法.园艺学报,2018,45(11):2254-2264.KANG H J,CHAI A L,SHI Y X,XIE X W,YUAN J H,LI B J.Quadruple PCR detection of Pseudomonas syringae pv.tomato,Clavibacter michiganensis subsp.michiganensis,Ralstonia solanacearum and Xanthomonas campestris pv.vesicatoria in infected tomato tissues.Acta Horticulturae Sinica,2018,45(11):2254-2264.(in Chinese)
[23]李淑芬,莫照兰,凡娜,朱明,茅云翔.一株紫菜腐霉的鉴定及其对条斑紫菜的致病性.中国海洋大学学报,2016,46(7):27-34.LI S F,MO Z L,FAN N,ZHU M,MAO Y X.Identification of a Pythrium porphyrae strain causing the red rot disease of Porphyra yezoensis.Periodical of Ocean University of China,2016,46(7):27-34.(in Chinese)
[24]高苇,李宝聚,王万立,郝永娟,石延霞.土壤中黄瓜棒孢叶斑病病原菌实时荧光定量PCR检测技术研究.华北农学报,2014,29(2):71-74.GAO W,LI B J,WANG W L,HAO Y J,SHI Y X.Detection of Corynespora cassiicola in soil with real-time quantitative PCR.Acta Agriculturae Boreali-Sinica,2014,29(2):71-74.(in Chinese)
[25]张真和,马兆红.我国设施蔬菜产业概况与“十三五”发展重点--中国蔬菜协会副会长张真和访谈录.中国蔬菜,2017(5):1-5.ZHANG Z H,MA Z H.Overview of China’s facility vegetable industry and key points of the 13th five-year plan--Interview with Zhang Zhenhe,vice president of China Vegetable Association.China Vegetables,2017(5):1-5.(in Chinese)
[26]何玮玲,张驰,杨静,黄明,杨军.食品中4种肉类成分多重PCR的快速鉴别方法.中国农业科学,2012,45(9):1873-1880.HE W L,ZHANG C,YANG J,HUANG M,YANG J.A quick multiplex PCR method for the identification of four meat ingredients in food products.Scientia Agricultura Sinica,2012,45(9):1873-1880.(in Chinese)
[27]HE Y,ZHAO Y,ZHOU G,HUANG M.Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from aged refuse for microbial community analysis.World Journal of Microbiology and Biotechnology,2009,25(11):2043-2051.
[28]何宛芹,付瑶,鲁雯璐,常小丽,杨文钰.大豆根腐病致病镰孢菌的多重PCR检测技术.植物保护学报,2017,44(4):609-616.HE W Q,FU Y,LU W L,CHANG X L,YANG W Y.A multiplex PCR detection technique for the pathogenic Fusarium species causing soybean root rot.Journal of Plant Protection,2017,44(4):609-616.(in Chinese)
[29]孙娟,陈舜胜,胡培龙,于子翔,于翠,杨翠云.同步检测棉黄萎病菌、枯萎病菌和炭疽病菌三重PCR方法的建立.上海农业学报,2014,30(4):10-15.SUN J,CHEN S S,HU P L,YU Z X,YU C,YANG C Y.A triplex PCR method to simultaneously detect Verticillium dahlia,Fusarium oxysporum f.sp.vasinfectum and Colletotrichum gloeosporioiles.Acta Agriculturae Shanghai,2014,30(4):10-15.(in Chinese)