红三叶根际促生菌中具生防效果菌株筛选、鉴定及特性研究
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摘要
【目的】从岷山红三叶根际分离的109株促生菌,鉴定、筛选了具有生防能力的菌种资源并研究其生防特性,为生产应用提供服务。【方法】采用平板对峙法筛选优良生防菌株,对其产铁载体能力进行定性、定量测定和16S r RNA基因序列分析鉴定。【结果】平板对峙试验显示,109株促生菌中有8株促生菌对3种病原真菌(立枯丝核菌、黄瓜枯萎菌、西瓜尖镰孢菌)具有拮抗效果,分别是菌株MHS3、MHS7、MHS9、MHS27、MHS31、MHS38、MHS39和MHS48。其中,菌株MHS27、MHS31发酵液对立枯丝核菌抑菌率分别为40.1%、47.1%;菌株MHS7和MHS48发酵液对黄瓜枯萎菌抑菌率分别为26.1%、22.8%;菌株MHS27和MHS31发酵液对西瓜尖镰孢菌抑制率分别为34.0%、30.5%;产嗜铁素是生防菌(MHS7、MHS27、MHS31、MHS48)拮抗3种病原真菌的主要途径之一。经16S r RNA基因序列分析初步鉴定,菌株MHS7为特基拉芽孢杆菌(Bacillus tequilensis),菌株MHS27为溶蛋白芽孢杆菌(Bacillus proteolyticus),菌株MHS31为Pseudomonas paralactis,菌株MHS48为克雷伯氏菌(Klebsiella oxytoca)。【结论】从岷山红三叶根际分离筛选出4株具有优良生防作用的PGPR菌株,为今后生物菌肥应用推广提供了菌种资源支持。
【Objectives】Biocontrol bacteria were screened from 109 strains of plant growth promoting rhizobacteria(PGPR) of Trifolium pratense in Minshan. Their promoting effects were identified and their biocontrol characteristics were studied for the practical use.【Methods】The plate confrontation method was used to screen the excellent biocontrol strains, and siderophore producing capacity of the strains was measured and the 16 S r RNA gene sequence of these strains was analyzed.【Results】Eight strains of biocontrol bacteria showed an antagonistic effect on 3 pathogenic fungus(Rhizoctonia solani, Fusarium oxysporium f. sp.Cucumerinum, and Fusarium oxysporum f. Niveum). The strains were MHS3, MHS7, MHS9, MHS27, MHS31,MHS38, MHS39 and MHS48, and among them, the inhibition rates of the strains of MHS27 and MHS31 against Rhizoctonia solani were 40.1% and 47.1%, the inhibition rates of the strains of MHS7 and MHS48 against Fusarium oxysporium f. sp. Cucumerinum were 26.1% and 22.8%, and the inhibition rates of the strains of MHS27 and MHS31 against Fusarium oxysporum f. Niveum were 34.0% and 30.5%. The siderophore produced was one of the main ways to antagonize 3 pathogenic fungi in biocontrol bacteria(MHS7, MHS27, MHS31,MHS48). The results of the 16 S r RNA gene sequence analysis showed that the strain MHS7 was Bacillus tequilensis, the strain MHS27 was Bacillus proteolyticus, the strain MHS31 was Pseudomonas paralactis, and the strain MHS48 was Klebsiella oxytoca.【Conclusions】These four excellent PGPR strains which were screened from the Trifolium pretense rhizosphere of Minshan can provide a support for the application of bio-fertilizer in the future.
【Objectives】Biocontrol bacteria were screened from 109 strains of plant growth promoting rhizobacteria(PGPR) of Trifolium pratense in Minshan. Their promoting effects were identified and their biocontrol characteristics were studied for the practical use.【Methods】The plate confrontation method was used to screen the excellent biocontrol strains, and siderophore producing capacity of the strains was measured and the 16 S r RNA gene sequence of these strains was analyzed.【Results】Eight strains of biocontrol bacteria showed an antagonistic effect on 3 pathogenic fungus(Rhizoctonia solani, Fusarium oxysporium f. sp.Cucumerinum, and Fusarium oxysporum f. Niveum). The strains were MHS3, MHS7, MHS9, MHS27, MHS31,MHS38, MHS39 and MHS48, and among them, the inhibition rates of the strains of MHS27 and MHS31 against Rhizoctonia solani were 40.1% and 47.1%, the inhibition rates of the strains of MHS7 and MHS48 against Fusarium oxysporium f. sp. Cucumerinum were 26.1% and 22.8%, and the inhibition rates of the strains of MHS27 and MHS31 against Fusarium oxysporum f. Niveum were 34.0% and 30.5%. The siderophore produced was one of the main ways to antagonize 3 pathogenic fungi in biocontrol bacteria(MHS7, MHS27, MHS31,MHS48). The results of the 16 S r RNA gene sequence analysis showed that the strain MHS7 was Bacillus tequilensis, the strain MHS27 was Bacillus proteolyticus, the strain MHS31 was Pseudomonas paralactis, and the strain MHS48 was Klebsiella oxytoca.【Conclusions】These four excellent PGPR strains which were screened from the Trifolium pretense rhizosphere of Minshan can provide a support for the application of bio-fertilizer in the future.
引文
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[15]Sayyed R,Chincholkar S.Siderophore-producing Alcaligenes feacalis exhibited more biocontrol potential Vis-a-Vis chemical fungicide[J].Current Microbiology,2009,58(1):47-51.
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[17]孙广正,侯栋,岳宏忠,等.番茄早疫病菌拮抗细菌的筛选及其抑制作用[J].草原与草坪,2015,35(1):32-36,43.Sun G Z,Hou D,Yue H Z,et al.Screening of bacteria antagonizing Altemaria solani and its inhibitory effects[J].Grassland and Turf,2015,35(1):32-36,43.
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[19]Sun G Z,Yao T,Feng C J,et al.Identification and biocontrol potential of antagonistic bacteria strains against Sclerotinia sclerotinia and their growth-promoting effects on Brassia napus[J].Biological Control,2017,104:35-43.
[20]韩立荣,张华姣,高保卫,等.放线菌11-3-1对油菜菌核病的防治作用与菌株鉴定[J].植物保护学报,2012,39(2):97-102.Han L R,Zhang H J,Gao B W,et al.Antifungal activity against rapeseed Sclerotinia stem rot and identification of actinomycete strain11-3-1[J].Journal of Plant Protection,2012,39(2):97-102.
[21]赵帅,田长彦,史应武,等.黄瓜枯萎病拮抗菌HD-087产抗菌物质条件的优化及抑菌作用初探[J].微生物学通报,2013,40(5):802-811.Zhao S,Tian C Y,Shi Y W,et al.Fermentation conditions for the biocontrol strain HD-087 against cucumber Fusarium wilt and its antibacterial effect[J].Microbiology China,2013,40(5):802-811.
[22]林超,郑服丛,贺春萍,等.嗜铁细菌C19对黄瓜种子发芽的影响及对芒果炭疽病生防效果初步评价[J].中国农学通报,2009,25(9):232-235.Lin C,Zheng F C,He C P,et al.The effect of siderophore-producing bacteria C19 on cucumber seed germination and biocontrol effects on mango anthracnose[J].Chinese Agricultural Science Bulletin,2009,25(9):232-235.
[23]Sajeli B A,Basha S A,Raghavendra G,et al.Isolation and characterization of antimicrobial cyclic dipeptides from Pseudomonas fluorescens and their efficacy on sorghum grain mold fungi[J].Chemistry and Biodiversity,2014,11(1):92-100.
[24]Yang M M,Wen S S,Mavrodi D V,et al.Biological control of wheat root diseases by the CLP-producing strain Pseudomonas fluorescens HC1-07[J].Phytopathology,2014,104(3):248-256.
[2]Ryu C M,Kim J,Choi O,et al.Improvement of biological control capacity of Paenibacillus polymyxa E681 by seed pelleting on sesame[J].Biological Control,2006,39(3):282-289.
[3]Compant S,Clement C,Sessitsch A.Plant growth-promoting bacteria in the rhizo and endosphere of plants:their role,colonization,mechanisms involved and prospects for utilization[J].Soil Biology&Biochemistry,2010,42(5):669-678.
[4]Compant S,Duffy B,Nowak J,et al.Use of plant growth-promoting bacteria for biocontrol of plant diseases:Principles,mechanisms of action,and future prospects[J].Applied and Environmental Microbiology,2005,71(9):4951-4959.
[5]Singh B K,Walker A,Morgan J A,et al.Effects of soil pH on the biodegradation of chlorpyrifos and isolation of a chlorpyrifosdegrading bacterium[J].Applied and Environmental Microbiology,2003,69(9):5198-5206.
[6]Britigan B E,Rasmussen G T,Cox C D.Augmentation of oxidant injury to human pulmon-ary epithelial cells by the Pseudomonas aeruginosa siderophore pyochelin[J].Infection and Immunity,1997,65(3):1071-1076.
[7]Matthijs S,Tehrani K A,Laus G,et al.Thioquinolobactin,a Pseudomonas siderophore with antifungal and anti-Pythium activity[J].Environmental Microbiology,2007,9(2):425-434.
[8]Youard Z A,Mislin G L,Majcherczyk P A,et al.Pseudomonas fluorescens CHAO produces enantio-pyochelin,the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin[J].Journal of Biological Chemistry,2007,282(49):35546-35553.
[9]Matthijs S,Budzikiewicz H,Schafer M,et al.Ornicorrugatin,a new siderophore from Pseudomonas fluorescens AF76[J].Zeitschrift Naturforschung,2008,63(11):8-12.
[10]Franza T,Mahe B,Expert D.Erwinia chrysanthemi requires a second iron transport route dependent of the siderophore achromobactin for extracellular growth and plant infection[J].Molecular Microbiology,2005,55(1):261-275.
[11]Jones A M,Lindow S E,Wildermuth M C.Salicylic acid,yersiniabactin,and pyoverdin production by the model phytopathogen Pseudomonas syringae pv.tomato DC3000:synthesis,regulation,and impact on tomato and Arabidopsis host plants[J].Journal of Bacteriology,2007,189(19):6773-6786.
[12]Sun G X,Zhou W Q,Zhong J J.Organotin decomposition by pyochelin,secreted by Pseudomonas aeruginosa even in an ironsufficient environment[J].Applied and Environmental Microbiology,2006,72(9):6411-6413.
[13]Petermann S R,Sherwood J S,Logue C M.The Yersinia high pathogenicity island is present in Salmonella enterica Subspecies Iisolated from turkeys[J].Microbial Pathogenesis,2008,45(2):110-114.
[14]Helmy M,Baddar D,Masry M H.Affinity purification of a siderophore that exhibits an antagonistic effect against soft rot bacterium[J].Biochemistry(Moscow),2008,73(7):776-782.
[15]Sayyed R,Chincholkar S.Siderophore-producing Alcaligenes feacalis exhibited more biocontrol potential Vis-a-Vis chemical fungicide[J].Current Microbiology,2009,58(1):47-51.
[16]Schwyn B,Neilands J B.Universal chemical assay for the detection and determination of siderophores[J].Analytical Biochemistry,1987,160(1):47-56.
[17]孙广正,侯栋,岳宏忠,等.番茄早疫病菌拮抗细菌的筛选及其抑制作用[J].草原与草坪,2015,35(1):32-36,43.Sun G Z,Hou D,Yue H Z,et al.Screening of bacteria antagonizing Altemaria solani and its inhibitory effects[J].Grassland and Turf,2015,35(1):32-36,43.
[18]孙广正,姚拓,侯栋,等.西葫芦根腐病菌拮抗细菌的防病促生作用[J].微生物学通报,2017,44(5):1121-1130.Sun G Z,Yao T,Hou D,et al.Biocontrol potential of antagonistic bacteria strains against Fusarium oxysporum and their growthpromoting effects on zucchini[J].Microbiology China,2017,44(5):1121-1130.
[19]Sun G Z,Yao T,Feng C J,et al.Identification and biocontrol potential of antagonistic bacteria strains against Sclerotinia sclerotinia and their growth-promoting effects on Brassia napus[J].Biological Control,2017,104:35-43.
[20]韩立荣,张华姣,高保卫,等.放线菌11-3-1对油菜菌核病的防治作用与菌株鉴定[J].植物保护学报,2012,39(2):97-102.Han L R,Zhang H J,Gao B W,et al.Antifungal activity against rapeseed Sclerotinia stem rot and identification of actinomycete strain11-3-1[J].Journal of Plant Protection,2012,39(2):97-102.
[21]赵帅,田长彦,史应武,等.黄瓜枯萎病拮抗菌HD-087产抗菌物质条件的优化及抑菌作用初探[J].微生物学通报,2013,40(5):802-811.Zhao S,Tian C Y,Shi Y W,et al.Fermentation conditions for the biocontrol strain HD-087 against cucumber Fusarium wilt and its antibacterial effect[J].Microbiology China,2013,40(5):802-811.
[22]林超,郑服丛,贺春萍,等.嗜铁细菌C19对黄瓜种子发芽的影响及对芒果炭疽病生防效果初步评价[J].中国农学通报,2009,25(9):232-235.Lin C,Zheng F C,He C P,et al.The effect of siderophore-producing bacteria C19 on cucumber seed germination and biocontrol effects on mango anthracnose[J].Chinese Agricultural Science Bulletin,2009,25(9):232-235.
[23]Sajeli B A,Basha S A,Raghavendra G,et al.Isolation and characterization of antimicrobial cyclic dipeptides from Pseudomonas fluorescens and their efficacy on sorghum grain mold fungi[J].Chemistry and Biodiversity,2014,11(1):92-100.
[24]Yang M M,Wen S S,Mavrodi D V,et al.Biological control of wheat root diseases by the CLP-producing strain Pseudomonas fluorescens HC1-07[J].Phytopathology,2014,104(3):248-256.