草莓枯萎病菌拮抗细菌JX-13的鉴定及生防效果评价
|
摘要
为明确生防菌株JX-13的分类地位,评价其对草莓枯萎病的生防效果,依据JX-13菌株形态、生理生化特性、16S rRNA和gyrB基因碱基序列同源性对其进行分析鉴定,采用菌丝生长速率法和田间试验评价其对草莓的促生作用和枯萎病生防效果。结果表明,JX-13菌株的形态特征和生理生化特性均与芽孢杆菌很接近,16S rRNA和gyrB基因碱基序列分析发现,JX-13菌株在系统发育树中与Paenibacillus polymyxa strain IIF5SW-B3属于一个类群,相似性高达99.00%。发酵加工的1×10~(10) CFU/g JX-13可湿性粉剂(WP)和1×10~(10) CFU/g枯草芽孢杆菌WP对草莓枯萎病菌菌丝生长的抑制中质量浓度(EC_(50))分别为19.977μg/ml和41.409μg/ml。对草莓繁苗田进行2次灌根处理,JX-13 WP 500倍液处理对草莓植株有明显促生作用,对草莓枯萎病的防治效果达96.03%。定植当天和第7 d对定植田进行2次灌根处理,药后50 d、80 d分别进行田间调查,JX-13 WP 500倍液对草莓枯萎病防治效果分别为100.00%和68.94%。菌株JX-13被鉴定为多黏类芽孢杆菌Paenibacillus polymyxa,其发酵液加工的1×10~(10) CFU/g WP在草莓繁苗田和定植田中具有防治枯萎病和促生的作用。
To identify the classification status and its effect on promoting strawberry growth and controlling fusarium wilt, JX-13 strain was analyzed by phenotypic characteristics observation, physiological and biochemical indices determination, 16 S rRNA and gyrB sequence assay, and the growth-promoting effect and biocontrol efficacy of JX-13 on strawberry fusarium wilt were evaluated by the mycelial growth inhibition rate method and field test. The results showed that morphological, physiological and biochemical characteristics of JX-13 strain were very close to those of Paenibacillus polymyxa, 16 S rRNA and gyrB gene sequences suggested that JX-13 strain was clustered together with Paenibacillus polymyxa strain IIF5 SW-B3 in phylogenetic tree, with the sequence similarity of 99.00%. The EC_(50) values of 1×10~(10) CFU/g JX-13 WP and 1×10~(10) CFU/g Bacillus subtilis WP against Fusarium oxysporum were 19.977 μg/ml and 41.409 μg/ml, respectively. Strawberry nursery was treated by twice root-irrigation, and the results indicated that JX-13 WP 500 not only effectively prevented strawberry fusarium wilt(96.03% control effect), but also significantly promoted seedling growth. Field investigation was conducted on 50 and 80 days after root irrigation, and the control efficacy of JX-13 WP 500 to strawberry fusarium wilt was 100.00% and 68.94%, respectively. The JX-13 strain was identified as Paenibacillus polymyxa, and 1×10~(10) CFU/g JX-13 WP had preventive efficacy to strawberry fusarium wilt and growth-promoting effect.
To identify the classification status and its effect on promoting strawberry growth and controlling fusarium wilt, JX-13 strain was analyzed by phenotypic characteristics observation, physiological and biochemical indices determination, 16 S rRNA and gyrB sequence assay, and the growth-promoting effect and biocontrol efficacy of JX-13 on strawberry fusarium wilt were evaluated by the mycelial growth inhibition rate method and field test. The results showed that morphological, physiological and biochemical characteristics of JX-13 strain were very close to those of Paenibacillus polymyxa, 16 S rRNA and gyrB gene sequences suggested that JX-13 strain was clustered together with Paenibacillus polymyxa strain IIF5 SW-B3 in phylogenetic tree, with the sequence similarity of 99.00%. The EC_(50) values of 1×10~(10) CFU/g JX-13 WP and 1×10~(10) CFU/g Bacillus subtilis WP against Fusarium oxysporum were 19.977 μg/ml and 41.409 μg/ml, respectively. Strawberry nursery was treated by twice root-irrigation, and the results indicated that JX-13 WP 500 not only effectively prevented strawberry fusarium wilt(96.03% control effect), but also significantly promoted seedling growth. Field investigation was conducted on 50 and 80 days after root irrigation, and the control efficacy of JX-13 WP 500 to strawberry fusarium wilt was 100.00% and 68.94%, respectively. The JX-13 strain was identified as Paenibacillus polymyxa, and 1×10~(10) CFU/g JX-13 WP had preventive efficacy to strawberry fusarium wilt and growth-promoting effect.
引文
[1] 吉沐祥,杨勇,彭燕琼,等.江苏草莓生产现状与消费需求调查分析及其发展建议[J].江苏农业科学,2017,45(16):336-340.
[2] 周游,李海梅,赵金山,等.乳酸菌对草莓生长和品质性状的影响[J].江苏农业学报,2017,33(5):1124-1128.
[3] 闵红,陈磊,呼世斌,等.大棚果蔬连作土壤肥力限制性因子研究[J].西北农林科技大学学报(自然科学版),2010,38(8):160-166.
[4] 吉沐祥,陈宏州,庄义庆,等.设施草莓土传病害无害化综合防治技术[J].江苏农业科学,2015,43(2):126-127.
[5] 刘奇志,李星月,刘艳斌,等.国内外草莓连作障碍与综合治理研究进展[J].中国果树,2012 (6):58-62.
[6] 刘喜更.草莓重茬土壤病害防治技术研究取得新突破[J].北京农业,1998(8):33.
[7] 申光辉.草莓连作根腐病发生机制与微生物及化学修复研究[D].杨凌:西北农林科技大学,2012.
[8] 顾春波,姜莉莉,王开运,等.抗戊唑醇草莓枯萎病菌 ZY-W 的诱导及其生物学特性[J].中国农业科学,2010,43(14):2897-2904.
[9] 伊海静,陈艳,刘正坪,等.草莓枯萎病菌的分离鉴定及防治药剂筛选[J].西北农业学报,2016,25(4):626-635.
[10] 杨焕青,王开运,范昆,等.草莓枯萎病的生物学特性及7种杀菌剂及其抑制作用[J].植物保护学报,2008,35(2):169-174.
[11] 顾春波,史晓斌,姜莉莉,等.草莓枯萎病菌对多菌灵的抗性及其抗性菌株生物学特性[J].植物保护学报,2010,37(3):266-272.
[12] LIU S,CHE Z,CHEN G.Multiple-fungicide resistance to carbendazim,diethofencarb,procymidone,and pyrimethanil in field isolates of Botrytis cinerea from tomato in Henan province,China[J].Crop Protection,2016,84:56-61.
[13] 段海明,余利,黄伟东,等.不同温度下 6 种化学杀菌剂对玉米茎腐病菌的抑制活性及与生防菌发酵上清液的混配[J].江苏农业学报,2018,34(1):41-49.
[14] 高旭利,李永腾,刘文宝,等.利用生防细菌防治黄瓜根结线虫病研究[J].山东农业科学,2018,50(8):116-119.
[15] LUO Y,CHENG Y,YI J,et al.Complete genome sequence of industrial biocontrol strain Paenibacillus polymyxa HY96-2 and further analysis of its biocontrol mechanism[J].Frontiers in Microbiology,2018,9:1-14.
[16] WESELOWSKI B,NATHOO N,EASTMAN A W,et al.Isolation,identification and characterization of Paenibacillus polymyxa CR1 with potentials for biopesticide,biofertilization,biomass degradation and biofuel production[J].BMC Microbiology,2016,16(1):244.
[17] 余小兰,邹立飞,邹雨坤,等.甜瓜枯萎病拮抗菌的筛选及鉴定[J].南方农业学报,2018,49(6):1118-1124.
[18] 王波,周涧楠,黄忠勤,等.一株多粘类芽孢杆菌 Paenibacillus polymyxa 对甘薯黑斑病的生物防治效果及作用机理初探[J].江西农业学报,2017,29(10):40-43.
[19] 曹远银,王婉琳,申璐岚,等.小麦白粉病生防菌拟诺卡氏菌属 TMG-8 菌株的筛选研究[J].江苏农业科学,2017,45(1):95-99.
[20] 周京龙.一株棉花内生蜡状芽孢杆菌对棉花黄、枯萎病的防治作用及机理[D].荆州:长江大学,2017.
[21] 吴祥,姚克兵,吉沐祥,等.句容地区草莓枯萎病病原菌的分离鉴定及田间防治[J].江苏农业学报,2015,31(4):764-770.
[22] 杜秉海.微生物学实验[M].北京:北京农业大学出版社,1994.
[23] 东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[24] AUSUBEL F M,BRENT R,KINGSTON R E,et al.Short protocols in molecular biology[M].3rd ed.Chichester:John Wiley&Sons,1995.
[25] LA DUC M T,SATOMI M,AGATA N,et al.gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group[J].Journal of Microbiological Methods,2004,56(3):383-394.
[26] KüPFER M,KUHNERT P,KORCZAK B M,et al.Genetic relationships of Aeromonas strains inferred from 16S rRNA,gyrB and rpoB gene sequences[J].International Journal of Systematic and Evolutionary Microbiology,2006,56(12):2743-2751.
[27] WANG H,SHI Y,WANG D,et al.A biocontrol strain of Bacillus subtilis WXCDD105 used to control tomato Botrytis cinerea and Cladosporium fulvum Cooke and promote the growth of seedlings[J].International Journal of Molecular Sciences,2018,19(5):1371.
[28] 许彦,罗丰,杨礼哲,等.几种杀菌剂对西瓜枯萎病的室内毒力测定[J].热带农业科学,2010,30(10):18-19,26.
[29] RYBAKOVA D,RACK-WETZLINGER U,CERNAVA T,et al.Aerial warfare:a volatile dialogue between the plant pathogen Verticillium longisporum and its antagonist Paenibacillus polymyxa[J].Frontiers in Plant Science,2017,8:1294.
[30] 郑雪芳,刘波,朱育菁,等.设施番茄连作障碍土壤修复及其对青枯病害的防治效果[J].中国生物防治学报,2018,34(1):117-123.
[31] 乔俊卿,陈志谊,梁雪杰,等.枯草芽孢杆菌 Bs916 防治番茄青枯病[J].中国生物防治学报,2016,32(2):229-234.
[32] 王刘庆,王秋影,廖美德.多粘类芽孢杆菌生物特性及其机理研究进展[J].中国农学通报,2013,29(11):158-163.
[33] DIJKSTERHUIS J,SANDERS M,GORRIS L G M,et al.Antibiosis plays a role in the context of direct interaction during antagonism of Paenibacillus polymyxa towards Fusarium oxysporum[J].Journal of Applied Microbiology,1999,86(1):13-21.
[34] ABD EL DAIM I A,H?GGBLOM P,KARLSSON M,et al.Paenibacillus polymyxa A26 Sfp-type PPTase inactivation limits bacterial antagonism against Fusarium graminearum but not of F.culmorum in kernel assay[J].Frontiers in Plant Science,2015,6:368.
[35] 范磊,张道敬,刘振华,等.多粘类芽孢杆菌HY96-2产脂肽类抗真菌物质的研究[J].天然产物研究与开发,2012(24):729-735.
[36] MEI L,LIANG Y,ZHANG L,et al.Induced systemic resistance and growth promotion in tomato by an indole-3-acetic acid-producing strain of Paenibacillus polymyxa[J].Annals of Applied Biology,2014,165(2):270-279.
[37] 马夙静.多粘类芽孢杆菌ZYPP18的分离鉴定与促生防病效果鉴定 [D].济南:山东农业大学,2018.
[38] SHI L,DU N,SHU S,et al.Paenibacillus polymyxa NSY50 suppresses Fusarium wilt in cucumbers by regulating the rhizospheric microbial community[J].Scientific Reports,2017,7:41234.
[39] DU N,SHI L,YUAN Y,et al.Isolation of a potential biocontrol agent Paenibacillus polymyxa NSY50 from vinegar waste compost and its induction of host defense responses against Fusarium wilt of cucumber[J].Microbiological Research,2017,202,1-10.
[40] 陈雪丽,王光华,金剑,等.多粘类芽孢杆菌 BRF-1 和枯草芽孢杆菌 BRF-2 对黄瓜和番茄枯萎病的防治效果[J].中国生态农业学报,2008,16(2):446-450.
[41] YAO Y E,YUAN J,YANG F,et al.PGPR strain Paenibacillus polymyxa SQR-21 potentially benefits watermelon growth by re-shaping root protein expression[J].AMB Express,2017,7:104.
[42] 朱金英.微生物菌剂在设施黄瓜和番茄上的应用效果研究[D].济南:山东农业大学,2014.
[43] PADDA K P,PURI A,CHANWAY C P.Effect of GFP tagging of Paenibacillus polymyxa P2b-2R on its ability to promote growth of canola and tomato seedlings[J].Biology and Fertility of Soils,2016,52(3):377-387.
[44] LI J,LIU W,LUO L,et al.Expression of Paenibacillus polymyxa β-1,3-1,4-glucanase in Streptomyces lydicus A01 improves its biocontrol effect against Botrytis cinerea[J].Biological Control,2015,90:141-147.
[45] 刘丹丹,李敏,刘润进.我国植物根围促生细菌研究进展[J].生态学杂志,2016,35(3):815-824.
[46] ABBASI M K,SHARIF S,KAZMI M,et al.Isolation of plant growth promoting rhizobacteria from wheat rhizosphere and their effect on improving growth,yield and nutrient uptake of plants[J].Plant Biosystems,2011,145(1):159-168.
[2] 周游,李海梅,赵金山,等.乳酸菌对草莓生长和品质性状的影响[J].江苏农业学报,2017,33(5):1124-1128.
[3] 闵红,陈磊,呼世斌,等.大棚果蔬连作土壤肥力限制性因子研究[J].西北农林科技大学学报(自然科学版),2010,38(8):160-166.
[4] 吉沐祥,陈宏州,庄义庆,等.设施草莓土传病害无害化综合防治技术[J].江苏农业科学,2015,43(2):126-127.
[5] 刘奇志,李星月,刘艳斌,等.国内外草莓连作障碍与综合治理研究进展[J].中国果树,2012 (6):58-62.
[6] 刘喜更.草莓重茬土壤病害防治技术研究取得新突破[J].北京农业,1998(8):33.
[7] 申光辉.草莓连作根腐病发生机制与微生物及化学修复研究[D].杨凌:西北农林科技大学,2012.
[8] 顾春波,姜莉莉,王开运,等.抗戊唑醇草莓枯萎病菌 ZY-W 的诱导及其生物学特性[J].中国农业科学,2010,43(14):2897-2904.
[9] 伊海静,陈艳,刘正坪,等.草莓枯萎病菌的分离鉴定及防治药剂筛选[J].西北农业学报,2016,25(4):626-635.
[10] 杨焕青,王开运,范昆,等.草莓枯萎病的生物学特性及7种杀菌剂及其抑制作用[J].植物保护学报,2008,35(2):169-174.
[11] 顾春波,史晓斌,姜莉莉,等.草莓枯萎病菌对多菌灵的抗性及其抗性菌株生物学特性[J].植物保护学报,2010,37(3):266-272.
[12] LIU S,CHE Z,CHEN G.Multiple-fungicide resistance to carbendazim,diethofencarb,procymidone,and pyrimethanil in field isolates of Botrytis cinerea from tomato in Henan province,China[J].Crop Protection,2016,84:56-61.
[13] 段海明,余利,黄伟东,等.不同温度下 6 种化学杀菌剂对玉米茎腐病菌的抑制活性及与生防菌发酵上清液的混配[J].江苏农业学报,2018,34(1):41-49.
[14] 高旭利,李永腾,刘文宝,等.利用生防细菌防治黄瓜根结线虫病研究[J].山东农业科学,2018,50(8):116-119.
[15] LUO Y,CHENG Y,YI J,et al.Complete genome sequence of industrial biocontrol strain Paenibacillus polymyxa HY96-2 and further analysis of its biocontrol mechanism[J].Frontiers in Microbiology,2018,9:1-14.
[16] WESELOWSKI B,NATHOO N,EASTMAN A W,et al.Isolation,identification and characterization of Paenibacillus polymyxa CR1 with potentials for biopesticide,biofertilization,biomass degradation and biofuel production[J].BMC Microbiology,2016,16(1):244.
[17] 余小兰,邹立飞,邹雨坤,等.甜瓜枯萎病拮抗菌的筛选及鉴定[J].南方农业学报,2018,49(6):1118-1124.
[18] 王波,周涧楠,黄忠勤,等.一株多粘类芽孢杆菌 Paenibacillus polymyxa 对甘薯黑斑病的生物防治效果及作用机理初探[J].江西农业学报,2017,29(10):40-43.
[19] 曹远银,王婉琳,申璐岚,等.小麦白粉病生防菌拟诺卡氏菌属 TMG-8 菌株的筛选研究[J].江苏农业科学,2017,45(1):95-99.
[20] 周京龙.一株棉花内生蜡状芽孢杆菌对棉花黄、枯萎病的防治作用及机理[D].荆州:长江大学,2017.
[21] 吴祥,姚克兵,吉沐祥,等.句容地区草莓枯萎病病原菌的分离鉴定及田间防治[J].江苏农业学报,2015,31(4):764-770.
[22] 杜秉海.微生物学实验[M].北京:北京农业大学出版社,1994.
[23] 东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[24] AUSUBEL F M,BRENT R,KINGSTON R E,et al.Short protocols in molecular biology[M].3rd ed.Chichester:John Wiley&Sons,1995.
[25] LA DUC M T,SATOMI M,AGATA N,et al.gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group[J].Journal of Microbiological Methods,2004,56(3):383-394.
[26] KüPFER M,KUHNERT P,KORCZAK B M,et al.Genetic relationships of Aeromonas strains inferred from 16S rRNA,gyrB and rpoB gene sequences[J].International Journal of Systematic and Evolutionary Microbiology,2006,56(12):2743-2751.
[27] WANG H,SHI Y,WANG D,et al.A biocontrol strain of Bacillus subtilis WXCDD105 used to control tomato Botrytis cinerea and Cladosporium fulvum Cooke and promote the growth of seedlings[J].International Journal of Molecular Sciences,2018,19(5):1371.
[28] 许彦,罗丰,杨礼哲,等.几种杀菌剂对西瓜枯萎病的室内毒力测定[J].热带农业科学,2010,30(10):18-19,26.
[29] RYBAKOVA D,RACK-WETZLINGER U,CERNAVA T,et al.Aerial warfare:a volatile dialogue between the plant pathogen Verticillium longisporum and its antagonist Paenibacillus polymyxa[J].Frontiers in Plant Science,2017,8:1294.
[30] 郑雪芳,刘波,朱育菁,等.设施番茄连作障碍土壤修复及其对青枯病害的防治效果[J].中国生物防治学报,2018,34(1):117-123.
[31] 乔俊卿,陈志谊,梁雪杰,等.枯草芽孢杆菌 Bs916 防治番茄青枯病[J].中国生物防治学报,2016,32(2):229-234.
[32] 王刘庆,王秋影,廖美德.多粘类芽孢杆菌生物特性及其机理研究进展[J].中国农学通报,2013,29(11):158-163.
[33] DIJKSTERHUIS J,SANDERS M,GORRIS L G M,et al.Antibiosis plays a role in the context of direct interaction during antagonism of Paenibacillus polymyxa towards Fusarium oxysporum[J].Journal of Applied Microbiology,1999,86(1):13-21.
[34] ABD EL DAIM I A,H?GGBLOM P,KARLSSON M,et al.Paenibacillus polymyxa A26 Sfp-type PPTase inactivation limits bacterial antagonism against Fusarium graminearum but not of F.culmorum in kernel assay[J].Frontiers in Plant Science,2015,6:368.
[35] 范磊,张道敬,刘振华,等.多粘类芽孢杆菌HY96-2产脂肽类抗真菌物质的研究[J].天然产物研究与开发,2012(24):729-735.
[36] MEI L,LIANG Y,ZHANG L,et al.Induced systemic resistance and growth promotion in tomato by an indole-3-acetic acid-producing strain of Paenibacillus polymyxa[J].Annals of Applied Biology,2014,165(2):270-279.
[37] 马夙静.多粘类芽孢杆菌ZYPP18的分离鉴定与促生防病效果鉴定 [D].济南:山东农业大学,2018.
[38] SHI L,DU N,SHU S,et al.Paenibacillus polymyxa NSY50 suppresses Fusarium wilt in cucumbers by regulating the rhizospheric microbial community[J].Scientific Reports,2017,7:41234.
[39] DU N,SHI L,YUAN Y,et al.Isolation of a potential biocontrol agent Paenibacillus polymyxa NSY50 from vinegar waste compost and its induction of host defense responses against Fusarium wilt of cucumber[J].Microbiological Research,2017,202,1-10.
[40] 陈雪丽,王光华,金剑,等.多粘类芽孢杆菌 BRF-1 和枯草芽孢杆菌 BRF-2 对黄瓜和番茄枯萎病的防治效果[J].中国生态农业学报,2008,16(2):446-450.
[41] YAO Y E,YUAN J,YANG F,et al.PGPR strain Paenibacillus polymyxa SQR-21 potentially benefits watermelon growth by re-shaping root protein expression[J].AMB Express,2017,7:104.
[42] 朱金英.微生物菌剂在设施黄瓜和番茄上的应用效果研究[D].济南:山东农业大学,2014.
[43] PADDA K P,PURI A,CHANWAY C P.Effect of GFP tagging of Paenibacillus polymyxa P2b-2R on its ability to promote growth of canola and tomato seedlings[J].Biology and Fertility of Soils,2016,52(3):377-387.
[44] LI J,LIU W,LUO L,et al.Expression of Paenibacillus polymyxa β-1,3-1,4-glucanase in Streptomyces lydicus A01 improves its biocontrol effect against Botrytis cinerea[J].Biological Control,2015,90:141-147.
[45] 刘丹丹,李敏,刘润进.我国植物根围促生细菌研究进展[J].生态学杂志,2016,35(3):815-824.
[46] ABBASI M K,SHARIF S,KAZMI M,et al.Isolation of plant growth promoting rhizobacteria from wheat rhizosphere and their effect on improving growth,yield and nutrient uptake of plants[J].Plant Biosystems,2011,145(1):159-168.