Screening of Antagonistic Bacteria Against Fusarium Wilt of Watermelon (Citrullus lanatus) and Its Antagonistic Properties
XU Wei-Hui1, WANG Heng-Xu1, ZHAO Jing-Ming2, WANG Zhi-Gang1,*, WANG Ke-Xin1
1 School of Life Science and Agriculture and Forestry, Qiqihar University, Qiqihar 161006, China; 2 Science-Engineering and Biotechnology Corporation, Harbin 150000, China
Abstract：Fusarium wilt of watermelon (Citrullus lanatus) is caused by Fusarium oxysporum f.sp. niveum (FON) which is considered as the most important soil-borne pathogens. Some safe antagonistic strain can control FON growth and reduce Fusarium wilt of watermelon. In order to screen an antagonistic strain against FON, the rhizosphere soil from vegetable crops on Qinxin farm in Qiqihar Heilongjiang province was collected and used in this study. The antagonistic strain was screened out using plate confrontation method, and identified by morphological, physiological, and biochemical and bio-control enzyme analyses, as well as 16S rDNA sequence analysis. Antifungal activities of sterile fermentation filtrate from different growth stage of antagonistic strain and the stability of sterile fermentation filtrate under different conditions were determined using growth rate method. FON spore morphology and cell membrane integrity were analyzed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The results showed that the antagonistic strain was classified as Bacillus methylotrophicus, named as WC. The WC strain could product amylase, glucanase, protease and siderophores. The sterile fermentation filtrate from the stationary phase had stronger antifungal activity against FON compared to those from the exponential stage and death phase, and the inhibition rate of FON colony growth was 61.57%. The stability tests showed that the sterile fermentation filtrate had stronger antifungal activity against FON under 10 ℃ for 30 min compared to other testing temperature, with inhibition rate of 62.78%. The antifungal activity from WC fermentation filtrate reached to 74.54% in pH 7, the stabilities of fermentation filtrate were poor in acid and base conditions. The filtrate could be stored with stable antifungal activity at 4 ℃ for 30 d and was insensitive to UV-irradiation. The analyses from SEM demonstrated that treatment with fermentation filtrate from WC led to wrinkling and depressed changes on the surface of FON spores. The CLSM revealed that the fermentation filtrate from WC damaged membrane integrity. The above results indicate that WC is a high-efficiency antagonistic strain against FON, and could be used to control Fusarium wilt of watermelon in future.
徐伟慧, 王恒煦, 赵井明, 王志刚, 王可昕. 西瓜枯萎病拮抗菌筛选及其拮抗性能研究[J]. 农业生物技术学报, 2019, 27(12): 2238-2247.
XU Wei-Hui, WANG Heng-Xu, ZHAO Jing-Ming, WANG Zhi-Gang, WANG Ke-Xin. Screening of Antagonistic Bacteria Against Fusarium Wilt of Watermelon (Citrullus lanatus) and Its Antagonistic Properties. 农业生物技术学报, 2019, 27(12): 2238-2247.
[1] 东秀珠, 蔡妙英. 2001. 常见细菌系统鉴定手册[M]. 北京: 科学出版社: 349-388. (Dong X Z, Cai M Y.2001. Handbook of Identification of Common Bacterial Systems[M]. Beijing: Science Press: 349-388.) [2] 胡忠亮, 郑催云, 田兴一, 等. 2017. 解淀粉芽孢杆菌HZM9菌株发酵液的抑菌谱及稳定性测定[J]. 南京林业大学学报(自然科学版), 41(03): 65-70. (Hu Z L, Zheng C Y, Tian X Y, et al.2017. Antibacterial spectrum and stability determination of fermentation broth of Bacillus amyloliquefaciens HZM9 strain[J]. Journal of Nanjing Forestry University, 41(03): 65-70.) [3] 康兴娇, 申红妙, 贾招闪, 等. 2016. 葡萄霜霉病生防菌甲基营养型芽胞杆菌T3的鉴定及其防治效果[J]. 中国生物防治学报, 32(06): 775-782. (Kang X J, Shen H M, Jia Z X, et al.2016. Identification and control effect of the strain of Bacillus methylotrophic T3 against grape downy mildew[J]. Chinese Journal of Biological Control, 32(06): 775-782.) [4] 李瑞芳, 徐怡, 赵玉峰, 等. 2008. 枯草芽孢杆菌不同生长时期抗稻瘟病菌活性研究[J]. 河南农业科学, (09): 76-78. (Li R F, Xu Y, Zhao Y F, et al. 2008. Study on the activity of resistance to rice blast fungus in different growth stages of Bacillus subtilis[J]. Henan Agricultural Sciences, (09): 76-78.) [5] 李玉聪, 李滨影, 油心怡, 等. 2018. 马铃薯疮痂病拮抗菌的筛选鉴定及防治效果初探[J]. 生物技术通报, 34(10): 122-127. (Li Y C, Li B Y, You X Y, et al.2018. Screening and identification of antagonistic bacteria against potato scab and its control effect[J]. Biotechnology Bulletin, 34(10): 122-127.) [6] 梁慎, 徐小利, 赵卫星, 等. 2013. 西瓜枯萎病菌的生防菌的分离筛选与鉴定[C]//, 中国园艺学会, 中国园艺学会2013年学术年会. 园艺学报, 北京, pp. 2702. (Liang S, Xu X L, Zhao W X, et al.2013. Isolation, screening and identification of biocontrol bacteria from Fusarium oxysporum f. sp.[C]//, Chinese horticultural society, 2013 academic annual meeting of Chinese horticultural society. Acta Horticulturae Sinica, Beijing, pp. 2702) [7] 刘海洋, 王琦, 王伟, 等. 2018. 拮抗菌AL7的鉴定及其生防特性的初步研究[J]. 植物保护, 44(02): 53-60. (Liu H Y, Wang Q, Wang W, et al.2018. Identification of antagonistic antimicrobial AL7 and preliminary study on its biocontrol characteristics[J]. Plant Protection, 44(02): 53-60.) [8] 刘金香, 蒲亚帅, 谢水波, 等. 2019. 灭活与非灭活条件下植物乳杆菌去除U(Ⅵ)的机理[J]. 中国环境科学, 39(07): 2880-2888. (Liu J X, Pu Y S, Xie S B, et al.2019. Mechanism of removal of U(Ⅵ) by Lactobacillus plantarum under inactivated and non-inactivated conditions[J]. Environmental Science of China, 39(07): 2880-2888.) [9] 吕智航, 徐伟慧, 王志刚, 等. 2018. 一株西瓜专化型尖孢镰刀菌抑制菌的分离鉴定及其抑菌活性[J]. 浙江农业学报, 30(12): 2065-2071. (Lü Z h, Xu W H, Wang Z G, et al.2018. Isolation, identification and antibacterial activity of a watermelon-specific Fusarium oxysporum strain[J]. Zhejiang Agricultural Journal, 30(12): 2065-2071.) [10] 陆楚月. 2015. 绿色木霉TV41对尖孢镰刀菌FW0在西瓜植株空间分布和枯萎病防控效果的影响[J]. 微生物学通报, 42(11): 2159-2167. (Lu C Y.2015. Effect of Trichoderma viride TV41 on the spatial distribution of Fusarium oxysporum FW0 in watermelon plants and the control effect of Fusarium wilt[J]. Bulletin of Microbiology, 42(11): 2159-2167.) [11] 任加庆, 薛守聪, 李锡宏, 等. 2015. 不同真菌菌剂对烟草病毒病的田间防治效果[J]. 中国烟草科学, 36(04): 96-101. (Ren J Q, Xue S C, Li X H, et al.2015. Field control effects of different fungal agents on tobacco virus disease[J]. Chinese Tobacco Science, 36(04): 96-101.) [12] 荣良燕, 姚拓, 赵桂琴, 等. 2011. 产铁载体PGPR菌筛选及其对病原菌的拮抗作用[J]. 植物保护, 37(01): 59-64. (Rong L Y, Yao T, Zhao G Q, et al.2011. Screening of PGPR strains for iron-producing carriers and their antagonistic effects on pathogenic bacteria[J]. Plant Protection, 37(01): 59-64.) [13] 申光辉, 冯孟, 张志清, 等. 2016. 一株蜂粮源拮抗细菌的分离鉴定及其抑菌物质特性[J]. 微生物学通报, 43(10):2197-2206. (Shen G H, Feng M, Zhang Z Q, et al.2016. Isolation and identification of an antagonistic bacteria from bee sources and their antibacterial properties[J]. Bulletin of Microbiology, 43(10): 2197-2206.) [14] 王卿, 林玲, 张昕, 等. 2013. 西瓜枯萎病生防细菌的筛选及鉴定[J]. 江苏农业科学, 41(8): 116-118. (Wang Q, Lin L, Zhang X, et al.2013. Screening and identification of biocontrol bacteria against watermelon wilt disease[J]. Jiangsu Agricultural Sciences, 41(8): 116-118.) [15] 王雪, 张丹妮, 王春伟, 等. 2019. 解淀粉芽孢杆菌FS6在人参体内的定殖特性及对人参诱导抗病性[J]. 西北农林科技大学学报(自然科学版), (07): 1-6. (Wang X, Zhang D N, Wang C W, et al.2019. Colonization characteristics of Bacillus amyloliquefaciens FS6 in ginseng and its resistance to ginseng induction[J]. Journal of Northwest A&F University (Natural Science Edition), (07): 1-6.) [16] 吴学宏, 卢志军, 陈倩, 等. 2010. 西瓜种传尖孢镰刀菌致病性及其粗毒素对种子发芽的影响[J]. 中国农业大学学报, 15(3): 50-56. (Wu X X, Lu L Z, Chen Q, et al.2010. Pathogenicity of Fusarium oxysporum and the effect of crude toxin on seed germination of watermelon[J]. Journal of China Agricultural University, 15(3): 50-56.) [17] 夏艳, 徐茜, 董瑜, 等. 2014. 烟草青枯病菌拮抗菌的筛选、鉴定及生防特性研究[J]. 中国生态农业学报, 22(2): 201-207. (Xia Y, Xu Q, Dong Y, et al.2014. Screening, identification and biocontrol characteristics of antagonistic bacteria against tobacco bacterial wilt pathogen[J]. Chinese Journal of Eco-Agriculture, 22(2): 201-207.) [18] 谢学文, 董瑞利, 石延霞, 等. 2016. 黄瓜炭疽病拮抗细菌的筛选及其抑制效果[J]. 中国生物防治学报, 32(02): 215-220. (Xie X W, Dong R L, Shi Y X, et al.2016. Screening of antagonistic bacteria against cucumber anthracnose and its inhibitory effect[J]. Chinese Journal of Biological Control, 32(02): 215-220.) [19] 杨敬辉, 文平兰, 庄义庆. 2013. 拮抗细菌的筛选及生防潜能评估[J]. 西南农业学报, 26(2): 565-571. (Yang J H, Wen P L, Zhang Y Q.2013. Screening of antagonistic bacteria and evaluation of biocontrol potential[J]. Southwest Agricultural Journal, 26(2): 565-571.) [20] 杨艳红, 余瑛, 胡永强, 等. 2017. 解淀粉芽孢杆菌AF1发酵液的抗菌活性与抗菌机理[J]. 生物技术通报, 33(09): 223-230. (Yang Y H, Yu Y, Hu Y Q, et al.2017. Antibacterial activity and antibacterial mechanism of Bacillus amyloliquefaciens AF1 fermentation broth[J]. Biotechnology Bulletin, 33(09): 223-230.) [21] 尹向田, 杨阳. 2018. 甲基营养型芽孢杆菌GSBM05产抗菌活性物质发酵条件优化[J]. 江苏农业科学, 46(20): 89-93. (Yin X T, Yang Y.2018. Optimization of fermentation conditions for antibacterial active substances produced by Bacillus methylotrophic GSBM05[J]. Jiangsu Agricultural Sciences, 46(20): 89-93.) [22] 游成真, 李平兰, 张志刚, 等. 2015. 黄瓜立枯病高效拮抗菌的筛选与鉴定[J]. 中国农业大学学报, 20(01): 90-95. (You C Z, Li P L, Zhang Z G, et al.2015. Screening and identification of high-efficiency antagonistic bacteria against cucumber blight[J]. Journal of China Agricultural University, 20(01): 90-95.) [23] 张宝俊, 张家榕, 韩巨才, 等. 2010. 内生解淀粉芽孢杆菌LP-5抗菌蛋白的分离纯化及特性[J]. 植物保护学报, 37(02): 143-147. (Zhang B J, Zhang J Y, Han J C, et al.2010. Isolation, purification and characterization of antibacterial protein from endophytic Bacillus amyloliquefaciens LP-5[J]. Journal of Plant Protection, 37(02): 143-147.) [24] 张洪涛, 赵国玉, 于频频, 等. 2007. 西瓜枯萎病高效拮抗菌XJUL-12的筛选与鉴定[J]. 生物技术, (17): 77-80. (Zhang H T, Zhao G Y, Yu P P, et al. 2007. Screening and identification of high-efficiency antagonistic XJUL-12 for watermelon wilt[J]. Biotechnology, (17): 77-80.) [25] 赵文珺, 葛蓓孛, 刘炳花, 等. 2018. 甲基营养型芽胞杆菌NKG-1对番茄白粉病的防病促生作用研究[J]. 中国农学通报, 34(23): 104-109. (Zhao W J, Ge W, Liu B H, et al.2018. Effect of Bacillus methylotrophic NKG-1 on disease prevention and growth of tomato powdery mildew[J]. Chinese Agricultural Science Bulletin, 34(23): 104-109.) [26] 周陈力, 万佳宁, 卢绪志, 等. 2019. 灵芝尿嘧啶营养缺陷型菌株的筛选和分子鉴定[J]. 微生物学通报, 46(10): 2657-2664. (Zhou C L, Wan J N, Lu X Z, et al.2019. Screening and molecular identification of uracil auxotrophic strains of Ganoderma lucidum[J]. Bulletin of Microbiology, 46(10): 2657-2664.) [27] Blanco E M, Little C, Baines A L D.2007. Variation in antibiotic inhibitory abilities among streptomycetes from south Texas agricultural soils[J]. Soil Biology & Biochemistry, 39(1): 268-275. [28] Benitez L B, Velho R V, Lisboa M P, et al.2010. Isolation and characterization of antifungal peptides produced by Bacillus amyloliquefaciens LBM5006[J]. Journal of Microbiology, 48(6): 791-797. [29] Chen Y, Wang J, Yang N, et al.2018. Wheat microbiome bacteria can reduce virulence of a plant pathogenic fungus by altering histone acetylation[J]. Nature Communications, 9(1): 3429. [30] Faheem M, Raza W, Zhong W, et al.Evaluation of the biocontrol potential of Streptomyces goshikiensis YCXU against Fusarium oxysporum f. sp. niveum[J]. Biological Control, 2014, 81: 101-110. [31] Liu G, Ren G, Zhao L, et al.2016. Antibacterial activity and mechanism of bifidocin A against Listeria monocytogenes[J]. Food Control, 73: 854-861. [32] Madhaiyan M, Poonguzhali S, Kwon S W, et al.2010. Bacillus methylotrophicus sp. nov. a methanol-utilizing, plant-growth-promoting bacterium isolated from rice rhizosphere soil[J]. International Journal of Systematic and Evolutionary Microbiology, 60(10): 2490-2495. [33] Moussa M, Perrier-Cornet J M, Gervais P.2007. Damage in Escherichia coli cellstreated with a combination of high hydrostatic pressure and subzero temperature[J]. Applied and Environmental Microbiology, 73(20): 6508-6518. [34] Romano A, Vitullo D, Di Pietro A, et al.2011. Antifungal Lipopeptides from Bacillus amyloliquefaciens strain BO7[J]. Journal of Natural Products, 74(2): 145-151. [35] Sun G, Yao T, Feng C, et al.2017. Identification and biocontrol potential of antagonistic bacteria strains against Sclerotinia sclerotiorum and their growth-promoting effects on Brassica napus[J]. Biological Control, 104: 35-43.
