玉米小斑病抗病鉴定接种体培养技术探讨
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摘要
【目的】研究不同谷物粒培养基配方及其培养条件对玉米小斑病菌产孢量的影响,为玉米小斑病大面积抗病鉴定工作提供保障。【方法】以玉米小斑病菌野生型菌株HX5为试材,探索18种植物组织培养基、9个温度梯度、9个培养时间段、7种高低温诱导和5种不同光照等条件对玉米小斑病菌分生孢子产量的影响。【结果】培养基的成分组合对玉米小斑病菌分生孢子产生起决定性作用,配方为高粱粒100 g+玉米叶15 g+MgSO40.2 g+Na3PO40.2 g的7号培养基产孢量最多,为16.8×104个/g;24℃条件下培养产孢量最多;在20和24℃黑暗条件下,培养35 d时的产孢量最多;35℃高温、4℃低温诱导对玉米小斑病菌产孢量均有一定的促进作用,但经高、低温诱导后产生的部分分生孢子一端连接1节分生孢子梗;光暗交替各12 h、光照强度为2000 lx的产孢量最多,为4.6×105个/g。【结论】高粱粒100 g+玉米叶15 g+MgSO40.2 g+Na3PO40.2 g为玉米小斑病菌产孢最佳培养基,该培养基可显著促进病菌产生分生孢子;在24℃、光照强度2000 lx光暗交替各12 h的培养条件下,病菌的产孢量最多;在35 d内培养病菌,培养时间与产孢量成正比;35℃高温和4℃低温诱导对产孢量均有一定的促进作用。
【Objective】The aim was to investigate the effects of different grain medium formulations and culture conditions on sporulation of Bipolaria maydis,and provide a guarantee for large-scale identification of resistance to southern corn leaf blight.【Method】The effects of eighteen plant tissue-based media,nine temperature gradients,nine culture times,seven high and lowtemperature inductions and five different light intensities on conidium formation of wild-type strain HX5 of B. maydis were studied.【Result】Component combinations of media played a decisive role in conidium formation. Medium No.7 containing 100 g sorghum grains + 15 g corn leaves + 0.2 g MgSO4+ 0.2 g Na3 PO4 showed the highest sporulation(16.8×104 spore/g). Under 24 ℃,the sporulation was the highest. Under darkness at 20 and 24 ℃,sporulation reached the peak on day 35. Induction at 35 and 4 ℃ promoted sporulation of B. maydis to a certain extent.However,after high and lowtemperature induction,one end of partial formed conidia connected to a conidial peduncle.Under light-dark 12 h alternation and light intensity of 2000 lx,sporulation was the highest(4.6×105 spore/g).【Conclusion】The optimal medium for sporulation of B. maydis is 100 g sorghum grains+15 g corn leaves+0.2 g MgSO4+0.2 g Na3 PO4,which can significantly promote the conidium formation of B. maydis. Under 24 ℃,light intensity of 2000 lx and light-dark 12 h alternation,the sporulation is the highest. Within 35 d,the culture time is proportional to sporulation. Induction at 35 and 4 ℃ promote sporulation of B. maydis to a certain extent.
【Objective】The aim was to investigate the effects of different grain medium formulations and culture conditions on sporulation of Bipolaria maydis,and provide a guarantee for large-scale identification of resistance to southern corn leaf blight.【Method】The effects of eighteen plant tissue-based media,nine temperature gradients,nine culture times,seven high and lowtemperature inductions and five different light intensities on conidium formation of wild-type strain HX5 of B. maydis were studied.【Result】Component combinations of media played a decisive role in conidium formation. Medium No.7 containing 100 g sorghum grains + 15 g corn leaves + 0.2 g MgSO4+ 0.2 g Na3 PO4 showed the highest sporulation(16.8×104 spore/g). Under 24 ℃,the sporulation was the highest. Under darkness at 20 and 24 ℃,sporulation reached the peak on day 35. Induction at 35 and 4 ℃ promoted sporulation of B. maydis to a certain extent.However,after high and lowtemperature induction,one end of partial formed conidia connected to a conidial peduncle.Under light-dark 12 h alternation and light intensity of 2000 lx,sporulation was the highest(4.6×105 spore/g).【Conclusion】The optimal medium for sporulation of B. maydis is 100 g sorghum grains+15 g corn leaves+0.2 g MgSO4+0.2 g Na3 PO4,which can significantly promote the conidium formation of B. maydis. Under 24 ℃,light intensity of 2000 lx and light-dark 12 h alternation,the sporulation is the highest. Within 35 d,the culture time is proportional to sporulation. Induction at 35 and 4 ℃ promote sporulation of B. maydis to a certain extent.
引文
艾堂顺,田志强,李会敏,邓策,丁俊强,张学林,刘海富,朱伟岭,李志敏.2018.玉米南方锈病抗病QTL鉴定和效应分析[J].河南农业大学学报,52(4):514-518.[Ai T S,Tian Z Q,Li H M,Deng C,Ding J Q,Zhang X L,Liu H F,Zhu W L,Li Z M.2018.Mapping and effectiveness analysis for resistance genes of southern corn rust in maize[J].Journal of Henan Agricultural University,52(4):514-518.]
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陈颖,郝丽梅,王立安.2003.不同培养基对玉米小斑病菌生长繁殖及致病力的影响[J].河北师范大学学报(自然科学版),27(2):192-194.[Chen Y,Hao L M,Wang L A.2003.Effects of different media on the growth reproduction and pathogenicity of Bipolaris maydis race C[J].Journal of Hebei Normal University(Natural Science Edition),27(2):192-194.]
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董怀玉,姜钰,王丽娟,徐秀德.2005.玉米种质资源抗灰斑病鉴定与评价[J].植物遗传资源学报,6(4):441-443.[Dong H Y,Jiang Y,Wang L J,Xu X D.2005.Evaluation on maize germplasm resources for resistanceto gray leaf spot[J].Journal on Plant Genetic Resources,6(4):441-443.]
方中达.1998.植病研究法[M].第3版.北京:中国农业出版社.[Fang Z D.1998.Plant Disease Study[M].The 3rdEdition.Beijing:China Agriculture Press.]
冯胜泽,刘星晨,王海祥,赵洁,赵立卿,郑亚男,巩校东,韩建民,谷守芹,董金皋.2017.玉米大斑病菌分生孢子形成的影响因素及GATA转录因子家族的表达分析[J].中国农业科学,50(7):1234-1241.[Feng S Z,Liu X C,Wang H X,Zhao J,Zhao L Q,Zheng Y N,Gong X D,Han J M,Gu S Q,Dong J G.2017.Influencing factors of conidiospore and expression analysis of GATA transcription factor gene family in Setosphaeria turcica[J].Scientia Agricultura Sinica,50(7):1234-1241.]
龚现丽.2010.夏玉米小斑病的发生与防治[J].河南农业,(17):19.[Gong X L.2010.Occurrence and prevention of small spot disease in summer maize[J].Agriculture of Henan,(17):19.]
兰成忠,刘裴清,李本金,陈庆河,翁启勇.2013.辣椒疫霉菌产孢培养基及诱导方法筛选[J].热带作物学报,34(9):1776-1780.[Lan C Z,Liu P Q,Li B J,Chen Q H,Weng QY.2013.Screening of medium and induction method for sporangia production of Phytophthora capsici[J].Chinese Journal of Tropical Crops,34(9):1776-1780.]
李洪连,徐敬友.2001.农业植物病理学实验实习指导[M].北京:中国农业出版社.[Li H L,Xu J Y.2001.Agricultural Plant Pathology Experiment Practice Guidance[M].Beijing:China Agriculture Press.]
刘静,李国华,周明,许丽月,肖春云.2014.橡胶树白粉菌分生孢子萌发条件及存活时间的研究[J].西南农业学报,27(1):151-155.[Lui J,Li G H,Zhou M,Xu L Y,Xiao CY.2014.Study on conditions of conidial germination and survival time of Oidium heveae[J].Southwest China Journal of Agricultural Sciences,27(1):151-155.]
刘丽丽,朱杰华,崔亚婧,杨志辉,徐进,路文雅.2014.培养条件对茄链格孢产孢的影响[J].菌物学报,33(3):659-667.[Lui L L,Zhu J H,Cui Y J,Yang Z H,Xu J,Lu W Y.2014.Effects of culture conditions on sporulation of Alternorio soloni[J].Mycosystema,33(3):659-667.]
王晓梅,吕平香,李莉莉,见德宝,杨信东.2007.玉米小斑病重要流行环节的初步定量研究Ⅱ病斑产孢、孢子飞散、杀菌剂筛选[J].吉林农业大学学报,29(2):128-132.[Wang X M,LüP X,Li L L,Jian D B,Yang X D.2007.Preliminary quantitative studies on important epidemic links of Bipolaris maydisⅡ.lesion spomlation,spore dispersion and fungicide screening[J].Journal of Jilin Agricultural U-niversity,29(2):128-132.]
王晓鸣,石洁,晋齐鸣,李晓,孙世贤.2010.玉米病虫害田间手册:病虫害鉴别与抗性鉴定[M].北京:中国农业科技出版社.[Wang X M,Shi J,Jin Q M,Li X,Sun S X.2010.Field Manual of Maize Diseases and Insect Pests:Identification of Pests and Diseases and Identification of Resistance[M].Beijing:China Agricultural Science and Technology Press.]
谢红辉.2010.玉米小斑病菌生物学特性研究[J].广西热带农业,(6):1-4.[Xie H H.2010.Study on the biological characteristics of Bipolaris maydis[J].Guangxi Tropical Agriculture,(6):1-4.]
杨丽敏.2012.影响玉米小斑病抗性鉴定的因子分析及病菌生物学特性研究[D].合肥:安微农业大学.[Yang L M.2012.Impact factor of resistance identification to slb and the biological characteristics of pathogenic fungi[D].Hefei:Anhui Agricultural University.]
杨秀娟,陈福如,甘林,阮宏椿,杜宜新,石妞妞.2018.一种玉米小斑病菌产孢培养基及其制备方法和应用:中国,104962510B[P].2018-04-24.[Yang X J,Chen F R,Gan L,Ruan H C,Du Y X,Shi N N.2018.The invention relates to a spore-producing culture medium of corn-spot bacteria and its preparation method and application:China,104962510B[P].2018-04-24.]
张立杰,张慧玲,哈矿武,王建设.2015.甜瓜蔓枯病病原菌分离与分生孢子的产孢诱导[J].宁夏大学学报(自然科学版),36(4):382-385.[Zhang L J,Zhang H L,Ha K W,Wang J S.2015.Isolation and identification of pathogen of didymellabryoniae and spore production induction of condium[J].Journal of Ningxia University(Natural Science Edition),36(4):382-385.]
赵红,王彩霞,陈晓忍,王海艳,李保华.2012.苹果腐烂病菌诱导产孢方法[J].中国农学通报,28(10):151-154.[Zhao H,Wang C X,Chen X R,Wang H Y,Li B H.2012.Methods of promoting sporulation of Valsa ceratosperma[J].Chinese Agricultural Science Bulletin,28(10):151-154.]
赵聚莹,屈振刚,贾海民,陈丹,李术臣.2010.不同玉米品种对小斑病强致病力菌的抗病性检测[J].河北农业科学,14(9):66-67.[Zhao J Y,Qu Z G,Jia H M,Chen D,Li S C.2010.Detection of resistance of maize varieties to virulent strain of southern leaf blight[J].Journal of Hebei Agricultural Sciences,14(9):66-67.]
庄义庆,乔广行,王源超,何东兵,郑小波.2008.蕉斑镰刀菌3-26菌株产孢条件研究[J].南京农业大学学报,31(4):77-81.[Zhuang Y Q,Qiao G X,Wang Y C,He D B,Zheng X B.2008.Conditions for mass production of Fusarium stoveri isolate 3-26 spore in liquid and solid culture[J].Journal of Nanjing Agricultural University,31(4):77-81.]
陈利峰.2002.农业植物病理学[M].北京:中国农业出版社:189.[Chen L F.2002.Agricultural Phytopathology[M].Beijing:China Agriculture Industry Press:189.]
陈颖,郝丽梅,王立安.2003.不同培养基对玉米小斑病菌生长繁殖及致病力的影响[J].河北师范大学学报(自然科学版),27(2):192-194.[Chen Y,Hao L M,Wang L A.2003.Effects of different media on the growth reproduction and pathogenicity of Bipolaris maydis race C[J].Journal of Hebei Normal University(Natural Science Edition),27(2):192-194.]
代玉立,甘林,阮宏椿,廖蕾,石妞妞,杜宜新,陈福如,杨秀娟.2016.福建省玉米小斑病菌的生物学特性研究[J].中国农学通报,32(31):131-137.[Dai Y L,Gan L,Ruan HC,Liao L,Shi N N,Du Y X,Chen F R,Yang X J.2016.Biological characteristics of Bipolaris maydis in Fujian Province[J].Chinese Agricultural Science Bulletin,32(31):131-137.]
董怀玉,姜钰,王丽娟,徐秀德.2005.玉米种质资源抗灰斑病鉴定与评价[J].植物遗传资源学报,6(4):441-443.[Dong H Y,Jiang Y,Wang L J,Xu X D.2005.Evaluation on maize germplasm resources for resistanceto gray leaf spot[J].Journal on Plant Genetic Resources,6(4):441-443.]
方中达.1998.植病研究法[M].第3版.北京:中国农业出版社.[Fang Z D.1998.Plant Disease Study[M].The 3rdEdition.Beijing:China Agriculture Press.]
冯胜泽,刘星晨,王海祥,赵洁,赵立卿,郑亚男,巩校东,韩建民,谷守芹,董金皋.2017.玉米大斑病菌分生孢子形成的影响因素及GATA转录因子家族的表达分析[J].中国农业科学,50(7):1234-1241.[Feng S Z,Liu X C,Wang H X,Zhao J,Zhao L Q,Zheng Y N,Gong X D,Han J M,Gu S Q,Dong J G.2017.Influencing factors of conidiospore and expression analysis of GATA transcription factor gene family in Setosphaeria turcica[J].Scientia Agricultura Sinica,50(7):1234-1241.]
龚现丽.2010.夏玉米小斑病的发生与防治[J].河南农业,(17):19.[Gong X L.2010.Occurrence and prevention of small spot disease in summer maize[J].Agriculture of Henan,(17):19.]
兰成忠,刘裴清,李本金,陈庆河,翁启勇.2013.辣椒疫霉菌产孢培养基及诱导方法筛选[J].热带作物学报,34(9):1776-1780.[Lan C Z,Liu P Q,Li B J,Chen Q H,Weng QY.2013.Screening of medium and induction method for sporangia production of Phytophthora capsici[J].Chinese Journal of Tropical Crops,34(9):1776-1780.]
李洪连,徐敬友.2001.农业植物病理学实验实习指导[M].北京:中国农业出版社.[Li H L,Xu J Y.2001.Agricultural Plant Pathology Experiment Practice Guidance[M].Beijing:China Agriculture Press.]
刘静,李国华,周明,许丽月,肖春云.2014.橡胶树白粉菌分生孢子萌发条件及存活时间的研究[J].西南农业学报,27(1):151-155.[Lui J,Li G H,Zhou M,Xu L Y,Xiao CY.2014.Study on conditions of conidial germination and survival time of Oidium heveae[J].Southwest China Journal of Agricultural Sciences,27(1):151-155.]
刘丽丽,朱杰华,崔亚婧,杨志辉,徐进,路文雅.2014.培养条件对茄链格孢产孢的影响[J].菌物学报,33(3):659-667.[Lui L L,Zhu J H,Cui Y J,Yang Z H,Xu J,Lu W Y.2014.Effects of culture conditions on sporulation of Alternorio soloni[J].Mycosystema,33(3):659-667.]
王晓梅,吕平香,李莉莉,见德宝,杨信东.2007.玉米小斑病重要流行环节的初步定量研究Ⅱ病斑产孢、孢子飞散、杀菌剂筛选[J].吉林农业大学学报,29(2):128-132.[Wang X M,LüP X,Li L L,Jian D B,Yang X D.2007.Preliminary quantitative studies on important epidemic links of Bipolaris maydisⅡ.lesion spomlation,spore dispersion and fungicide screening[J].Journal of Jilin Agricultural U-niversity,29(2):128-132.]
王晓鸣,石洁,晋齐鸣,李晓,孙世贤.2010.玉米病虫害田间手册:病虫害鉴别与抗性鉴定[M].北京:中国农业科技出版社.[Wang X M,Shi J,Jin Q M,Li X,Sun S X.2010.Field Manual of Maize Diseases and Insect Pests:Identification of Pests and Diseases and Identification of Resistance[M].Beijing:China Agricultural Science and Technology Press.]
谢红辉.2010.玉米小斑病菌生物学特性研究[J].广西热带农业,(6):1-4.[Xie H H.2010.Study on the biological characteristics of Bipolaris maydis[J].Guangxi Tropical Agriculture,(6):1-4.]
杨丽敏.2012.影响玉米小斑病抗性鉴定的因子分析及病菌生物学特性研究[D].合肥:安微农业大学.[Yang L M.2012.Impact factor of resistance identification to slb and the biological characteristics of pathogenic fungi[D].Hefei:Anhui Agricultural University.]
杨秀娟,陈福如,甘林,阮宏椿,杜宜新,石妞妞.2018.一种玉米小斑病菌产孢培养基及其制备方法和应用:中国,104962510B[P].2018-04-24.[Yang X J,Chen F R,Gan L,Ruan H C,Du Y X,Shi N N.2018.The invention relates to a spore-producing culture medium of corn-spot bacteria and its preparation method and application:China,104962510B[P].2018-04-24.]
张立杰,张慧玲,哈矿武,王建设.2015.甜瓜蔓枯病病原菌分离与分生孢子的产孢诱导[J].宁夏大学学报(自然科学版),36(4):382-385.[Zhang L J,Zhang H L,Ha K W,Wang J S.2015.Isolation and identification of pathogen of didymellabryoniae and spore production induction of condium[J].Journal of Ningxia University(Natural Science Edition),36(4):382-385.]
赵红,王彩霞,陈晓忍,王海艳,李保华.2012.苹果腐烂病菌诱导产孢方法[J].中国农学通报,28(10):151-154.[Zhao H,Wang C X,Chen X R,Wang H Y,Li B H.2012.Methods of promoting sporulation of Valsa ceratosperma[J].Chinese Agricultural Science Bulletin,28(10):151-154.]
赵聚莹,屈振刚,贾海民,陈丹,李术臣.2010.不同玉米品种对小斑病强致病力菌的抗病性检测[J].河北农业科学,14(9):66-67.[Zhao J Y,Qu Z G,Jia H M,Chen D,Li S C.2010.Detection of resistance of maize varieties to virulent strain of southern leaf blight[J].Journal of Hebei Agricultural Sciences,14(9):66-67.]
庄义庆,乔广行,王源超,何东兵,郑小波.2008.蕉斑镰刀菌3-26菌株产孢条件研究[J].南京农业大学学报,31(4):77-81.[Zhuang Y Q,Qiao G X,Wang Y C,He D B,Zheng X B.2008.Conditions for mass production of Fusarium stoveri isolate 3-26 spore in liquid and solid culture[J].Journal of Nanjing Agricultural University,31(4):77-81.]