兰州市娃娃菜褐腐病菌生物学特性及融合群鉴定
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摘要
为给病害科学防治提供基础信息,对分离自兰州市红古区和永登县武胜驿镇罹病娃娃菜病株上的8株立枯丝核菌菌株的形态、生长温度、致病性、对杀菌剂的敏感性等生物学特性进行研究,对其菌丝融合群归属进行分子生物学鉴定。采用温度梯度法测定病菌适宜生长温度,同时观察试验菌株微菌核产生情况;采用番红O和KOH染色法观测试验菌株细胞核数目;采用离体叶片接种法测定试验菌株的致病力;采用含药平板法测定试验菌株对5种杀菌剂的敏感性;以试验菌株的5.8S rDNA-ITS区序列构建系统发育树,进行融合群的分子生物学鉴定。结果表明,除菌株Rh-5适宜生长温度为15～20℃外,其余试验菌株适宜生长温度均为25℃。试验菌株在5～30℃培养均可形成微菌核。菌株Rh-1～Rh-8的细胞核数目分别为3～6、6～14、3～18、4～15、3～10、4～10、3～10、4～8个。接种Rh-2～Rh-6的娃娃菜离体叶片在20℃保湿培养2天内发病显症,7天发病率达75%～100%;接种Rh-1～Rh-8的娃娃菜离体叶片在25℃保湿培养1～3天内发病显症,7天发病率达25%～100%;接种Rh-1、Rh-7和Rh-8的娃娃菜离体叶片在28℃保湿培养2～3天发病显症,7天发病率达100%。在试验浓度下,8个试验菌株对5种杀菌剂的敏感性由大到小依次为多菌灵、代森锰锌、速克灵、三唑酮、苯醚甲环唑。除Rh-1与Rhizoctonia solani AG 4 HG-II聚为一枝外,其余试验菌株(Rh-6除外,因Rh-6测序未果)均与R. solani AG 2-1聚为一枝。依据试验菌株的5.8S rDNA-ITS区序列分析所得结果,引起兰州市娃娃菜褐腐病的立枯丝核菌包括AG 2-1和AG 4HG-II融合群。试验菌株在菌落形态、细胞核数目、适宜生长温度、生长速度、侵染温度、致病性及对杀菌剂的敏感性等方面呈现出丰富的多样性特征。在试验浓度下,多菌灵和代森锰锌对试验菌株的抑制率≥80%,可作为田间病害防治试验的推荐药剂。
To provide information for the disease control, 8 strains of Rhizoctonia solani isolated from diseased mini Chinese cabbage in Honggu and Yongdeng of Lanzhou were used for testing morphology, cardinal temperature, pathogenicity, fungicide sensibility, and anastomosis group identification. The optimal growth temperatures were evaluated by temperature gradient method, and microsclerotia forming status was observed in the meantime. Numbers of nuclei were observed by staining with safranin O and KOH. The pathogenicity was determined by inoculation to detached leaves. Fungicide sensitivity of R. solani strains was assessed by amendment plate method, autoclaved PDA media were amended prior to pouring with 5 different fungicides.Phylogenetic tree was established based on rDNA-ITS(ITS1-5.8 S-ITS2) sequences of test strains. The results showed that: the optimal growth temperature of all other strains was 25℃, except Rh-5, whose was 15-20℃. All tested strains could produce microsclerotia cultured on PDA plate at 5-30℃. Numbers of nuclei of strain Rh-1-Rh-8 was 3-6, 6-14, 3-18, 4-15, 3-10, 4-10, 3-10 and 4-8, respectively. Detached leaves of mini Chinese cabbage inoculated by strain Rh-2-Rh-6, infected in 2 d at 20℃, had the incidence of 75%-100% in 7 d after inoculation; the leaves inoculated by strain Rh-1-Rh-8, infected in 1-3 d at 25℃, had the incidence of 25%-100% in 7 d after inoculation; the leaves inoculated by strain Rh-1, Rh-7 and Rh-8, infected in 2-3 d at 28℃,had the incidence of 100% in 7 d after inoculation. Under test concentrations, the sensitivity of the eight tested strains of R. solani to five fungicides was successively: carbendazim> mancozeb> procymidone> triadimefon>difenoconazole. Rh-1 aligned with R. solani AG 4 HG-II, other test strains(except Rh-6, since sequencing failure) aligned with R. solani AG 2-1. According to rDNA-ITS(ITS1-5.8 S-ITS2) sequences analysis of the test strains, the pathogens causing mini Chinese cabbage brown rot in Lanzhou include R. solani AG 2-1 and AG 4 HG-II. Tested strains of R. solani show the diversified characteristics in colony morphology, numbers of nuclei,optimal growth temperatures, growth speeds, infection temperatures, pathogenicity and fungicide sensitivity. At the tested concentrations, all strains show higher sensitivity to carbendazim and mancozeb(inhibition rate≥80%), suggesting that these two fungicides could be used in field trials on control of mini Chinese cabbage brown rot disease.
To provide information for the disease control, 8 strains of Rhizoctonia solani isolated from diseased mini Chinese cabbage in Honggu and Yongdeng of Lanzhou were used for testing morphology, cardinal temperature, pathogenicity, fungicide sensibility, and anastomosis group identification. The optimal growth temperatures were evaluated by temperature gradient method, and microsclerotia forming status was observed in the meantime. Numbers of nuclei were observed by staining with safranin O and KOH. The pathogenicity was determined by inoculation to detached leaves. Fungicide sensitivity of R. solani strains was assessed by amendment plate method, autoclaved PDA media were amended prior to pouring with 5 different fungicides.Phylogenetic tree was established based on rDNA-ITS(ITS1-5.8 S-ITS2) sequences of test strains. The results showed that: the optimal growth temperature of all other strains was 25℃, except Rh-5, whose was 15-20℃. All tested strains could produce microsclerotia cultured on PDA plate at 5-30℃. Numbers of nuclei of strain Rh-1-Rh-8 was 3-6, 6-14, 3-18, 4-15, 3-10, 4-10, 3-10 and 4-8, respectively. Detached leaves of mini Chinese cabbage inoculated by strain Rh-2-Rh-6, infected in 2 d at 20℃, had the incidence of 75%-100% in 7 d after inoculation; the leaves inoculated by strain Rh-1-Rh-8, infected in 1-3 d at 25℃, had the incidence of 25%-100% in 7 d after inoculation; the leaves inoculated by strain Rh-1, Rh-7 and Rh-8, infected in 2-3 d at 28℃,had the incidence of 100% in 7 d after inoculation. Under test concentrations, the sensitivity of the eight tested strains of R. solani to five fungicides was successively: carbendazim> mancozeb> procymidone> triadimefon>difenoconazole. Rh-1 aligned with R. solani AG 4 HG-II, other test strains(except Rh-6, since sequencing failure) aligned with R. solani AG 2-1. According to rDNA-ITS(ITS1-5.8 S-ITS2) sequences analysis of the test strains, the pathogens causing mini Chinese cabbage brown rot in Lanzhou include R. solani AG 2-1 and AG 4 HG-II. Tested strains of R. solani show the diversified characteristics in colony morphology, numbers of nuclei,optimal growth temperatures, growth speeds, infection temperatures, pathogenicity and fungicide sensitivity. At the tested concentrations, all strains show higher sensitivity to carbendazim and mancozeb(inhibition rate≥80%), suggesting that these two fungicides could be used in field trials on control of mini Chinese cabbage brown rot disease.
引文
[1]张凤兰.大力发展大白菜生产和消费新模式[J].中国蔬菜,2008(1):9.
[2]彭建姝,杨晓菊.7个娃娃菜品种在红古区的引种试验初报[J].甘肃农业科技,2012(12):25-26.
[3]李业贵.红古区娃娃菜产业发展现状及发展途径[J].甘肃农业,2009(11):66-67.
[4]戚佩坤,白金铠,朱桂香.吉林省栽培植物真菌病害志[M].北京:科学出版社,1966:111.
[5]北方大白菜病害综防技术研究与应用协作组.我国北方大白菜病害现状及发展的浅见[J].中国蔬菜,1993(4):38-40.
[6]苗国辉,闻凤英,张耀伟.津黑两地大白菜褐腐病病原菌鉴定及生物学特性的比较研究[J].植物保护,2012,38(4):89-95.
[7]李明远.北京小油菜褐腐病日趋严重[J].植物保护,1993,19(4):50-51.
[8]周慧敏,谢学文,石延霞,等.大白菜褐腐病(茎基腐病)的病原菌鉴定[J].中国蔬菜,2012(22):70-74.
[9]张耀伟,苗国辉.大白菜对褐腐病抗性的快速鉴定方法研究[J].植物保护,2014,40(3):117-121.
[10]段春芳,杨根华,尼章光,等.立枯丝核菌AG-1 IB引起白菜、薄荷、莴苣叶腐病的研究[J].云南农业大学学报,2008,23(3):422-425.
[11]Carling D E.Grouping in Rhizoctonia solani by hyphal anastomosis reaction[A].//In:Sneh B,Jabaji-Hare S,Neate S,et al.Rhizoctonia species:Taxonomy,Molecular Biology,Ecology,Pathology and Disease Control[M].Dordrecht:Kluwer Academic Publishers,1996:37-47.
[12]Ogoshi A.Introduction-The genus Rhizoctonia[A].//In:Sneh B,Jabaji-Hare S,Neate S,et al.Rhizoctonia species:Taxonomy,Molecular Biology,Ecology,Pathology and Disease Control[M].Dordrecht:Kluwer Academic Publishers,1996:1-9.
[13]Kuninaga S,Natsuaki T,Takeuchi T,et al.Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani[J].Current Genetics,1997,32(3):237-243.
[14]Salazar O,Julian M C,Rubio V.Primers based on specific rDNA-ITS sequences for PCR detection of Rhizoctonia solani AG 2subgroups and ecological types,and binucleate Rhizoctonia[J].Mycological Research,2000,104(3):281-285.
[15]Lübeck M,Poulsen H.UP-PCR cross-blot hybridization as a tool for identification of anastomosis groups in the Rhizoctonia solani complex[J].FEMS Microbiology Letters,2001,201(1):83-89.
[16]Carling D E,Kuninaga S,Brainard K A.Hyphal anastomosis reactions,r DNA-internal transcribed spacers sequences,and virulence levels among subsets of Rhizoctonia solani anastomosis group 2(AG-2)and AG BI[J].Phytopathology,2002,92(1):43-50.
[17]Toda T,Mushika T,Hyakumachi M.Development of specific PCRprimers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting largepatch symptoms on zoysia grass[J].FEMS Microbiology Letters,2004,232(1):67-74.
[18]Grosch R,Schneider J H M,Peth A,et al.Development of a specific PCR assay for the detection of Rhizoctonia solani AG 1-IBusing SCAR primers[J].Journal of Applied Microbiology,2007,102(3):806-819.
[19]Abbas S J,Ahmad B,Karlovskyp P.Real-time PCR(QPCR)assay for Rhizoctonia solani anastomoses group AG2-2 IIIB[J].Pakistan Journal of Botany,2014,46(1):353-356.
[20]Amaradasa B S,Lakshman D,Horvath B J,et al.Development of SCAR markers and UP-PCR cross-hybridization method for specific detection of four major subgroups of Rhizoctonia from infected turfgrasses[J].Mycologia,2014,106(1):163-172.
[21]淮稳霞,程衬衬,李东林,等.中国局部地区草坪立枯丝核菌的rDNA ITS序列分析.中国农学通报,2012,28(4):200-205.
[22]李晓妮,徐娜娜,于金凤.中国北方马铃薯黑痣病立枯丝核菌的融合群鉴定[J].菌物学报,2014,33(3):584-593.
[23]方中达.植病研究方法.第三版.北京:中国农业出版社,1998:122-126.
[24]Bandoni R J.Safranin O as a rapid stain for fungi[J].Mycologia,1979,71(4):873-874.
[25]白滨,何苏琴,荆卓琼,等.分离自黄瓜的多主棒孢霉不同表型菌株对杀菌剂的敏感性[J].微生物学通报,2016,43(2):322-329.
[26]White T J,Bruns T,Lee S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[A].//In:Innis M A,Gelfand D H,Sninsky J J,et al.PCR Protocols:AGuide to Methods and Applications[M].San Diego,CA:Academic Press,1990:315-322.
[27]Parmeter J R,Whitney H S.Taxonomy and nomenclature of the imperfect state[A].//In:Parmeter J R.Rhizoctonia solani:Biology and Pathology[M].Berkeley,Los Angeles and London:University of California Press,1970:7-19.
[2]彭建姝,杨晓菊.7个娃娃菜品种在红古区的引种试验初报[J].甘肃农业科技,2012(12):25-26.
[3]李业贵.红古区娃娃菜产业发展现状及发展途径[J].甘肃农业,2009(11):66-67.
[4]戚佩坤,白金铠,朱桂香.吉林省栽培植物真菌病害志[M].北京:科学出版社,1966:111.
[5]北方大白菜病害综防技术研究与应用协作组.我国北方大白菜病害现状及发展的浅见[J].中国蔬菜,1993(4):38-40.
[6]苗国辉,闻凤英,张耀伟.津黑两地大白菜褐腐病病原菌鉴定及生物学特性的比较研究[J].植物保护,2012,38(4):89-95.
[7]李明远.北京小油菜褐腐病日趋严重[J].植物保护,1993,19(4):50-51.
[8]周慧敏,谢学文,石延霞,等.大白菜褐腐病(茎基腐病)的病原菌鉴定[J].中国蔬菜,2012(22):70-74.
[9]张耀伟,苗国辉.大白菜对褐腐病抗性的快速鉴定方法研究[J].植物保护,2014,40(3):117-121.
[10]段春芳,杨根华,尼章光,等.立枯丝核菌AG-1 IB引起白菜、薄荷、莴苣叶腐病的研究[J].云南农业大学学报,2008,23(3):422-425.
[11]Carling D E.Grouping in Rhizoctonia solani by hyphal anastomosis reaction[A].//In:Sneh B,Jabaji-Hare S,Neate S,et al.Rhizoctonia species:Taxonomy,Molecular Biology,Ecology,Pathology and Disease Control[M].Dordrecht:Kluwer Academic Publishers,1996:37-47.
[12]Ogoshi A.Introduction-The genus Rhizoctonia[A].//In:Sneh B,Jabaji-Hare S,Neate S,et al.Rhizoctonia species:Taxonomy,Molecular Biology,Ecology,Pathology and Disease Control[M].Dordrecht:Kluwer Academic Publishers,1996:1-9.
[13]Kuninaga S,Natsuaki T,Takeuchi T,et al.Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani[J].Current Genetics,1997,32(3):237-243.
[14]Salazar O,Julian M C,Rubio V.Primers based on specific rDNA-ITS sequences for PCR detection of Rhizoctonia solani AG 2subgroups and ecological types,and binucleate Rhizoctonia[J].Mycological Research,2000,104(3):281-285.
[15]Lübeck M,Poulsen H.UP-PCR cross-blot hybridization as a tool for identification of anastomosis groups in the Rhizoctonia solani complex[J].FEMS Microbiology Letters,2001,201(1):83-89.
[16]Carling D E,Kuninaga S,Brainard K A.Hyphal anastomosis reactions,r DNA-internal transcribed spacers sequences,and virulence levels among subsets of Rhizoctonia solani anastomosis group 2(AG-2)and AG BI[J].Phytopathology,2002,92(1):43-50.
[17]Toda T,Mushika T,Hyakumachi M.Development of specific PCRprimers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting largepatch symptoms on zoysia grass[J].FEMS Microbiology Letters,2004,232(1):67-74.
[18]Grosch R,Schneider J H M,Peth A,et al.Development of a specific PCR assay for the detection of Rhizoctonia solani AG 1-IBusing SCAR primers[J].Journal of Applied Microbiology,2007,102(3):806-819.
[19]Abbas S J,Ahmad B,Karlovskyp P.Real-time PCR(QPCR)assay for Rhizoctonia solani anastomoses group AG2-2 IIIB[J].Pakistan Journal of Botany,2014,46(1):353-356.
[20]Amaradasa B S,Lakshman D,Horvath B J,et al.Development of SCAR markers and UP-PCR cross-hybridization method for specific detection of four major subgroups of Rhizoctonia from infected turfgrasses[J].Mycologia,2014,106(1):163-172.
[21]淮稳霞,程衬衬,李东林,等.中国局部地区草坪立枯丝核菌的rDNA ITS序列分析.中国农学通报,2012,28(4):200-205.
[22]李晓妮,徐娜娜,于金凤.中国北方马铃薯黑痣病立枯丝核菌的融合群鉴定[J].菌物学报,2014,33(3):584-593.
[23]方中达.植病研究方法.第三版.北京:中国农业出版社,1998:122-126.
[24]Bandoni R J.Safranin O as a rapid stain for fungi[J].Mycologia,1979,71(4):873-874.
[25]白滨,何苏琴,荆卓琼,等.分离自黄瓜的多主棒孢霉不同表型菌株对杀菌剂的敏感性[J].微生物学通报,2016,43(2):322-329.
[26]White T J,Bruns T,Lee S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[A].//In:Innis M A,Gelfand D H,Sninsky J J,et al.PCR Protocols:AGuide to Methods and Applications[M].San Diego,CA:Academic Press,1990:315-322.
[27]Parmeter J R,Whitney H S.Taxonomy and nomenclature of the imperfect state[A].//In:Parmeter J R.Rhizoctonia solani:Biology and Pathology[M].Berkeley,Los Angeles and London:University of California Press,1970:7-19.