水稻内生放线菌OsiRt-1的分离鉴定及对稻瘟病的防治作用
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摘要
【目的】从水稻中分离、筛选并鉴定出对稻瘟病真菌具有拮抗性能的内生放线菌,对高抗菌株进行稻瘟病生防效果评定,初步探讨其作用机制。【方法】采用4种分离培养基对水稻内生放线菌进行分离,用平板对峙法筛选出对稻瘟病菌拮抗性能最好的菌株,结合其菌落形态、生理生化及16S rRNA基因序列分析进行鉴定;用环境扫描电镜(SEM)观察拮抗菌对稻瘟病菌菌丝形态影响,用菌丝生长速率法对拮抗菌发酵滤液进行抗菌活性评价;大田条件下,喷洒拮抗菌孢子液于水稻,检测其稻瘟病控制效果;同时,对拮抗菌进行产酶和次级代谢产物分析,检测其聚酮合酶基因(PKSⅡ型)和非核糖体多肽合成酶基因(NRPS)。【结果】从1 800个水稻样品中分离出117株内生放线菌,从中筛选出拮抗性能最好的菌株Osi Rt-1,鉴定为米修链霉菌Streptomyces misionensis。该菌株使稻瘟病菌菌丝出现畸形,其无菌滤液对病菌菌丝生长的抑制率为28.06%;大田条件下,Osi Rt-1对水稻苗瘟、穗瘟均有较好防效,其中对苗瘟防效为7.76%,穗瘟防效高达25.65%,损失率降低了17.46%。与之对应,Osi Rt-1处理过的水稻地上部分,Osi Rt-1所占内生放线菌的比例明显高于未处理水稻。该菌株具有可能降解真菌细胞壁的纤维素酶、蛋白酶活性,同时可产生植物生长促进剂铁载体、IAA、ACC脱氨酶。菌株Osi Rt-1呈现PKSⅡ型和NRPS阳性。【结论】菌株Osi Rt-1是一株具生防潜力的内生放线菌,在农业上具有实际研发价值。
[Objective] To isolate, screen and identify rice endophytic actinomyces with antagonistic activity against rice blast pathogen, to evaluate the disease control effect of the selected antagonist and to study its potential antagonistic mechanism. [Methods] We used 4 isolation media to isolate endophytic actinomycetes from surface sterilized rice. Using dual culture assay, we screened the strain with antagonistic activity and identified it by morphological, physiological and biochemical characteristics and 16 S rRNA gene sequence analysis. We observed the effect of the antagonist on the hyphae of pathogen under scanning electron microscopy and tested the in vitro antagonistic activity of fermentation broth by growth rate method. In field condition, we evaluated the disease control efficiency after spraying the antagonist spore solution on rice. Meanwhile, we analyzed enzymes and secondary metabolites production of the antagonist, and amplified its polyketide synthases(PKSⅡ) and nonribosomal peptide synthetases(NRPS) genes. [Results] From 1 800 rice samples, a total of 117 endophytic actinomycetes were isolated, among which, OsiRt-1 exhibited remarkable antagonistic activity. OsiRt-1 was identified as Streptomyces misionensis. The pathogen Magnaporthe oryzae treated by OsiRt-1 revealed significant morphological alterations in hyphe and its mycelial growth was inhibited by culture filtrates of OsiRt-1 with a percent of 28.06%. In field condition, OsiRt-1 spore solution could reduce seedling blast severity by 7.76%, panicle blast disease severity by 25.65% and lose rate by 17.46%. The separation rate of OsiRt-1 from above-ground portion of rice treated with OsiRt-1 is higher than the not treated ones. Besides, OsiRt-1 is positive for PKSⅡ and NRPS genes and can produce cellulase, protease, siderophore, IAA and ACC deaminase. [Conclusion] Endophytic Streptomyces misionensis OsiRt-1 has the application potential in agriculture as a biocontrol agent against rice blast disease.
[Objective] To isolate, screen and identify rice endophytic actinomyces with antagonistic activity against rice blast pathogen, to evaluate the disease control effect of the selected antagonist and to study its potential antagonistic mechanism. [Methods] We used 4 isolation media to isolate endophytic actinomycetes from surface sterilized rice. Using dual culture assay, we screened the strain with antagonistic activity and identified it by morphological, physiological and biochemical characteristics and 16 S rRNA gene sequence analysis. We observed the effect of the antagonist on the hyphae of pathogen under scanning electron microscopy and tested the in vitro antagonistic activity of fermentation broth by growth rate method. In field condition, we evaluated the disease control efficiency after spraying the antagonist spore solution on rice. Meanwhile, we analyzed enzymes and secondary metabolites production of the antagonist, and amplified its polyketide synthases(PKSⅡ) and nonribosomal peptide synthetases(NRPS) genes. [Results] From 1 800 rice samples, a total of 117 endophytic actinomycetes were isolated, among which, OsiRt-1 exhibited remarkable antagonistic activity. OsiRt-1 was identified as Streptomyces misionensis. The pathogen Magnaporthe oryzae treated by OsiRt-1 revealed significant morphological alterations in hyphe and its mycelial growth was inhibited by culture filtrates of OsiRt-1 with a percent of 28.06%. In field condition, OsiRt-1 spore solution could reduce seedling blast severity by 7.76%, panicle blast disease severity by 25.65% and lose rate by 17.46%. The separation rate of OsiRt-1 from above-ground portion of rice treated with OsiRt-1 is higher than the not treated ones. Besides, OsiRt-1 is positive for PKSⅡ and NRPS genes and can produce cellulase, protease, siderophore, IAA and ACC deaminase. [Conclusion] Endophytic Streptomyces misionensis OsiRt-1 has the application potential in agriculture as a biocontrol agent against rice blast disease.
引文
[1]Pooja K,Katoch A.Past,present and future of rice blast management[J].Plant Science Today,2014,1(3):165-173
[2]Kunova A,Pizzatti C,Cortesi P.Impact of tricyclazole and azoxystrobin on growth,sporulation and secondary infection of the rice blast fungus,Magnaporthe oryzae[J].Pest Management Science,2013,69(2):278-284
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[5]Hyakumachi M,Takahashi H,Matsubara Y,et al.Recent studies on biological control of plant diseases in Japan[J].Journal of General Plant Pathology,2014,80(4):287-302
[6]Boukaew S,Chuenchit S,Petcharat V.Evaluation of Streptomyces spp.for biological control of Sclerotium root and stem rot and Ralstonia wilt of chili pepper[J].Biocontrol,2011,56(3):365-374
[7]Chung WC,Wu RS,Hsu CP,et al.Application of antagonistic rhizobacteria for control of Fusarium seedling blight and basal rot of lily[J].Australasian Plant Pathology,2011,40(3):269-276
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[9]Kobayashi YO,Kobayashi A,Maeda M,et al.Biological control of potato scab and antibiosis by antagonistic Streptomyces sp.Wo Rs-501[J].Journal of General Plant Pathology,2015,81(6):439-448
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[11]Hardoim PR,van Overbeek LS,Berg G,et al.The hidden world within plants:ecological and evolutionary considerations for defining functioning of microbial endophytes[J].Microbiology and Molecular Biology Reviews,2015,79(3):293-320
[12]Reinhold-Hurek B,Hurek T.Living inside plants:bacterial endophytes[J].Current Opinion in Plant Biology,2011,14(4):435-443
[13]Li XL,Huang P,Wang Q,et al.Staurosporine from the endophytic Streptomyces sp.strain CNS-42 acts as a potential biocontrol agent and growth elicitor in cucumber[J].Antonie van Leeuwenhoek,2014,106(3):515-525
[14]Goudjal Y,Toumatia O,Yekkour A,et al.Biocontrol of Rhizoctonia solani damping-off and promotion of tomato plant growth by endophytic actinomycetes isolated from native plants of Algerian Sahara[J].Microbiological Research,2014,169(1):59-65
[15]El-Tarabily KA,Nassar AH,Hardy GSJ,et al.Plant growth promotion and biological control of Pythium aphanidermatum,a pathogen of cucumber,by endophytic actinomycetes[J].Journal of Applied Microbiology,2009,106(1):13-26
[16]Coombs JT,Franco CMM.Isolation and identification of actinobacteria from surface-sterilized wheat roots[J].Applied and Environmental Microbiology,2003,69(9):5603-5608
[17]Shirling EB,Gottlieb D.Methods for characterization of Streptomyces species[J].International Journal of Systematic and Evolutionary Microbiology,1966,16(3):313-340
[18]Xiong XQ,Liao HD,Ma JS,et al.Isolation of a rice endophytic bacterium,Pantoea sp.Sd-1,with ligninolytic activity and characterization of its rice straw degradation ability[J].Letters in Applied Microbiology,2014,58(2):123-129
[19]Kaewkla O,Franco CMM.Rational approaches to improving the isolation of endophytic actinobacteria from Australian native trees[J].Microbial Ecology,2013,65(2):384-393
[20]Williams ST,Goodfellow M,Alderson G,et al.Numerical classification of Streptomyces and related genera[J].Journal of General Microbiology,1983,129(6):1743-1813
[21]Agrawal T,Kotasthane AS.Chitinolytic assay of indigenous Trichoderma isolates collected from different geographical locations of Chhattisgarh in central India[J].Springer Plus,2012,1(1):73
[22]Gopalakrishnan S,Pande S,Sharma M,et al.Evaluation of actinomycete isolates obtained from herbal vermicompost for the biological control of Fusarium wilt of chickpea[J].Crop Protection,2011,30(8):1070-1078
[23]Penrose DM,Glick BR.Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria[J].Physiologia Plantarum,2003,118(1):10-15
[24]Nautiyal CS.An efficient microbiological growth medium for screening phosphate solubilizing microorganisms[J].FEMS Microbiology Letters,1999,170(1):265-270
[25]Ayuso-Sacido A,Genilloud O.New PCR primers for the screening of NRPS and PKS-I systems in actinomycetes:detection and distribution of these biosynthetic gene sequences in major taxonomic groups[J].Microbial Ecology,2005,49(1):10-24
[26]Cercos AP,Eilberg BL,Goyena JG,et al.Misionina:antibiotico polienico producido por Streptomyces misionensis n.sp.[J].Revista de Investigaciones Agricolas,1962,17:5-27
[27]Qin S,Li J,Chen HH,et al.Isolation,diversity,and antimicrobial activity of rare actinobacteria from medicinal plants of tropical rain forests in Xishuangbanna,China[J].Applied and Environmental Microbiology,2009,75(19):6176-6186
[28]Yuan WM,Crawford DL.Characterization of Streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots[J].Applied and Environmental Microbiology,1995,61(8):3119-3128
[29]Zarandi ME,Bonjar GS,Dehkaei FP,et al.Biological control of rice blast(Magnaporthe oryzae)by use of Streptomyces sindeneusis isolate 263 in greenhouse[J].American Journal of Applied Sciences,2009,6(1):194-199
[30]Zhang CN,Huang MJ,Mao N.The control effect of griseofulvin on Magnaporthe grisea[J].Chinese Agricultural Science Bulletin,2015,31(4):190-194(in Chinese)张传能,黄铭杰,毛宁.灰黄霉素对水稻稻瘟病菌的防治效果研究[J].中国农学通报,2015,31(4):190-194
[31]Prapagdee B,Kuekulvong C,Mongkolsuk S.Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus against phytopathogenic fungi[J].International Journal of Biological Sciences,2008,4(5):330-337
[32]Palaniyandi SA,Yang SH,Zhang LX,et al.Effects of actinobacteria on plant disease suppression and growth promotion[J].Applied Microbiology and Biotechnology,2013,97(22):9621-9636
[33]Shirling EB,Gottlieb D.Cooperative description of type cultures of Streptomyces.IV.Species descriptions from the second,third and fourth studies[J].International Journal of Systematic and Evolutionary Microbiology,1969,19(4):391-512
[34]Lu LM,Du DC,Cheng BP,et al.Isolation and identification of marine actinomyces A3202 and its control efficacy against postharvest citrus diseases[J].Acat Phytophylacica Sinica,2014,41(3):327-334(in Chinese)鹿连明,杜丹超,程保平,等.海洋放线菌A3202的分离鉴定及其对柑橘采后病害的防效[J].植物保护学报,2014,41(3):327-334
[2]Kunova A,Pizzatti C,Cortesi P.Impact of tricyclazole and azoxystrobin on growth,sporulation and secondary infection of the rice blast fungus,Magnaporthe oryzae[J].Pest Management Science,2013,69(2):278-284
[3]Chuma I,Isobe C,Hotta Y,et al.Multiple translocation of the AVR-Pita effector gene among chromosomes of the rice blast fungus Magnaporthe oryzae and related species[J].PLo S Pathogens,2011,7(7):e1002147
[4]Miah G,Rafii MY,Ismail MR,et al.Blast resistance in rice:a review of conventional breeding to molecular approaches[J].Molecular Biology Reports,2013,40(3):2369-2388
[5]Hyakumachi M,Takahashi H,Matsubara Y,et al.Recent studies on biological control of plant diseases in Japan[J].Journal of General Plant Pathology,2014,80(4):287-302
[6]Boukaew S,Chuenchit S,Petcharat V.Evaluation of Streptomyces spp.for biological control of Sclerotium root and stem rot and Ralstonia wilt of chili pepper[J].Biocontrol,2011,56(3):365-374
[7]Chung WC,Wu RS,Hsu CP,et al.Application of antagonistic rhizobacteria for control of Fusarium seedling blight and basal rot of lily[J].Australasian Plant Pathology,2011,40(3):269-276
[8]Van Hop D,Phuong Hoa PT,Quang ND,et al.Biological control of Xanthomonas oryzae pv.Oryzae causing rice bacterial blight disease by Streptomyces toxytricini VN08-A-12,isolated from soil and leaf-litter samples in Vietnam[J].Biocontrol Science,2014,19(3):103-111
[9]Kobayashi YO,Kobayashi A,Maeda M,et al.Biological control of potato scab and antibiosis by antagonistic Streptomyces sp.Wo Rs-501[J].Journal of General Plant Pathology,2015,81(6):439-448
[10]Li QL,Jiang YH,Ning P,et al.Suppression of Magnaporthe oryzae by culture filtrates of Streptomyces globisporus JK-1[J].Biological Control,2011,58(2):139-148
[11]Hardoim PR,van Overbeek LS,Berg G,et al.The hidden world within plants:ecological and evolutionary considerations for defining functioning of microbial endophytes[J].Microbiology and Molecular Biology Reviews,2015,79(3):293-320
[12]Reinhold-Hurek B,Hurek T.Living inside plants:bacterial endophytes[J].Current Opinion in Plant Biology,2011,14(4):435-443
[13]Li XL,Huang P,Wang Q,et al.Staurosporine from the endophytic Streptomyces sp.strain CNS-42 acts as a potential biocontrol agent and growth elicitor in cucumber[J].Antonie van Leeuwenhoek,2014,106(3):515-525
[14]Goudjal Y,Toumatia O,Yekkour A,et al.Biocontrol of Rhizoctonia solani damping-off and promotion of tomato plant growth by endophytic actinomycetes isolated from native plants of Algerian Sahara[J].Microbiological Research,2014,169(1):59-65
[15]El-Tarabily KA,Nassar AH,Hardy GSJ,et al.Plant growth promotion and biological control of Pythium aphanidermatum,a pathogen of cucumber,by endophytic actinomycetes[J].Journal of Applied Microbiology,2009,106(1):13-26
[16]Coombs JT,Franco CMM.Isolation and identification of actinobacteria from surface-sterilized wheat roots[J].Applied and Environmental Microbiology,2003,69(9):5603-5608
[17]Shirling EB,Gottlieb D.Methods for characterization of Streptomyces species[J].International Journal of Systematic and Evolutionary Microbiology,1966,16(3):313-340
[18]Xiong XQ,Liao HD,Ma JS,et al.Isolation of a rice endophytic bacterium,Pantoea sp.Sd-1,with ligninolytic activity and characterization of its rice straw degradation ability[J].Letters in Applied Microbiology,2014,58(2):123-129
[19]Kaewkla O,Franco CMM.Rational approaches to improving the isolation of endophytic actinobacteria from Australian native trees[J].Microbial Ecology,2013,65(2):384-393
[20]Williams ST,Goodfellow M,Alderson G,et al.Numerical classification of Streptomyces and related genera[J].Journal of General Microbiology,1983,129(6):1743-1813
[21]Agrawal T,Kotasthane AS.Chitinolytic assay of indigenous Trichoderma isolates collected from different geographical locations of Chhattisgarh in central India[J].Springer Plus,2012,1(1):73
[22]Gopalakrishnan S,Pande S,Sharma M,et al.Evaluation of actinomycete isolates obtained from herbal vermicompost for the biological control of Fusarium wilt of chickpea[J].Crop Protection,2011,30(8):1070-1078
[23]Penrose DM,Glick BR.Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria[J].Physiologia Plantarum,2003,118(1):10-15
[24]Nautiyal CS.An efficient microbiological growth medium for screening phosphate solubilizing microorganisms[J].FEMS Microbiology Letters,1999,170(1):265-270
[25]Ayuso-Sacido A,Genilloud O.New PCR primers for the screening of NRPS and PKS-I systems in actinomycetes:detection and distribution of these biosynthetic gene sequences in major taxonomic groups[J].Microbial Ecology,2005,49(1):10-24
[26]Cercos AP,Eilberg BL,Goyena JG,et al.Misionina:antibiotico polienico producido por Streptomyces misionensis n.sp.[J].Revista de Investigaciones Agricolas,1962,17:5-27
[27]Qin S,Li J,Chen HH,et al.Isolation,diversity,and antimicrobial activity of rare actinobacteria from medicinal plants of tropical rain forests in Xishuangbanna,China[J].Applied and Environmental Microbiology,2009,75(19):6176-6186
[28]Yuan WM,Crawford DL.Characterization of Streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots[J].Applied and Environmental Microbiology,1995,61(8):3119-3128
[29]Zarandi ME,Bonjar GS,Dehkaei FP,et al.Biological control of rice blast(Magnaporthe oryzae)by use of Streptomyces sindeneusis isolate 263 in greenhouse[J].American Journal of Applied Sciences,2009,6(1):194-199
[30]Zhang CN,Huang MJ,Mao N.The control effect of griseofulvin on Magnaporthe grisea[J].Chinese Agricultural Science Bulletin,2015,31(4):190-194(in Chinese)张传能,黄铭杰,毛宁.灰黄霉素对水稻稻瘟病菌的防治效果研究[J].中国农学通报,2015,31(4):190-194
[31]Prapagdee B,Kuekulvong C,Mongkolsuk S.Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus against phytopathogenic fungi[J].International Journal of Biological Sciences,2008,4(5):330-337
[32]Palaniyandi SA,Yang SH,Zhang LX,et al.Effects of actinobacteria on plant disease suppression and growth promotion[J].Applied Microbiology and Biotechnology,2013,97(22):9621-9636
[33]Shirling EB,Gottlieb D.Cooperative description of type cultures of Streptomyces.IV.Species descriptions from the second,third and fourth studies[J].International Journal of Systematic and Evolutionary Microbiology,1969,19(4):391-512
[34]Lu LM,Du DC,Cheng BP,et al.Isolation and identification of marine actinomyces A3202 and its control efficacy against postharvest citrus diseases[J].Acat Phytophylacica Sinica,2014,41(3):327-334(in Chinese)鹿连明,杜丹超,程保平,等.海洋放线菌A3202的分离鉴定及其对柑橘采后病害的防效[J].植物保护学报,2014,41(3):327-334