黄瓜茄病镰刀菌拮抗芽孢杆菌的筛选、鉴定及促生效果

doi:10.13560/j.cnki.biotech.bull.1985.2022-0522

摘要/Abstract

摘要：

茄病镰刀菌（Fusarium solani）引起的根腐病严重影响黄瓜产业的可持续发展，为获得高效拮抗黄瓜根腐病的芽孢杆菌并明确其促生效果，从黄瓜根际分离芽孢杆菌，筛选高抗茄病镰刀菌的菌株，并对其进行形态、生理生化、遗传特性及植株促生特性的评价。结果表明，菌株XY-1、XY-13、XY-53抑制茄病镰刀菌效果显著，抑菌率分别为65.90%、66.13%、60.83%，鉴定XY-1、XY-13为解淀粉芽孢杆菌（Bacillus amyloliquefaciens），XY-53为枯草芽孢杆菌（B. subtilis），3个菌株均具有释磷、固氮能力，产蛋白酶和ACC脱氨酶。幼苗促生试验中，接种菌株XY-1、XY-13、XY-53有效促进黄瓜幼苗生长，与对照相比幼苗株高分别增加了37.17%、27.14%、34.94%，叶绿素含量分别增加了31.37%、40.48%、43.43%，地上部鲜重分别增加了33.03%、38.81%、51.52%，地上部干重分别增加了28.66%、29.03%、46.27%。盆栽防效试验中，接种菌株XY-1、XY-13和XY-53 不同浓度发酵液10 d后，10⁸CFU/mL防治效果最优，对黄瓜根腐病防效分别为65.12%、72.09%和82.86%，此外，病原菌侵染下菌株XY-1、XY-13、XY-53促进了幼苗株高、叶绿素含量的增加。总之，XY-1、XY-13和XY-53可作为防控黄瓜苗期茄病镰刀菌引起的根腐病、促进幼苗生长有潜力的生物防治资源用于黄瓜可持续高效生产。

关键词: 生物防治, 芽孢杆菌, 茄病镰刀菌, 黄瓜根腐病, 促生

Abstract:

Cucumber root rot caused by Fusarium solani seriously affects the sustainable development of cucumber industry. In order to obtain high-efficiency antagonistic Bacillus spp. against cucumber root rot and to clarify its growth-promoting effect, Bacillus strains were isolated from the rhizosphere soil of cucumber plants, and the strains with high antagonistics to F. solani were screened, and the morphological, physiological and biochemical, genetic characteristics and plant growth-promoting characteristics were evaluated. The results showed that strain XY-1, XY-13 and XY-53 demonstrated significant inhibitory effects on F. solani, with inhibitory rates of 65.90%, 66.13% and 60.83%, respectively. XY-1 and XY-13 were identified as B. amyloliquefaciens, and XY-53 as B. subtilis. All the three strains showed phosphate-solubilizing ability, nitrogen-fixing ability, and produced protease and ACC deaminase. In the seedlings growth promotion test, inoculation with strain XY-1, XY-13 and XY-53 effectively promoted the growth of cucumber seedlings, and the seedlings’ stem lengths increased by 37.17%, 27.14% and 34.94%, respectively compared with control, and the chlorophyll content increased by 31.37%, 40.48% and 43.43%, the fresh weight of above ground increased by 33.03%, 38.81% and 51.52%, and the dry weight of above ground increased by 28.66%, 29.03% and 46.27%, respectively. In pot experiment for control efficiency, the concentrations of 10⁸ CFU/mL showed the best control efficiency on the cucumber root rot after 10 d of inoculation with XY-1, XY-13 and XY-53 different concentrations bacteria suspensions, and it was 65.12%, 72.09% and 82.86%, respectively. Moreover, the strain XY-1, XY-13 and XY-53 promoted the increase of seedlings stem length and chlorophyll content under the infection of pathogenic fungi. In conclusion, XY-1, XY-13 and XY-53 can be used as potential biocontrol resources to control cucumber seedlings root rot caused by F. solani and to simultaneously promote the growth of seedlings for sustainable and efficient cucumber production.

Key words: biological control, Bacillus spp., Fusarium solani, cucumber root rot, growth promotion

杨东亚, 祁瑞雪, 李昭轩, 林薇, 马慧, 张雪艳. 黄瓜茄病镰刀菌拮抗芽孢杆菌的筛选、鉴定及促生效果[J]. 生物技术通报, 2023, 39(2): 211-220.

YANG Dong-ya, QI Rui-xue LI, Zhao-xuan , LIN Wei, MA Hui, ZHANG Xue-yan. Screening, Identification and Growth-promoting Effect of Antagonistic Bacillus spp. Against Cucumber Fusarium solani[J]. Biotechnology Bulletin, 2023, 39(2): 211-220.

图/表 10

参考文献 44

[1]	朱绍坤, 赵文东, 孙凌俊, 等. 连作障碍及缓解措施研究进展[J]. 北方果树, 2018(4): 1-3, 11.
	Zhu SK, Zhao WD, Sun LJ, et al. Advances in alleviating the replant problem[J]. North Fruits, 2018(4): 1-3, 11.
[2]	王素亭. 设施蔬菜栽培中连作障碍及防治措施[J]. 吉林蔬菜, 2014(8): 29-30.
	Wang ST. Continuous cropping obstacles and prevention measures in the cultivation of protected vegetables[J]. Jilin Vegetable, 2014(8): 29-30.
[3]	史宣杰, 程俊跃, 刘杰, 等. 温室黄瓜根腐病的发生与综合防治[J]. 河南农业科学, 2010, 39(3): 69-70.
	Shi XJ, Cheng JY, Liu J, et al. Occurrence and integrated control of cucumber root rot in greenhouse[J]. J Henan Agric Sci, 2010, 39(3): 69-70.
[4]	刘心刚, 杨成德, 王振. 西藏设施西(黄)瓜根腐病的分离与鉴定[J]. 甘肃农业大学学报, 2018, 53(2): 80-85.
	Liu XG, Yang CD, Wang Z. Isolation and identification of root rot pathogens on protected cultivation watermelons and cucumbers in Tibet[J]. J Gansu Agric Univ, 2018, 53(2): 80-85.
[5]	刘洋. 茄病镰刀菌瓜类专化型的鉴定及其侵染途径研究[D]. 北京: 中国农业科学院, 2010.
	Liu Y. Studies on identification and infection routes of Fusarium solani f. sp. cucurbitae[D]. Beijing: Chinese Academy of Agricultural Sciences, 2010.
[6]	Sallam NA, Riad SN, Mohamed MS, et al. Formulations of Bacillus spp. and Pseudomonas fluorescens for biocontrol of cantaloupe root rot caused by Fusarium solani[J]. J Plant Prot Res, 2013, 53(3): 295-300. doi: 10.2478/jppr-2013-0044 URL
[7]	Agarwal M, Dheeman S, Dubey RC, et al. Differential antagonistic responses of Bacillus pumilus MSUA3 against Rhizoctonia solani and Fusarium oxysporum causing fungal diseases in Fagopyrum escul Moench[J]. Microbiol Res, 2017, 205: 40-47. doi: S0944-5013(17)30534-7 pmid: 28942843
[8]	程园园. 苜蓿根际芽孢杆菌的分离鉴定及特性分析[D]. 哈尔滨: 哈尔滨师范大学, 2015.
	Cheng YY. Isolation, identification and characteristics of Bacillus spp. from rhizosphere of alfalfa[D]. Harbin: Harbin Normal University, 2015.
[9]	Kim YG, Kang HK, Kwon KD, et al. Antagonistic activities of novel peptides from Bacillus amyloliquefaciens PT14 against Fusarium solani and Fusarium oxysporum[J]. J Agric Food Chem, 2015, 63(48): 10380-10387. doi: 10.1021/acs.jafc.5b04068 URL
[10]	Mnif I, Hammami I, Triki MA, et al. Antifungal efficiency of a lipopeptide biosurfactant derived from Bacillus subtilis SPB1 versus the phytopathogenic fungus, Fusarium solani[J]. Environ Sci Pollut Res Int, 2015, 22(22): 18137-18147. doi: 10.1007/s11356-015-5005-6 URL
[11]	杨晓燕, 王钰鑫, 叶伟伟, 等. 枯草芽孢杆菌对几种植物病原真菌的抑菌活性[J]. 工业微生物, 2018, 48(6): 32-38.
	Yang XY, Wang YX, Ye WW, et al. Antifungal activity of Bacillus subtilis strain against several plant pathogenic fungi[J]. Ind Microbiol, 2018, 48(6): 32-38.
[12]	Anjum MZ, Ghazanfar MU, Hussain I. Bio-efficacy of Trichoderma isolates and Bacillus subtilis against root rot of muskmelon Cucumis melo L. caused by Phytophthora drechsleri under controlled and field conditions[J]. Pak J Bot, 2019, 51(5): 1877-1882.
[13]	Punja ZK, Tirajoh A, Collyer D, et al. Efficacy of Bacillus subtilis strain QST 713(Rhapsody)against four major diseases of greenhouse cucumbers[J]. Crop Prot, 2019, 124: 104845. doi: 10.1016/j.cropro.2019.104845 URL
[14]	Freitas MA, Medeiros FHV, Melo IS, et al. Stem inoculation with bacterial strains Bacillus amyloliquefaciens(GB03)and Microbacterium imperiale(MAIIF2a)mitigates Fusarium root rot in cassava[J]. Phytoparasitica, 2019, 47(1): 135-142. doi: 10.1007/s12600-018-0706-2
[15]	林壁润, 伍尚忠. 土传病害抑病土研究进展[J]. 生物防治通报, 1992, 8(3): 137-140.
	Lin BR, Wu SZ. A review on research progress of suppressive soil against soil-borne diseases[J]. Chin J Biol Control, 1992, 8(3): 137-140.
[16]	周敬伟. 芽孢杆菌属诱导真菌形成厚垣孢子的环肽抗生素的分离及鉴定[D]. 云南: 云南大学, 2010.
	ZhouJW. Isolation and identification of cyclopeptide antibiotics that induce fungi to form chlamydospores by Bacillus[D]. Yunnan: Yunnan University, 2010.
[17]	杨革. 微生物学实验教程[M]. 2版. 北京: 科学出版社, 2010.
	Yang G. Microbiology experiment[M]. Beijing: Science Press, 2010.
[18]	蒋凯丽, 周新丽, 高海燕. 一株具有拮抗作用的解淀粉芽孢杆菌的筛选、鉴定及生物学特性研究[J]. 工业微生物, 2020, 50(1): 8-13.
	Jiang KL, Zhou XL, Gao HY. Screening, identification and biological characteristics of an antagonistic Bacillus Amyloliquefaciens[J]. Ind Microbiol, 2020, 50(1): 8-13.
[19]	Yu XM, Ai CX, Xin L, et al. The siderophore-producing bacterium, Bacillus subtilis CAS15, has a biocontrol effect on Fusarium wilt and promotes the growth of pepper[J]. Eur J Soil Biol, 2011, 47(2): 138-145. doi: 10.1016/j.ejsobi.2010.11.001 URL
[20]	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.
	Dong XZ, Cai MY. Common bacterial system identification manual[M]. Beijing: Science Press, 2001.
[21]	Wu X, Xie Y, Qiao J, et al. Rhizobacteria strain from a hypersaline environment promotes plant growth of Kengyilia thoroldiana[J]. Microbiology, 2019, 88(2): 220-231. doi: 10.1134/S0026261719020127 URL
[22]	Nautiyal CS. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms[J]. FEMS Microbiol Lett, 1999, 170(1): 265-270. doi: 10.1111/j.1574-6968.1999.tb13383.x pmid: 9919677
[23]	韩泽宇. 黄瓜高效耐盐促生菌株筛选鉴定及复合菌剂的制备[D]. 银川: 宁夏大学, 2019.
	Han ZY. The screening and identification of effective salt-tolerant and growth-promoting bacteria strains of cucumber and the preparation of the bacterial compound[D]. Yinchuan: Ningxia University, 2019.
[24]	Milagres AM, Machuca A, Napoleão D. Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S(CAS)agar plate assay[J]. J Microbiol Methods, 1999, 37(1): 1-6. pmid: 10395458
[25]	要雅倩, 成娜娜, 李培根, 等. 解淀粉芽胞杆菌Bacillus amyloliquefaciens T-6的分离鉴定及抗病促生潜力[J]. 生物技术通报, 2020, 36(9): 202-210. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0847
	Yao YQ, Cheng NN, Li PG, et al. Isolation and identification of Bacillus amyloliquefaciens T-6 and its potential of resisting disease and promoting growth[J]. Biotechnol Bull, 2020, 36(9): 202-210. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0847
[26]	Cao FM, Shen DL, Li J, et al. Multiplex-PCR approach to identify Bacillus species applied in microbial fertilizers[J]. Acta Microbiologica Sinica, 2008, 48(5): 651-656.
[27]	Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Mol Biol Evol, 2016, 33(7): 1870-1874. doi: 10.1093/molbev/msw054 pmid: 27004904
[28]	Li BJ, Liu Y, Shi YX, et al. First report of crown rot of grafted cucumber caused by Fusarium solani in China[J]. Plant Dis, 2010, 94(11): 1377. doi: 10.1094/PDIS-03-10-0217 pmid: 30743650
[29]	江欢欢, 程凯, 杨兴明, 等. 辣椒青枯病拮抗菌的筛选及其生物防治效应[J]. 土壤学报, 2010, 47(6): 1225-1231.
	Jiang HH, Cheng K, Yang XM, et al. Isolation and biological effect of capsicum wilt antagonist(a45)[J]. Acta Pedol Sin, 2010, 47(6): 1225-1231.
[30]	Chauhan AK, Maheshwari DK, Kim K, et al. Termitarium-inhabiting Bacillus endophyticus TSH42 and Bacillus cereus TSH77 colonizing Curcuma longa L.: isolation, characterization, and evaluation of their biocontrol and plant-growth-promoting activities[J]. Can J Microbiol, 2016, 62(10): 880-892. pmid: 27604298
[31]	李姝江, 朱天辉, 谯天敏, 等. 花椒根腐病生防芽孢杆菌的筛选鉴定及定殖和防治效果[J]. 西北农林科技大学学报: 自然科学版, 2016, 44(4): 114-122.
	Li SJ, Zhu TH, Qiao TM, et al. Screening, identification, colonization and control effect of biocontrol Bacillus sp. against root rot of Zanthoxylum bungeanum Maxim[J]. J Northwest A ＆ F Univ Nat Sci Ed, 2016, 44(4): 114-122.
[32]	Chen QQ, Liu B, Wang JP, et al. Antifungal lipopeptides produced by Bacillus sp. FJAT-14262 isolated from rhizosphere soil of the medicinal plant Anoectochilus roxburghii[J]. Appl Biochem Biotechnol, 2017, 182(1): 155-167. doi: 10.1007/s12010-016-2317-z URL
[33]	梁丽琼, 黄少莉, 邵杭, 等. 水稻基腐病菌拮抗菌解淀粉芽孢杆菌E3菌株的鉴定及抑菌活性[J]. 华南农业大学学报, 2021, 42(4): 51-62.
	Liang LQ, Huang SL, Shao H, et al. Identification of an antagonistic strain Bacillus amyloliquefaciens E3 against Dickeya Zeae and its antimicrobial activity[J]. J South China Agric Univ, 2021, 42(4): 51-62.
[34]	Parveen Rani R, Anandharaj M, Hema S, et al. Purification of antilisterial peptide(subtilosin A)from novel Bacillus tequilensis FR9 and demonstrate their pathogen invasion protection ability using human carcinoma cell line[J]. Front Microbiol, 2016, 7: 1910.
[35]	史一然, 徐伟慧, 吕智航, 等. 解淀粉芽孢杆菌LZN01对西瓜专化型尖孢镰刀菌的抑制效应[J]. 江苏农业科学, 2019, 47(12): 141-145.
	Shi YR, Xu WH, Lv ZH, et al. Inhibiting effects of Bacillus amyloliquefaciens LZN01 on Fusarium oxysporum f. sp. niveum[J]. Jiangsu Agric Sci, 2019, 47(12): 141-145.
[36]	Haas D, Défago G. Biological control of soil-borne pathogens by fluorescent pseudomonads[J]. Nat Rev Microbiol, 2005, 3(4): 307-319. doi: 10.1038/nrmicro1129 pmid: 15759041
[37]	杨倩, 裴红宾, 高振峰, 等. 芽孢杆菌ZJM-P5与磷肥互作对红小豆根系及产量的影响[J]. 西北植物学报, 2020, 40(7): 1192-1200.
	Yang Q, Pei HB, Gao ZF, et al. Effect of the interaction between Bacillus ZJM-P5 and phosphate fertilizer on root system and yield of adzuki bean[J]. Acta Bot Boreali Occidentalia Sin, 2020, 40(7): 1192-1200.
[38]	王永强. 解淀粉芽孢杆菌SDTB009的分离鉴定及其对番茄枯萎病的防治研究[D]. 泰安: 山东农业大学, 2020.
	Wang YQ. Isolation and identification of Bacillus amyloliquefaciens SDTB009 and its control effect on tomato Fusarium wilt[D]. Tai'an: Shandong Agricultural University, 2020.
[39]	武利勤, 顾海科, 王青, 等. 石斛内生甲基营养芽胞杆菌的拮抗和促生作用研究[J]. 生物技术通报, 2016, 32(8): 200-206. doi: 10.13560/j.cnki.biotech.bull.1985.2016.08.029
	Wu LQ, Gu HK, Wang Q, et al. Antagonistic efficacy and growth-promoting effect of Bacillus methylotrophicus isolated from Dendrobium huoshanense[J]. Biotechnol Bull, 2016, 32(8): 200-206.
[40]	张莹, 秦宇轩, 尚庆茂, 等. 解淀粉芽孢杆菌L-H15的促生与抗病特性研究[J]. 农业机械学报, 2017, 48(12): 284-291, 298.
	Zhang Y, Qin YX, Shang QM, et al. Characteristics of growth-promotion and antibiosis by Bacillus amyloliquefaciens L-H15[J]. Trans Chin Soc Agric Mach, 2017, 48(12): 284-291, 298.
[41]	张翠绵, 贾楠, 马佳, 等. 番茄根际多功能益生芽孢杆菌的筛选与鉴定[J]. 河北农业科学, 2019, 23(4): 47-52.
	Zhang CM, Jia N, Ma J, et al. Screening and identification of multifunctional beneficial Bacillus in tomato rhizosphere soil[J]. J Hebei Agric Sci, 2019, 23(4): 47-52.
[42]	Agbodjato NA, Noumavo PA, Adjanohoun A, et al. Synergistic effects of plant growth promoting rhizobacteria and chitosan on in vitro seeds germination, greenhouse growth, and nutrient uptake of maize(Zea mays L.)[J]. Biotechnol Res Int, 2016, 2016: 7830182.
[43]	王娟娟. 肥效微生物筛选及对小麦促生效果的研究[D]. 杨凌: 西北农林科技大学, 2019.
	Wang JJ. Fertilizer-functional microbes screen and the study of growth-promoting efficacy on wheat[D]. Yangling: Northwest A & F University, 2019.
[44]	王琪媛, 王甲辰, 叶磊, 等. 含ACC脱氨酶的根际细菌提高植物抗盐性的研究进展[J]. 生物技术通报, 2021, 37(2): 174-186. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0831
	Wang QY, Wang JC, Ye L, et al. Research advances on enhancement of plant resistance to salinity stress by rhizobacteria containing ACC deaminase[J]. Biotechnol Bull, 2021, 37(2): 174-186.

抑菌率Inhibition rate/%	菌株数目Number of strains
60-70	5
50-60	59
40-50	36
30-40	51
30以下	192

抑菌率Inhibition rate/%	菌株数目Number of strains
60-70	5
50-60	59
40-50	36
30-40	51
30以下	192

菌株Strain	菌落直径Colony diameter/mm	抑菌率Inhibition rate/%
XY-1	24.67±0.17b	65.90±0.23a
XY-13	24.50±0.29b	66.13±0.40a
XY-53	28.33±0.17a	60.83±0.23b

菌株Strain	菌落直径Colony diameter/mm	抑菌率Inhibition rate/%
XY-1	24.67±0.17b	65.90±0.23a
XY-13	24.50±0.29b	66.13±0.40a
XY-53	28.33±0.17a	60.83±0.23b

菌株 Strain	颜色 Color	菌落形状 Colony shape	边缘 Edge	表面光滑凸起与否 Surface is smooth and bump or not	表面干湿 Surface is dry or wet	色素产生 Pigment
XY-1	白色	圆形	光滑	表面粗糙不规则，隆起有褶皱	干燥	无色素产生
XY-13	白色	圆形	光滑	表面隆起有褶皱	湿润	无色素产生
XY-53	乳白色	圆形	光滑	表面凸起	湿润	无色素产生