1株水蜜桃采后病害拮抗细菌的鉴定及抑菌作用
|
摘要
为开发高效安全的生物保鲜剂,充分发掘和利用有益微生物资源,对1株从豆腐乳中分离到的对水蜜桃采后病害具有较好防治效果的拮抗细菌CF-2,通过培养性状与形态特征、生理生化特征及16S rDNA同源序列比对分析进行鉴定,并测定其生长曲线及最适生长条件,同时采用平板对峙法及实体实验研究CF-2及其代谢产物对交链孢霉、桃褐腐菌、复端孢霉与丝核菌的抑制作用。结果表明:菌株CF-2经鉴定为枯草芽孢杆菌(Bacillus subtilis),培养24～48 h后生长最旺盛,最适生长pH值为7.0,最适生长温度为37℃;CF-2对4?种病原真菌在体外均有抑制作用,平均抑制率为(38.70±9.22)%,其中对复端孢霉的抑制作用最大,抑制率为(52.30±3.67)%;其24 h发酵液及其无菌滤液与菌悬液对4种病原菌亦有较强的抑制效果,其中无菌滤液对4种病原真菌的平均抑制率为(68.76±5.77)%,对交链孢霉的抑制作用最大,抑制率为(77.41±1.91)%;经CF-2无菌滤液处理后,孢子萌发时间延长了62～64 h,12 d后孢子萌发率平均降低了87.93%;在实体实验中,25℃室温下CF-2 24 h发酵液及无菌滤液与菌悬液可有效提升水蜜桃好果率,其中无菌滤液的效果最好,6 d时好果率提升了29%。以上结果表明,生防菌CF-2及其代谢产物对水蜜桃采后4种病原菌有较强的抑制作用。
This study aimed to develop an effective and safe biological control agent and to sufficiently explore and make use of beneficial microorganisms. One bacterial strain named CF-2, which was separated from fermented bean curd and showed inhibitory effect on postharvest diseases of honey peaches, was identified based on morphological features, physiological and biochemical characteristics, and 16 S rDNA sequence homology analysis. The growth curve of CF-2 and its optimal growth conditions were determined. Subsequently, the antimicrobial activity of CF-2 and its metabolites against four main postharvest fungal pathogens of honey peach (Alternaria alternata, Monilinia fructicola, Cephalothecium sp. and Rhizoctonia sp.) was assessed by plate confrontation method and in vivo experiments. The results showed that strain CF-2 was identified as Bacillus subtilis. At 24-48 h of culture, the growth rate of CF-2 reached a peak, and the optimal pH and temperature for its growth were determined as 7.0 and 37 ℃, respectively. CF-2 showed inhibitory effect on the four fungal pathogens in vitro with an average percentage inhibition of (38.70 ± 9.22)%, and it exhibited the strongest inhibitory effect on Cephalothecium sp. with a percentage inhibition of (52.30 ± 3.67)%. The 24 h fermented broth and its cell-free filtrate and cell suspension also had strong inhibitory effect on all four pathogenic fungi. The average percentage inhibition of the cellfree filtrate was(68.76 ± 5.77)% and it showed the strongest inhibitory effect on A. alternata with a percentage inhibition of (77.41 ± 1.91)%. Compared to the control group, treatment with the cell-free filtrate delayed spore germination of the fungal pathogens by 62-64 h and decreased spore germination rate by 87.93% on average after 12 d. In in vivo experiments, the 24 h fermented broth of CF-2 and its cell-free filtrate and cell suspension all increased the percentage of marketable fruit compared to the control group, especially the cell-free filtrate, which showed the strongest effect, increasing the percentage of marketable fruit by 29% after 6 d. The above findings indicated that CF-2 and its metabolites had strong inhibitory effect on four main fungal pathogens in honey peach.
This study aimed to develop an effective and safe biological control agent and to sufficiently explore and make use of beneficial microorganisms. One bacterial strain named CF-2, which was separated from fermented bean curd and showed inhibitory effect on postharvest diseases of honey peaches, was identified based on morphological features, physiological and biochemical characteristics, and 16 S rDNA sequence homology analysis. The growth curve of CF-2 and its optimal growth conditions were determined. Subsequently, the antimicrobial activity of CF-2 and its metabolites against four main postharvest fungal pathogens of honey peach (Alternaria alternata, Monilinia fructicola, Cephalothecium sp. and Rhizoctonia sp.) was assessed by plate confrontation method and in vivo experiments. The results showed that strain CF-2 was identified as Bacillus subtilis. At 24-48 h of culture, the growth rate of CF-2 reached a peak, and the optimal pH and temperature for its growth were determined as 7.0 and 37 ℃, respectively. CF-2 showed inhibitory effect on the four fungal pathogens in vitro with an average percentage inhibition of (38.70 ± 9.22)%, and it exhibited the strongest inhibitory effect on Cephalothecium sp. with a percentage inhibition of (52.30 ± 3.67)%. The 24 h fermented broth and its cell-free filtrate and cell suspension also had strong inhibitory effect on all four pathogenic fungi. The average percentage inhibition of the cellfree filtrate was(68.76 ± 5.77)% and it showed the strongest inhibitory effect on A. alternata with a percentage inhibition of (77.41 ± 1.91)%. Compared to the control group, treatment with the cell-free filtrate delayed spore germination of the fungal pathogens by 62-64 h and decreased spore germination rate by 87.93% on average after 12 d. In in vivo experiments, the 24 h fermented broth of CF-2 and its cell-free filtrate and cell suspension all increased the percentage of marketable fruit compared to the control group, especially the cell-free filtrate, which showed the strongest effect, increasing the percentage of marketable fruit by 29% after 6 d. The above findings indicated that CF-2 and its metabolites had strong inhibitory effect on four main fungal pathogens in honey peach.
引文
[1]姚良辉,尹京苑,何惠霞,等.生物保鲜剂C06对水蜜桃采后的保鲜效果[J].农业工程学报,2010,26(4):357-361.DOI:10.3969/j.issn.1002-6819.2010.04.061.
[2]ABATA L K,VIERA W F,PAZ I A,et al.First report of alternaria rot caused by Alternaria alternata on peach in ecuador[J].Plant Disease,2016,100(11):2323.DOI:10.1094/PDIS-03-16-0318-PDN.
[3]LI G J,ZHU S H,WU W X,et al.Exogenous nitric oxide induces disease resistance against Monilinia fructicola through activating the phenylpropanoid pathway in peach fruit[J].Journal of the Science of Food and Agriculture,2017,97(9):3030-3038.DOI:10.1002/jsfa.8146.
[4]GAO H Y,XU X X,DAI Y W,et al.Isolation,Identification and characterization of Bacillus subtilis CF-3,a bacterium from fermented bean curd for controlling postharvest diseases of peach fruit[J].Food Science and Technology Research,2016,22(3):377-385.DOI:10.3136/fstr.22.377.
[5]SCHREINEMACHERS P,TIPRAQSA P.Agricultural pesticides and land use intensification in high,middle and low income countries[J].Food Policy,2012,37(6):616-626.DOI:10.1016/j.foodpol.2012.06.003.
[6]HOSSAIN M I,SADEKUZZAMAN M,HA S D.Probiotics as potential alternative biocontrol agents in the agriculture and food industries:a review[J].Food Research International(Ottawa,Ont.),2017,100(1):63-73.DOI:10.1016/j.foodres.2017.07.077.
[7]何惠霞,高海燕,曹玉明,等.桃采后保鲜技术研究进展[J].食品与发酵工业,2009,35(2):130-133.DOI:10.13995/j.cnki.11-1802/ts.2009.02.029.
[8]ZHANG Z Q,CHEN J,LI B Q,et al.Influence of oxidative stress on biocontrol activity of Cryptococcus laurentii against blue mold on peach fruit[J].Frontiers in Microbiology,2017,8:151.DOI:10.3389/fmicb.2017.00151.
[9]韦灵林,李小礼,李卿,等.水蜜桃采后生物保鲜初步研究[J].上海交通大学学报(农业科学版),2011,29(4):45-49.DOI:10.3969/j.issn.1671-9964.2011.04.011.
[10]张丽,郁志芳,姜丽,等.淀粉液化芽孢杆菌ES-2发酵产物对贮藏期间“湖锦蜜露”水蜜桃品质和生理特性的影响[J].食品科学,2009,30(20):421-425.
[11]王亦佳,李建龙,刚诚成,等.利用拮抗菌常温保鲜凤凰水蜜桃效果系统研究[J].食品工业科技,2012,33(2):384-388.DOI:10.13386/j.issn1002-0306.2012.02.009.
[12]LI X H,ZHANG Y Z,WEI Z W,et al.Antifungal activity of isolated Bacillus amyloliquefaciens SYBC H47 for the biocontrol of peach gummosis[J].PLoS ONE,2016,11(9):e0162125.DOI:10.1371/journal.pone.0162125.
[13]GOTOR-VILA A,TEIXIDO N,CASALS C,et al.Biological control of brown rot in stone fruit using Bacillus amyloliquefaciens CPA-8 under field conditions[J].Crop Protection,2017,102:72-80.DOI:10.1016/j.cropro.2017.08.010.
[14]GAO H Y,XU X X,ZENG Q,et al.Optimization of headspace solidphase microextraction for GC-MS analysis of volatile compounds produced by biocontrol strain Bacillus subtilis CF-3 using response surface methodology[J].Food Science and Technology Research,2017,23(4):583-593.DOI:10.3136/fstr.23.583.
[15]林钧安,高锦梁,洪健.实用生物电子显微技术[M].沈阳:辽宁科学技术出版社,1998.
[16]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.
[17]布坎南?R E,吉本斯?N E.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984.
[18]WEISBURG W G,BAMS S M,PELLETIER D A,et al.16SRibosomal DNA amplification for phylogentic study[J].Journal of Bacteriology,1991,173(2):697-703.DOI:10.1128/jb.173.2.697-703.1991.
[19]赵圣明,赵岩岩,马汉军,等.1株广谱抗菌活性菌株的筛选鉴定及其抗菌蛋白的分离纯化[J].食品科学,2018,39(2):170-176.DOI:10.7506/spkx1002-6630-201802027.
[20]关一鸣,潘晓曦,王莹,等.哈茨木霉菌、枯草芽孢杆菌对人参灰霉病和根腐病病原菌的拮抗作用[J].江苏农业科学,2014,42(5):123-125.DOI:10.15889/j.issn.1002-1302.2014.05.057.
[21]韩雨桐,刘烨,张淋淋,等.稻瘟病菌拮抗细菌的筛选及其防效作用研究[J].东北农业科学,2016,41(3):67-72.DOI:10.16423/j.cnki.1003-8701.2016.03.014.
[22]ARROYAVE-TORO J J,MOSQUERA S,VILLEGAS-ESCOBARV.Biocontrol activity of Bacillus subtilis EA-CB0015 cells and lipopeptides against postharvest fungal pathogens[J].Biological Control,2017,114:195-200.DOI:10.1016/j.biocontrol.2017.08.014.
[23]KILANI-FEKI O,KHEDHER S B,DAMMAK M,et al.Improvement of antifungal metabolites production by Bacillus subtilis V26 for biocontrol of tomato postharvest disease[J].Biological Control,2016,95:73-82.DOI:10.1016/j.biocontrol.2016.01.005.
[24]KHEDHER S B,KILANI-FEKI O,DAMMAK M,et al.Efficacy of Bacillus subtilis V26 as a biological control agent against Rhizoctonia solani on potato[J].Comptes Rendus Biologies,2015,338(12):784-792.DOI:10.1016/j.crvi.2015.09.005.
[25]刘诗胤.生防菌MF-91对水稻主要病害防治效果、根际微生物多样性及稻米品质的影响[D].杭州:杭州师范大学,2012:19-20.
[26]李晶,杨谦,赵丽华,等.生防枯草芽孢杆菌B29菌株抗菌物质的初步研究[J].中国生物工程杂志,2008,28(2):59-65.DOI:10.13523/j.cb.20080211.
[27]侯旭,张国庆,胡晓,等.桃褐腐病菌拮抗性内生细菌的筛选及其抑病效果[J].微生物学通报,2017,44(8):1874-1881.DOI:10.13344/j.microbiol.china.160860.
[2]ABATA L K,VIERA W F,PAZ I A,et al.First report of alternaria rot caused by Alternaria alternata on peach in ecuador[J].Plant Disease,2016,100(11):2323.DOI:10.1094/PDIS-03-16-0318-PDN.
[3]LI G J,ZHU S H,WU W X,et al.Exogenous nitric oxide induces disease resistance against Monilinia fructicola through activating the phenylpropanoid pathway in peach fruit[J].Journal of the Science of Food and Agriculture,2017,97(9):3030-3038.DOI:10.1002/jsfa.8146.
[4]GAO H Y,XU X X,DAI Y W,et al.Isolation,Identification and characterization of Bacillus subtilis CF-3,a bacterium from fermented bean curd for controlling postharvest diseases of peach fruit[J].Food Science and Technology Research,2016,22(3):377-385.DOI:10.3136/fstr.22.377.
[5]SCHREINEMACHERS P,TIPRAQSA P.Agricultural pesticides and land use intensification in high,middle and low income countries[J].Food Policy,2012,37(6):616-626.DOI:10.1016/j.foodpol.2012.06.003.
[6]HOSSAIN M I,SADEKUZZAMAN M,HA S D.Probiotics as potential alternative biocontrol agents in the agriculture and food industries:a review[J].Food Research International(Ottawa,Ont.),2017,100(1):63-73.DOI:10.1016/j.foodres.2017.07.077.
[7]何惠霞,高海燕,曹玉明,等.桃采后保鲜技术研究进展[J].食品与发酵工业,2009,35(2):130-133.DOI:10.13995/j.cnki.11-1802/ts.2009.02.029.
[8]ZHANG Z Q,CHEN J,LI B Q,et al.Influence of oxidative stress on biocontrol activity of Cryptococcus laurentii against blue mold on peach fruit[J].Frontiers in Microbiology,2017,8:151.DOI:10.3389/fmicb.2017.00151.
[9]韦灵林,李小礼,李卿,等.水蜜桃采后生物保鲜初步研究[J].上海交通大学学报(农业科学版),2011,29(4):45-49.DOI:10.3969/j.issn.1671-9964.2011.04.011.
[10]张丽,郁志芳,姜丽,等.淀粉液化芽孢杆菌ES-2发酵产物对贮藏期间“湖锦蜜露”水蜜桃品质和生理特性的影响[J].食品科学,2009,30(20):421-425.
[11]王亦佳,李建龙,刚诚成,等.利用拮抗菌常温保鲜凤凰水蜜桃效果系统研究[J].食品工业科技,2012,33(2):384-388.DOI:10.13386/j.issn1002-0306.2012.02.009.
[12]LI X H,ZHANG Y Z,WEI Z W,et al.Antifungal activity of isolated Bacillus amyloliquefaciens SYBC H47 for the biocontrol of peach gummosis[J].PLoS ONE,2016,11(9):e0162125.DOI:10.1371/journal.pone.0162125.
[13]GOTOR-VILA A,TEIXIDO N,CASALS C,et al.Biological control of brown rot in stone fruit using Bacillus amyloliquefaciens CPA-8 under field conditions[J].Crop Protection,2017,102:72-80.DOI:10.1016/j.cropro.2017.08.010.
[14]GAO H Y,XU X X,ZENG Q,et al.Optimization of headspace solidphase microextraction for GC-MS analysis of volatile compounds produced by biocontrol strain Bacillus subtilis CF-3 using response surface methodology[J].Food Science and Technology Research,2017,23(4):583-593.DOI:10.3136/fstr.23.583.
[15]林钧安,高锦梁,洪健.实用生物电子显微技术[M].沈阳:辽宁科学技术出版社,1998.
[16]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.
[17]布坎南?R E,吉本斯?N E.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984.
[18]WEISBURG W G,BAMS S M,PELLETIER D A,et al.16SRibosomal DNA amplification for phylogentic study[J].Journal of Bacteriology,1991,173(2):697-703.DOI:10.1128/jb.173.2.697-703.1991.
[19]赵圣明,赵岩岩,马汉军,等.1株广谱抗菌活性菌株的筛选鉴定及其抗菌蛋白的分离纯化[J].食品科学,2018,39(2):170-176.DOI:10.7506/spkx1002-6630-201802027.
[20]关一鸣,潘晓曦,王莹,等.哈茨木霉菌、枯草芽孢杆菌对人参灰霉病和根腐病病原菌的拮抗作用[J].江苏农业科学,2014,42(5):123-125.DOI:10.15889/j.issn.1002-1302.2014.05.057.
[21]韩雨桐,刘烨,张淋淋,等.稻瘟病菌拮抗细菌的筛选及其防效作用研究[J].东北农业科学,2016,41(3):67-72.DOI:10.16423/j.cnki.1003-8701.2016.03.014.
[22]ARROYAVE-TORO J J,MOSQUERA S,VILLEGAS-ESCOBARV.Biocontrol activity of Bacillus subtilis EA-CB0015 cells and lipopeptides against postharvest fungal pathogens[J].Biological Control,2017,114:195-200.DOI:10.1016/j.biocontrol.2017.08.014.
[23]KILANI-FEKI O,KHEDHER S B,DAMMAK M,et al.Improvement of antifungal metabolites production by Bacillus subtilis V26 for biocontrol of tomato postharvest disease[J].Biological Control,2016,95:73-82.DOI:10.1016/j.biocontrol.2016.01.005.
[24]KHEDHER S B,KILANI-FEKI O,DAMMAK M,et al.Efficacy of Bacillus subtilis V26 as a biological control agent against Rhizoctonia solani on potato[J].Comptes Rendus Biologies,2015,338(12):784-792.DOI:10.1016/j.crvi.2015.09.005.
[25]刘诗胤.生防菌MF-91对水稻主要病害防治效果、根际微生物多样性及稻米品质的影响[D].杭州:杭州师范大学,2012:19-20.
[26]李晶,杨谦,赵丽华,等.生防枯草芽孢杆菌B29菌株抗菌物质的初步研究[J].中国生物工程杂志,2008,28(2):59-65.DOI:10.13523/j.cb.20080211.
[27]侯旭,张国庆,胡晓,等.桃褐腐病菌拮抗性内生细菌的筛选及其抑病效果[J].微生物学通报,2017,44(8):1874-1881.DOI:10.13344/j.microbiol.china.160860.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |