荧光假单胞菌ZX对采后锦橙绿霉病的防治及其抑菌机制
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摘要
【目的】采后柑橘极易受指状青霉(Penicillium digitatum)侵染而发生严重的绿霉病腐烂,生物防治因具有安全、有效、环保等特点近年来备受关注。论文旨在研究荧光假单胞菌(Pseudomonas fluorescens)ZX对采后柑橘绿霉病的防治效果,揭示P.fluorescensZX对P.digitatum可能存在的作用机制。【方法】以"北碚447"锦橙果实为试材,先分别接种20μL拮抗菌培养液、滤液(培养液离心后,上清经0.22μm滤膜过滤)、菌悬液(培养液离心后,菌体用无菌水反复洗涤并用无菌水重悬)和热杀死液(培养液高温高压灭菌),2 h后接种20μL P. digitatum孢子悬浮液(1×10~4spores/m L),所有果实于20oC、90%相对湿度环境下恒温恒湿培养8 d后,测定果实的发病率和病斑直径;制备柑橘皮培养基,进行平板抑菌试验,探索P. fluorescens ZX对P. digitatum孢子发芽情况的影响;采用两板对扣法和生物熏蒸法研究P.fluorescensZX挥发性次级代谢产物的抑菌作用;利用插入式细胞培养皿等分析P.fluorescensZX和P.digitatum之间竞争的营养物质;同时,测定P.fluorescensZX的生长曲线,利用结晶紫染色法评估P. fluorescens ZX的生物膜形成能力。【结果】P. fluorescens ZX不同处理液之间对采后锦橙绿霉病的作用效果差异显著,菌悬液抑菌效果最好,经菌悬液处理的果实,发病率和病斑直径分别仅为40.83%和1.78 cm;不论是在柑橘皮固体培养基上对峙培养还是在液体培养基中混合培养,菌悬液和原液的作用效果较好,固体平板上,相对抑制率达到了35%–45%,液体培养基中,P. digitatum孢子12 h后的发芽率不超过27%;P. fluorescens ZX产生的挥发性物质具有抑菌作用,经P. fluorescensZX熏蒸处理的锦橙果实,发病率和病斑直径都显著降低;营养竞争试验结果表明,P. fluorescens ZX能更快速有效地消耗柑橘皮培养基中的营养,并和P. digitatum竞争葡萄糖、果糖、蔗糖、天冬氨酸、苏氨酸、丝氨酸、亮氨酸、精氨酸和脯氨酸等营养物质;同时,P. fluorescens ZX生命力强,培养4 h后即进入对数生长期,约24 h后形成成熟的生物膜。【结论】P. fluorescens ZX可能通过抑制P. digitatum孢子发芽、营养与空间竞争、形成生物膜、产生抑菌物质等方式抑制P.digitatum的生长繁殖,有效防治采后锦橙绿霉病。
[Objective] Researches were carried out for biocontrol of green mold decay in citrus by Pseudomonas fluorescens ZX. [Methods] The biocontrol effectiveness in fruits and effects on Penicillium digitatum growth were evaluated along with the antifungal activity of volatile organic compounds(VOCs). Meanwhile, assays of competitive grow between Pseudomonas fluorescens ZX and Penicillium digitatum were conducted, as well as the growth curve and biofilm formation ability of Pseudomonas fluorescens ZX. [Results] Pseudomonas fluorescens ZX suspension showed potent ability for the depression of Penicillium digitatum, so did the VOCs. In addition, Pseudomonas fluorescens ZX made better use of nutrients than Penicillium digitatum, and it entered the logarithmic growth stage quickly(4 h) and formed an effective biofilm after 24 h. [Conclusion] The application of Pseudomonas fluorescens ZX was effective to control green mold, and the potential mechanisms included inhibition of spore germination, competition for nutrients and space, and formation of biofilm.
[Objective] Researches were carried out for biocontrol of green mold decay in citrus by Pseudomonas fluorescens ZX. [Methods] The biocontrol effectiveness in fruits and effects on Penicillium digitatum growth were evaluated along with the antifungal activity of volatile organic compounds(VOCs). Meanwhile, assays of competitive grow between Pseudomonas fluorescens ZX and Penicillium digitatum were conducted, as well as the growth curve and biofilm formation ability of Pseudomonas fluorescens ZX. [Results] Pseudomonas fluorescens ZX suspension showed potent ability for the depression of Penicillium digitatum, so did the VOCs. In addition, Pseudomonas fluorescens ZX made better use of nutrients than Penicillium digitatum, and it entered the logarithmic growth stage quickly(4 h) and formed an effective biofilm after 24 h. [Conclusion] The application of Pseudomonas fluorescens ZX was effective to control green mold, and the potential mechanisms included inhibition of spore germination, competition for nutrients and space, and formation of biofilm.
引文
[1]邓秀新,彭抒昂.柑橘学.北京:中国农业出版社,2013.
[2]Ballester AR,Lafuente MT,Forment J,Gadea J,de Vos RCH,Bovy AG,González-Candelas L.Transcriptomic profiling of citrus fruit peel tissues reveals fundamental effects of phenylpropanoids and ethylene on induced resistance.Molecular Plant Pathology,2011,12(9):879-897,doi:10.1111/j.1364-3703.2011.00721.x.
[3]路来风.海洋拮抗酵母Rhodosporidium paludigenum对柑橘果实抗性的增强效应及其生物学机理研究.浙江大学博士学位论文,2015.
[4]毕阳.果蔬采后病害:原理与控制.北京:科学出版社,2016.
[5]Sánchez-Torres P,Tuset JJ.Molecular insights into fungicide resistance in sensitive and resistant Penicillium digitatum strains infecting citrus.Postharvest Biology and Technology,2011,59(2):159-165,doi:10.1016/j.postharvbio.2010.08.017.
[6]Wallace RL.Biological control of common postharvest diseases of apples with Pseudomonas fluorescens and potential modes of action.Doctor Dissertation of The University of British Columbia,2018.
[7]王一非.海洋拮抗酵母Rhodosporidium paludigenum对果实采后病害生物防治的研究.浙江大学博士学位论文,2008.
[8]Sun GZ,Yao T,Zhao GQ,Lu H,Ma WB.Research progress and prospects for controlling plant diseases using Pseudomonas fluorescens.Acta Prataculturae Sinica,2015,24(4):174-190,doi:10.11686/cyxb20150421.(in Chinese)孙广正,姚拓,赵桂琴,卢虎,马文彬.荧光假单胞菌防治植物病害研究现状与展望.草业学报,2015,24(4):174-190,doi:10.11686/cyxb20150421.
[9]El-Gamal NG,Shehata AN,Hamed ER,Shehata HS.Improvement of lytic enzymes producing Pseudomonas fluorescens and Bacillus subtilis isolates for enhancing their biocontrol potential against root rot disease in tomato plants.Research Journal of Pharmaceutical,Biological and Chemical Sciences,2016,7(1):1393-1400.
[10]Gro?kinsky DK,Tafner R,Moreno MV,Stenglein SA,de Salamone IEG,Nelson LM,Novák O,Strnad M,van der Graaff E,Roitsch T.Cytokinin production by Pseudomonas fluorescens G20-18 determines biocontrol activity against Pseudomonas syringae in Arabidopsis.Scientific Reports,2016,6:23310,doi:10.1038/srep23310.
[11]Prabhukarthikeyan SR,Keerthana U,Raguchander T.Antibiotic-producing Pseudomonas fluorescens mediates rhizome rot disease resistance and promotes plant growth in turmeric plants.Microbiological Research,2018,210:65-73.
[12]Ilhan K,Karabulut OA.Efficacy and population monitoring of bacterial antagonists for gray mold(Botrytis cinerea Pers.ex.Fr.)infecting strawberries.Bio Control,2013,58(4):457-470,doi:10.1007/s10526-012-9503-x.
[13]Ghazanfar MU,Hussain M,Hamid MI,Ansari SU.Utilization of biological control agents for the management of postharvest pathogens of tomato.Pakistan Journal of Botany,2016,48(5):2093-2100.
[14]Mostafavi HA,Mirmajlessi SM,Fathollahi H,Shahbazi S,Mirjalili SM.Integrated effect of gamma radiation and biocontrol agent on quality parameters of apple fruit:An innovative commercial preservation method.Radiation Physics and Chemistry,2013,91:193-199.
[15]Peeran MF,Krishnan N,Thangamani PR,Gandhi K,Thiruvengadam R,Kuppusamy P.Development and evaluation of water-in-oil formulation of Pseudomonas fluorescens(FP7)against Colletotrichum musae incitant of anthracnose disease in banana.European Journal of Plant Pathology,2014,138(1):167-180,doi:10.1007/s10658-013-0320-6.
[16]Wang ZR,Jiang MY,Chen KW,Wang KT,Du MY,Zalán Z,Hegyi F,Kan JQ.Biocontrol of Penicillium digitatum on postharvest citrus fruits by Pseudomonas fluorescens.Journal of Food Quality,2018,2018:2910481.
[17]Wang ZR,Jiang MY,Du MY,Wang KT,Zalán Z,Hegyi F,Kan JQ.Investigation on effects of treatments with Pseudomonas fluorescens on controlling blue mold caused by Penicillium italicum and storage quality in Jincheng citrus fruits.Food and Fermentation Industries,2018,44(11):176-184,doi:10.13995/j.cnki.11-1802/ts.017668.(in Chinese)王智荣,江孟遥,杜木英,汪开拓,Zalán Z,Hegyi F,阚建全.荧光假单胞菌对采后锦橙青霉病的防治和贮藏品质的影响.食品与发酵工业,2018,44(11):176-184,doi:10.13995/j.cnki.11-1802/ts.017668.
[18]王友升.拮抗酵母菌与果蔬采后病害防治.北京:知识产权出版社,2012.
[19]Raza W,Ling N,Liu DY,Wei Z,Huang QW,Shen QR.Volatile organic compounds produced by Pseudomonas fluorescens WR-1 restrict the growth and virulence traits of Ralstonia solanacearum.Microbiological Research,2016,192:103-113.
[20]Janisiewicz WJ,Tworkoski TJ,Sharer C.Characterizing the mechanism of biological control of postharvest diseases on fruits with a simple method to study competition for nutrients.Biological Control,2000,90(11):1196-1200,doi:10.1094/PHYTO.2000.90.11.1196.
[21]罗丽.柠檬形克勒克酵母(34-9)对柑橘意大利青霉抑菌机理的研究.华中农业大学硕士学位论文,2010.
[22]周雅涵.水杨酸、膜醭毕赤酵母、壳寡糖诱导柑橘果实抗病性及其生物学机制研究.西南大学博士学位论文,2017.
[23]Fu Q,Cheng LK,Miao LZ,Wang Y,He C,Shen ZQ.Determination of growth curve and screening of medium for A type Naso trachea.China Poultry,2017,39(11):60-63.(in Chinese)付强,程立坤,苗立中,王艳,何诚,沈志强.A型鼻气管鸟杆菌生长曲线的测定和培养基筛选.中国家禽,2017,39(11):60-63.
[24]刘正强.利用核糖体工程技术提高荧光假单胞菌Pf-5菌株活性次级代谢产物产量.湖南师范大学硕士学位论文,2016.
[25]Wei HL,Zhang LQ.Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24.Antonie van Leeuwenhoek,2006,89(2):267-280,doi:10.1007/s10482-005-9028-8.
[26]Sahu B,Singh J,Shankar G,Pradhan A.Pseudomonas fluorescens PGPR bacteria as well as biocontrol agent:Areview.International Journal of Chemical Studies,2018,6(2):1-7.
[27]Arseneault T,Goyer C,Filion M.Biocontrol of potato common scab is associated with high Pseudomonas fluorescens LBUM223 populations and phenazine-1-carboxylic acid biosynthetic transcript accumulation in the potato geocaulosphere.Phytopathology,2016,106(9):963-970,doi:10.1094/PHYTO-01-16-0019-R.
[28]Dukare AS,Paul S,Nambi VE,Gupta RK,Singh R,Sharma K,Vishwakarma RK.Exploitation of microbial antagonists for the control of postharvest diseases of fruits:A review.Critical Reviews in Food Science and Nutrition,2018,2:1-16.
[29]Peekate PL,Abu GO.Optimizing C:N ratio,C:P ratio,and p H for biosurfactant production by Pseudomonas fluorescens.Journal of Advances in Microbiology,2017,7(2):JAMB.38199.
[30]Peekate PL,Abu GO,Ogugbue CJ.Investigating the effectiveness between using Pseudomonas fluorescens and its biosurfactant in bioremediation of petroleum hydrocarbon contaminated soil.Asian Journal of Biotechnology and Bioresource Technology,2018,3(2):AJB2T.40528,doi:10.9734/AJB2T/2018/40528.
[31]Park YS,Dutta S,Ann M,Raaijmakers JM,Park K.Promotion of plant growth by Pseudomonas fluorescens strain SS101 via novel volatile organic compounds.Biochemical and Biophysical Research Communications,2015,461(2):361-365.
[32]Hernández-León R,Rojas-Solís D,Contreras-Pérez M,Orozco-Mosqueda MDC,Macías-Rodríguez LI,Reyes-de la Cruz H,Valencia-Cantero E,Santoyo G.Characterization of the antifungal and plant growth-promoting effects of diffusible and volatile organic compounds produced by Pseudomonas fluorescens strains.Biological Control,2015,81:83-92.
[33]Sapers GM,Gorny JR,Yousef AE.果蔬微生物学.陈卫,田丰伟,译.北京:中国轻工业出版社,2011.
[34]黄迅辰.荧光假单胞菌分离鉴定、生物膜形成及免疫磁珠检测技术的研究.中国计量学院硕士学位论文,2015.
[35]Allen A,Habimana O,Casey E.The effects of extrinsic factors on the structural and mechanical properties of Pseudomonas fluorescens biofilms:A combined study of nutrient concentrations and shear conditions.Colloids and Surfaces B:Biointerfaces,2018,165:127-134.
[2]Ballester AR,Lafuente MT,Forment J,Gadea J,de Vos RCH,Bovy AG,González-Candelas L.Transcriptomic profiling of citrus fruit peel tissues reveals fundamental effects of phenylpropanoids and ethylene on induced resistance.Molecular Plant Pathology,2011,12(9):879-897,doi:10.1111/j.1364-3703.2011.00721.x.
[3]路来风.海洋拮抗酵母Rhodosporidium paludigenum对柑橘果实抗性的增强效应及其生物学机理研究.浙江大学博士学位论文,2015.
[4]毕阳.果蔬采后病害:原理与控制.北京:科学出版社,2016.
[5]Sánchez-Torres P,Tuset JJ.Molecular insights into fungicide resistance in sensitive and resistant Penicillium digitatum strains infecting citrus.Postharvest Biology and Technology,2011,59(2):159-165,doi:10.1016/j.postharvbio.2010.08.017.
[6]Wallace RL.Biological control of common postharvest diseases of apples with Pseudomonas fluorescens and potential modes of action.Doctor Dissertation of The University of British Columbia,2018.
[7]王一非.海洋拮抗酵母Rhodosporidium paludigenum对果实采后病害生物防治的研究.浙江大学博士学位论文,2008.
[8]Sun GZ,Yao T,Zhao GQ,Lu H,Ma WB.Research progress and prospects for controlling plant diseases using Pseudomonas fluorescens.Acta Prataculturae Sinica,2015,24(4):174-190,doi:10.11686/cyxb20150421.(in Chinese)孙广正,姚拓,赵桂琴,卢虎,马文彬.荧光假单胞菌防治植物病害研究现状与展望.草业学报,2015,24(4):174-190,doi:10.11686/cyxb20150421.
[9]El-Gamal NG,Shehata AN,Hamed ER,Shehata HS.Improvement of lytic enzymes producing Pseudomonas fluorescens and Bacillus subtilis isolates for enhancing their biocontrol potential against root rot disease in tomato plants.Research Journal of Pharmaceutical,Biological and Chemical Sciences,2016,7(1):1393-1400.
[10]Gro?kinsky DK,Tafner R,Moreno MV,Stenglein SA,de Salamone IEG,Nelson LM,Novák O,Strnad M,van der Graaff E,Roitsch T.Cytokinin production by Pseudomonas fluorescens G20-18 determines biocontrol activity against Pseudomonas syringae in Arabidopsis.Scientific Reports,2016,6:23310,doi:10.1038/srep23310.
[11]Prabhukarthikeyan SR,Keerthana U,Raguchander T.Antibiotic-producing Pseudomonas fluorescens mediates rhizome rot disease resistance and promotes plant growth in turmeric plants.Microbiological Research,2018,210:65-73.
[12]Ilhan K,Karabulut OA.Efficacy and population monitoring of bacterial antagonists for gray mold(Botrytis cinerea Pers.ex.Fr.)infecting strawberries.Bio Control,2013,58(4):457-470,doi:10.1007/s10526-012-9503-x.
[13]Ghazanfar MU,Hussain M,Hamid MI,Ansari SU.Utilization of biological control agents for the management of postharvest pathogens of tomato.Pakistan Journal of Botany,2016,48(5):2093-2100.
[14]Mostafavi HA,Mirmajlessi SM,Fathollahi H,Shahbazi S,Mirjalili SM.Integrated effect of gamma radiation and biocontrol agent on quality parameters of apple fruit:An innovative commercial preservation method.Radiation Physics and Chemistry,2013,91:193-199.
[15]Peeran MF,Krishnan N,Thangamani PR,Gandhi K,Thiruvengadam R,Kuppusamy P.Development and evaluation of water-in-oil formulation of Pseudomonas fluorescens(FP7)against Colletotrichum musae incitant of anthracnose disease in banana.European Journal of Plant Pathology,2014,138(1):167-180,doi:10.1007/s10658-013-0320-6.
[16]Wang ZR,Jiang MY,Chen KW,Wang KT,Du MY,Zalán Z,Hegyi F,Kan JQ.Biocontrol of Penicillium digitatum on postharvest citrus fruits by Pseudomonas fluorescens.Journal of Food Quality,2018,2018:2910481.
[17]Wang ZR,Jiang MY,Du MY,Wang KT,Zalán Z,Hegyi F,Kan JQ.Investigation on effects of treatments with Pseudomonas fluorescens on controlling blue mold caused by Penicillium italicum and storage quality in Jincheng citrus fruits.Food and Fermentation Industries,2018,44(11):176-184,doi:10.13995/j.cnki.11-1802/ts.017668.(in Chinese)王智荣,江孟遥,杜木英,汪开拓,Zalán Z,Hegyi F,阚建全.荧光假单胞菌对采后锦橙青霉病的防治和贮藏品质的影响.食品与发酵工业,2018,44(11):176-184,doi:10.13995/j.cnki.11-1802/ts.017668.
[18]王友升.拮抗酵母菌与果蔬采后病害防治.北京:知识产权出版社,2012.
[19]Raza W,Ling N,Liu DY,Wei Z,Huang QW,Shen QR.Volatile organic compounds produced by Pseudomonas fluorescens WR-1 restrict the growth and virulence traits of Ralstonia solanacearum.Microbiological Research,2016,192:103-113.
[20]Janisiewicz WJ,Tworkoski TJ,Sharer C.Characterizing the mechanism of biological control of postharvest diseases on fruits with a simple method to study competition for nutrients.Biological Control,2000,90(11):1196-1200,doi:10.1094/PHYTO.2000.90.11.1196.
[21]罗丽.柠檬形克勒克酵母(34-9)对柑橘意大利青霉抑菌机理的研究.华中农业大学硕士学位论文,2010.
[22]周雅涵.水杨酸、膜醭毕赤酵母、壳寡糖诱导柑橘果实抗病性及其生物学机制研究.西南大学博士学位论文,2017.
[23]Fu Q,Cheng LK,Miao LZ,Wang Y,He C,Shen ZQ.Determination of growth curve and screening of medium for A type Naso trachea.China Poultry,2017,39(11):60-63.(in Chinese)付强,程立坤,苗立中,王艳,何诚,沈志强.A型鼻气管鸟杆菌生长曲线的测定和培养基筛选.中国家禽,2017,39(11):60-63.
[24]刘正强.利用核糖体工程技术提高荧光假单胞菌Pf-5菌株活性次级代谢产物产量.湖南师范大学硕士学位论文,2016.
[25]Wei HL,Zhang LQ.Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24.Antonie van Leeuwenhoek,2006,89(2):267-280,doi:10.1007/s10482-005-9028-8.
[26]Sahu B,Singh J,Shankar G,Pradhan A.Pseudomonas fluorescens PGPR bacteria as well as biocontrol agent:Areview.International Journal of Chemical Studies,2018,6(2):1-7.
[27]Arseneault T,Goyer C,Filion M.Biocontrol of potato common scab is associated with high Pseudomonas fluorescens LBUM223 populations and phenazine-1-carboxylic acid biosynthetic transcript accumulation in the potato geocaulosphere.Phytopathology,2016,106(9):963-970,doi:10.1094/PHYTO-01-16-0019-R.
[28]Dukare AS,Paul S,Nambi VE,Gupta RK,Singh R,Sharma K,Vishwakarma RK.Exploitation of microbial antagonists for the control of postharvest diseases of fruits:A review.Critical Reviews in Food Science and Nutrition,2018,2:1-16.
[29]Peekate PL,Abu GO.Optimizing C:N ratio,C:P ratio,and p H for biosurfactant production by Pseudomonas fluorescens.Journal of Advances in Microbiology,2017,7(2):JAMB.38199.
[30]Peekate PL,Abu GO,Ogugbue CJ.Investigating the effectiveness between using Pseudomonas fluorescens and its biosurfactant in bioremediation of petroleum hydrocarbon contaminated soil.Asian Journal of Biotechnology and Bioresource Technology,2018,3(2):AJB2T.40528,doi:10.9734/AJB2T/2018/40528.
[31]Park YS,Dutta S,Ann M,Raaijmakers JM,Park K.Promotion of plant growth by Pseudomonas fluorescens strain SS101 via novel volatile organic compounds.Biochemical and Biophysical Research Communications,2015,461(2):361-365.
[32]Hernández-León R,Rojas-Solís D,Contreras-Pérez M,Orozco-Mosqueda MDC,Macías-Rodríguez LI,Reyes-de la Cruz H,Valencia-Cantero E,Santoyo G.Characterization of the antifungal and plant growth-promoting effects of diffusible and volatile organic compounds produced by Pseudomonas fluorescens strains.Biological Control,2015,81:83-92.
[33]Sapers GM,Gorny JR,Yousef AE.果蔬微生物学.陈卫,田丰伟,译.北京:中国轻工业出版社,2011.
[34]黄迅辰.荧光假单胞菌分离鉴定、生物膜形成及免疫磁珠检测技术的研究.中国计量学院硕士学位论文,2015.
[35]Allen A,Habimana O,Casey E.The effects of extrinsic factors on the structural and mechanical properties of Pseudomonas fluorescens biofilms:A combined study of nutrient concentrations and shear conditions.Colloids and Surfaces B:Biointerfaces,2018,165:127-134.