枯草芽胞杆菌WY8-7的溶磷、抑菌及促生长作用
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摘要
[目的]本文旨在筛选兼有溶磷和抑菌作用的微生物菌株,分析其在水体和土壤中的溶磷效果以及对植物生长的作用,同时分析其抑菌活性,鉴定其抑菌物质。[方法]采用稀释涂平板法,从茶园土壤中分离具有溶磷和抑菌作用的菌株WY8-7,通过形态学、生理生化和分子生物学分析,确定菌株WY8-7的分类地位;检测菌株WY8-7在土壤和培养液中对难溶性无机磷的降解作用;检测其对苗期玉米植株生长的影响;对峙培养法分析菌株WY8-7对不同病原真菌的抑菌作用;超高效液相色谱四级杆飞行时间质谱鉴定抑菌功能物质。[结果]经鉴定,菌株WY8-7为枯草芽胞杆菌(Bacillus subtilis),在固体培养基、液体培养基和土壤中均可转化难溶性无机磷,提升可溶性磷含量;液体振荡培养20 d后,可溶性磷含量达512.77 mg·L~(-1),为对照的174倍;菌株WY8-7可提升土壤中可溶性磷含量,并促进苗期玉米的生长,与对照组相比,在叶长、叶宽、单叶叶面积、株高和鲜质量等指标均有显著增长(P<0.05),增长率分别达17.68%、22.08%、42.62%、20.34%和20.59%;质谱分析表明,菌株WY8-7可产生伊枯草菌素A和丰原素A两类抑菌物质,对3种真菌具有高效广谱抑菌作用,对禾谷炭疽菌的抑菌率最高,达87.34%。[结论]分离自茶园的枯草芽胞杆菌WY8-7,在土壤和液体振荡培养中可高效转化难溶性无机磷为可溶性磷,促进苗期玉米植株生长,同时WY8-7还可产生伊枯草菌素A和丰原素A两种脂肽类物质,抑制多种病原真菌生长。WY8-7具有高效溶磷和抑菌作用,为新型微生物菌肥的研发提供重要材料。
[Objectives]The purpose of our study was screening microorganism with phosphate solubilizing(P-solubilizing)and antifungal activity,analyzing the P-solubilizing,antifungal and plant growth promoting effect,and identifying the primary antifungal compounds. [Methods]Strain WY8-7 with P-solubilizing and antifungal activity was isolated from tea rhizosphere soil through gradient dilution method. The isolated bacteria WY8-7 was identified through morphology,biochemistry and molecular methods. WY8-7 was inoculated to liquid medium and soil to analyze the P-solubilizing activity,and co-cultured with maize seedling to detect the plant growth promoting activity. Additionally,WY8-7 was co-cultured with different fungi to test the broad antifungal activity,and the effective compounds were identified through ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q/TOF-MS). [Results]Strain WY8-7 was identified to be Bacillus subtilis through morphology,biochemical character and molecular methods. It could dissolve phosphate in solid and liquid medium,as well as in soil conditions. In liquid medium,the concentration of dissolved phosphate was up to 512.77 mg·L~(-1),which was 174 times to the control treatment in 20 d. In soil,strain WY8-7 could increase the concentration of soluble phosphate,and promote the growth of maize seedlings in the aspect of leaf length,leaf width,single leaf area,plant height,fresh weight. The increased rate was 17.68%,22.08%,42.62%,20.34% and 20.59% compared to the control treatment with significant differences(P<0.05),respectively. Moreover,WY8-7 could also inhibit the growth of three different fungal pathogens in dual cultural tests by the production of two kinds of lipopeptides. WY8-7 showed better antifungal activity to Colletotrichum graminicola than other two strains,and the inhibitory rate was 87.34%. The lipopeptides were identified to be iturin A and fengycin A through UPLC-Q/TOF-MS methods. [Conclusions]Bacillus subtilis WY8-7 isolated from tea garden could dissolve phosphate into soluble in liquid medium and soil conditions,and promote the growth of maize seedling. Additionally,strain WY8-7 could produce iturin A and fengycin A with broad antifungal activity in dual cultural tests. Hence,strain WY8-7 with effective P-solubilizing and antifungal activity will provide important materials for the application of biofertilizer in future.
[Objectives]The purpose of our study was screening microorganism with phosphate solubilizing(P-solubilizing)and antifungal activity,analyzing the P-solubilizing,antifungal and plant growth promoting effect,and identifying the primary antifungal compounds. [Methods]Strain WY8-7 with P-solubilizing and antifungal activity was isolated from tea rhizosphere soil through gradient dilution method. The isolated bacteria WY8-7 was identified through morphology,biochemistry and molecular methods. WY8-7 was inoculated to liquid medium and soil to analyze the P-solubilizing activity,and co-cultured with maize seedling to detect the plant growth promoting activity. Additionally,WY8-7 was co-cultured with different fungi to test the broad antifungal activity,and the effective compounds were identified through ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q/TOF-MS). [Results]Strain WY8-7 was identified to be Bacillus subtilis through morphology,biochemical character and molecular methods. It could dissolve phosphate in solid and liquid medium,as well as in soil conditions. In liquid medium,the concentration of dissolved phosphate was up to 512.77 mg·L~(-1),which was 174 times to the control treatment in 20 d. In soil,strain WY8-7 could increase the concentration of soluble phosphate,and promote the growth of maize seedlings in the aspect of leaf length,leaf width,single leaf area,plant height,fresh weight. The increased rate was 17.68%,22.08%,42.62%,20.34% and 20.59% compared to the control treatment with significant differences(P<0.05),respectively. Moreover,WY8-7 could also inhibit the growth of three different fungal pathogens in dual cultural tests by the production of two kinds of lipopeptides. WY8-7 showed better antifungal activity to Colletotrichum graminicola than other two strains,and the inhibitory rate was 87.34%. The lipopeptides were identified to be iturin A and fengycin A through UPLC-Q/TOF-MS methods. [Conclusions]Bacillus subtilis WY8-7 isolated from tea garden could dissolve phosphate into soluble in liquid medium and soil conditions,and promote the growth of maize seedling. Additionally,strain WY8-7 could produce iturin A and fengycin A with broad antifungal activity in dual cultural tests. Hence,strain WY8-7 with effective P-solubilizing and antifungal activity will provide important materials for the application of biofertilizer in future.
引文
[1] 车升国.区域作物专用复合(混)肥料配方制定方法与应用[D].北京:中国农业大学,2015.Che S G.Design method and application of formula of regional crop-based compound fertilizer[D].Beijing:China Agricultural University,2015(in Chinese with English abstract).
[2] 鲍朋,许章峰.巨大芽胞杆菌在生物肥料上的研究现状与发展方向[J].农技服务,2013,30(6):601-602.Bao P,Xu Z F.Research status and prospect of giant bacillus on biological fertilizer[J].Agricultural Extension Service,2013,30(6):601-602(in Chinese).
[3] 张炳火,李汉全,罗娟艳,等.放线菌JXJ-0136对白菜和豇豆生长的影响及其解磷作用[J].中国农业科学,2016,49(16):3152-3161.Zhang B H,Li H Q,Luo J Y,et al.Influences of Actinomycete strain JXJ-0136 on the growth of Brassica chinensis and Vigna unguiculata and its phosphate solubilization[J].Scientia Agriculture Sinica,2016,49(16):3152-3161(in Chinese with English abstract).
[4] Tian D,Wang W C,Su M,et al.Remediation of lead-contaminated water by geological fluorapatite and fungus Penicillium oxalicum[J].Environmental Science and Pollution Research,2018,25(21):21118-21126.
[5] Gaind S,Nain L.Soil-Phosphorus mobilization potential of phytate mineralizing fungi[J].Journal of Plant Nutrition,2015,38(14):2159-2175.
[6] Nevita T,Sharma G D,Pandey P.Composting of rice-residues using lignocellulolytic plant-probiotic Stenotrophomonas maltophilia and its evaluation for growth enhancement of Oryza sativa L.[J].Environmental Sustainability,2018,1(2):185-196.
[7] 银婷婷,王敬敬,柳影,等.高效解磷菌的筛选及其促生机制的初步研究[J].生物技术通报,2015,31(12):234-242.Yin T T,Wang J J,Liu Y,et al.The screening of efficient phosphorus-solubilizing bacteria and the primary study on its mechanism of plant-growth-promoting[J].Biotechnology Bulletin,2015,31(12):234-242(in Chinese with English abstract).
[8] Ludueňa L M,Anzuay M S,Magallanes-Noguera C,et al.Effects of P limitation and molecules from peanut root exudates on pqqE gene expression and pqq promoter activity in the phosphate-solubilizing strain Serratia sp.S119[J].Research in Microbiology,2017,168(8):710-721.
[9] Cerozi B D S,Fitzsimmons K.Use of Bacillus spp.to enhance phosphorus availability and serve as a plant growth promoter in aquaponics systems[J].Scientia Horticulturae,2016,211:277-282.
[10] 黄伟,俞新玲,林勇明,等.两株球形赖氨酸芽胞杆菌对巨桉幼苗生长及光合特性的影响[J].应用与环境生物学报,2016,22(5):839-844.Huang W,Yu X L,Lin Y M,et al.Effects of two strains of Lysinibacillus sphaericus on growth and photosynthetic characteristics of Eucalyptus grandis seedlings[J].Chinese Journal of Applied & Environmental Biology,2016,22(5):839-844(in Chinese with English abstract).
[11] 邢芳芳,高明夫,禚优优,等.玉米根际高效溶磷菌的筛选、鉴定及溶磷特性研究[J].中国农学通报,2016,32(9):119-124.Xing F F,Gao M F,Zhuo Y Y,et al.Screening and identification of phosphate solubilizing bacteria in maize rhizosphere and their characteristics of phosphate solubilizing[J].Chinese Agricultural Science Bulletin,2016,32(9):119-124(in Chinese with English abstract).
[12] 张爱民.解磷解钾特异菌株 CX-7 的筛选及其应用试验研究[D].保定:河北农业大学,2014.Zhang A M.Screening of the specific solubilizing phosphate and poassium CX-7 strain and research on its applying experiment[D].Baoding:Hebei Agricultural University,2014(in Chinese with English abstract).
[13] 代志,高俊明.兼具解磷解钾功能生防菌分离鉴定及效果评价[J].山西农业科学,2018,46(4):627-633.Dai Z,Gao J M.Isolation,identification and effect evaluation of bio-control bacteria with phosphorus and potassium dissolving function[J].Journal of Shanxi Agricultural Sciences,2018,46(4):627-633.
[14] Wang L T,Lee F L,Tai C J,et al.Comparison of gyrB gene sequences,16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group[J].International Journal of Systematic and Evolutionary Microbiology,2007,57(8):1846-1850.
[15] 李晓娜,刘尚义,张国芳.一种基于连续流动分析仪的土壤全磷检测方法:201710100449.9[P].2017-02-23.Li X N,Liu S Y,Zhang G F.A soil total phosphorus detection method based on continuous flow analyzer:201710100449.9[P].2017-02-23(in Chinese).
[16] Schneider K D,Voroney R P,Lynch D H,et al.Microbially-mediated P fluxes in calcareous soils as a function of water-extractable phosphate[J].Soil Biology & Biochemistry,2017,106:51-60.
[17] Gong A D,Li H P,Yuan Q S,et al.Antagonistic mechanism of iturin A and plipastatin A from Bacillus amyloliquefaciens S76-3 from wheat spikes against Fusarium graminearum[J].PLoS One,2015,10(2):e0116871.
[18] 张云霞,雷鹏,许宗奇,等.一株高效解磷菌Bacillus subtilis JT-1 的筛选及其对土壤微生态和小麦生长的影响[J].江苏农业学报,2016,32(5):1073-1080.Zhang Y X,Lei P,Xu Z Q,et al.Screening of a high-efficiency phosphate solubilizing bacterium Bacillus subtilis JT-1 and its effects on soil microecology and wheat growth[J].Jiangsu Journal of Agricultural Sciences,2016,32(5):1073-1080(in Chinese with English abstract).
[19] 余贤美,王义,沈奇宾,等.解磷细菌 PSB3 的筛选及拮抗作用的研究[J].微生物学通报,2008,35(9):1398-1403.Yu X M,Wang Y,Shen Q B,et al.The screening of phosphorus solubilizing bacteria PSB3 and the study of its antagonism[J].Microbiology,2008,35(9):1398-1403(in Chinese with English abstract).
[20] 戴沈艳,申卫收,贺云举,等.一株高效解磷细菌的筛选及其在红壤性水稻土中的施用效果[J].应用与环境生物学报,2011,17(5):678-683.Dai S Y,Shen W S,He Y J,et al.Screening of efficient phosphate-solubilizing bacterial strain and its application in red paddy soil to rice cultivation[J].Chinese Journal of Applied & Environmental Biology,2011,17(5):678-683(in Chinese with English abstract).
[21] 王琰.解磷芽胞杆菌的筛选鉴定及其对玉米促生机理的研究[D].广州:华南农业大学,2016.Wang Y.Study on isolation of phosphate-solubilizing Bacillus and their impact of growth-promoting for maize[D].Guangzhou:South China Agricultural University,2016(in Chinese with English abstract).
[22] Vimal S R,Sigh J S,Arora N K,et al.Soil-plant-microbe interactions in stressed agriculture management:a review[J].Pedosphere,2017,27(2):177-192.
[23] Ongena M,Jourdan E,Adam A,et al.Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants[J].Environmental Microbiology,2007,9(4):1084-1090.
[24] Saechow S,Thammasittirong A,Kittakoop P,et al.Antagonistic activity against dirty panicle rice fungal pathogens and plant growth-promoting activity of Bacillus amyloliquefaciens BAS23[J].Journal of Microbiology and Biotechnology,2018,28(9):1527-1535.
[25] Rosier A,Medeiros F H V,Bais H P.Defining plant growth promoting rhizobacteria molecular and biochemical networks in beneficial plant-microbe interactions[J].Plant and Soil,2018:,428(1/2):35-55.
[26] Kloepper J W,Ryu C M,Zhang S A.Induced systemic resistance and promotion of plant growth by Bacillus spp.[J].Phytopathology,2004,94(11):1259-1266.
[2] 鲍朋,许章峰.巨大芽胞杆菌在生物肥料上的研究现状与发展方向[J].农技服务,2013,30(6):601-602.Bao P,Xu Z F.Research status and prospect of giant bacillus on biological fertilizer[J].Agricultural Extension Service,2013,30(6):601-602(in Chinese).
[3] 张炳火,李汉全,罗娟艳,等.放线菌JXJ-0136对白菜和豇豆生长的影响及其解磷作用[J].中国农业科学,2016,49(16):3152-3161.Zhang B H,Li H Q,Luo J Y,et al.Influences of Actinomycete strain JXJ-0136 on the growth of Brassica chinensis and Vigna unguiculata and its phosphate solubilization[J].Scientia Agriculture Sinica,2016,49(16):3152-3161(in Chinese with English abstract).
[4] Tian D,Wang W C,Su M,et al.Remediation of lead-contaminated water by geological fluorapatite and fungus Penicillium oxalicum[J].Environmental Science and Pollution Research,2018,25(21):21118-21126.
[5] Gaind S,Nain L.Soil-Phosphorus mobilization potential of phytate mineralizing fungi[J].Journal of Plant Nutrition,2015,38(14):2159-2175.
[6] Nevita T,Sharma G D,Pandey P.Composting of rice-residues using lignocellulolytic plant-probiotic Stenotrophomonas maltophilia and its evaluation for growth enhancement of Oryza sativa L.[J].Environmental Sustainability,2018,1(2):185-196.
[7] 银婷婷,王敬敬,柳影,等.高效解磷菌的筛选及其促生机制的初步研究[J].生物技术通报,2015,31(12):234-242.Yin T T,Wang J J,Liu Y,et al.The screening of efficient phosphorus-solubilizing bacteria and the primary study on its mechanism of plant-growth-promoting[J].Biotechnology Bulletin,2015,31(12):234-242(in Chinese with English abstract).
[8] Ludueňa L M,Anzuay M S,Magallanes-Noguera C,et al.Effects of P limitation and molecules from peanut root exudates on pqqE gene expression and pqq promoter activity in the phosphate-solubilizing strain Serratia sp.S119[J].Research in Microbiology,2017,168(8):710-721.
[9] Cerozi B D S,Fitzsimmons K.Use of Bacillus spp.to enhance phosphorus availability and serve as a plant growth promoter in aquaponics systems[J].Scientia Horticulturae,2016,211:277-282.
[10] 黄伟,俞新玲,林勇明,等.两株球形赖氨酸芽胞杆菌对巨桉幼苗生长及光合特性的影响[J].应用与环境生物学报,2016,22(5):839-844.Huang W,Yu X L,Lin Y M,et al.Effects of two strains of Lysinibacillus sphaericus on growth and photosynthetic characteristics of Eucalyptus grandis seedlings[J].Chinese Journal of Applied & Environmental Biology,2016,22(5):839-844(in Chinese with English abstract).
[11] 邢芳芳,高明夫,禚优优,等.玉米根际高效溶磷菌的筛选、鉴定及溶磷特性研究[J].中国农学通报,2016,32(9):119-124.Xing F F,Gao M F,Zhuo Y Y,et al.Screening and identification of phosphate solubilizing bacteria in maize rhizosphere and their characteristics of phosphate solubilizing[J].Chinese Agricultural Science Bulletin,2016,32(9):119-124(in Chinese with English abstract).
[12] 张爱民.解磷解钾特异菌株 CX-7 的筛选及其应用试验研究[D].保定:河北农业大学,2014.Zhang A M.Screening of the specific solubilizing phosphate and poassium CX-7 strain and research on its applying experiment[D].Baoding:Hebei Agricultural University,2014(in Chinese with English abstract).
[13] 代志,高俊明.兼具解磷解钾功能生防菌分离鉴定及效果评价[J].山西农业科学,2018,46(4):627-633.Dai Z,Gao J M.Isolation,identification and effect evaluation of bio-control bacteria with phosphorus and potassium dissolving function[J].Journal of Shanxi Agricultural Sciences,2018,46(4):627-633.
[14] Wang L T,Lee F L,Tai C J,et al.Comparison of gyrB gene sequences,16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group[J].International Journal of Systematic and Evolutionary Microbiology,2007,57(8):1846-1850.
[15] 李晓娜,刘尚义,张国芳.一种基于连续流动分析仪的土壤全磷检测方法:201710100449.9[P].2017-02-23.Li X N,Liu S Y,Zhang G F.A soil total phosphorus detection method based on continuous flow analyzer:201710100449.9[P].2017-02-23(in Chinese).
[16] Schneider K D,Voroney R P,Lynch D H,et al.Microbially-mediated P fluxes in calcareous soils as a function of water-extractable phosphate[J].Soil Biology & Biochemistry,2017,106:51-60.
[17] Gong A D,Li H P,Yuan Q S,et al.Antagonistic mechanism of iturin A and plipastatin A from Bacillus amyloliquefaciens S76-3 from wheat spikes against Fusarium graminearum[J].PLoS One,2015,10(2):e0116871.
[18] 张云霞,雷鹏,许宗奇,等.一株高效解磷菌Bacillus subtilis JT-1 的筛选及其对土壤微生态和小麦生长的影响[J].江苏农业学报,2016,32(5):1073-1080.Zhang Y X,Lei P,Xu Z Q,et al.Screening of a high-efficiency phosphate solubilizing bacterium Bacillus subtilis JT-1 and its effects on soil microecology and wheat growth[J].Jiangsu Journal of Agricultural Sciences,2016,32(5):1073-1080(in Chinese with English abstract).
[19] 余贤美,王义,沈奇宾,等.解磷细菌 PSB3 的筛选及拮抗作用的研究[J].微生物学通报,2008,35(9):1398-1403.Yu X M,Wang Y,Shen Q B,et al.The screening of phosphorus solubilizing bacteria PSB3 and the study of its antagonism[J].Microbiology,2008,35(9):1398-1403(in Chinese with English abstract).
[20] 戴沈艳,申卫收,贺云举,等.一株高效解磷细菌的筛选及其在红壤性水稻土中的施用效果[J].应用与环境生物学报,2011,17(5):678-683.Dai S Y,Shen W S,He Y J,et al.Screening of efficient phosphate-solubilizing bacterial strain and its application in red paddy soil to rice cultivation[J].Chinese Journal of Applied & Environmental Biology,2011,17(5):678-683(in Chinese with English abstract).
[21] 王琰.解磷芽胞杆菌的筛选鉴定及其对玉米促生机理的研究[D].广州:华南农业大学,2016.Wang Y.Study on isolation of phosphate-solubilizing Bacillus and their impact of growth-promoting for maize[D].Guangzhou:South China Agricultural University,2016(in Chinese with English abstract).
[22] Vimal S R,Sigh J S,Arora N K,et al.Soil-plant-microbe interactions in stressed agriculture management:a review[J].Pedosphere,2017,27(2):177-192.
[23] Ongena M,Jourdan E,Adam A,et al.Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants[J].Environmental Microbiology,2007,9(4):1084-1090.
[24] Saechow S,Thammasittirong A,Kittakoop P,et al.Antagonistic activity against dirty panicle rice fungal pathogens and plant growth-promoting activity of Bacillus amyloliquefaciens BAS23[J].Journal of Microbiology and Biotechnology,2018,28(9):1527-1535.
[25] Rosier A,Medeiros F H V,Bais H P.Defining plant growth promoting rhizobacteria molecular and biochemical networks in beneficial plant-microbe interactions[J].Plant and Soil,2018:,428(1/2):35-55.
[26] Kloepper J W,Ryu C M,Zhang S A.Induced systemic resistance and promotion of plant growth by Bacillus spp.[J].Phytopathology,2004,94(11):1259-1266.