棉花内生蜡状芽孢杆菌YUPP-10对棉花黄萎病的防治作用及机制

doi:10.3864/j.issn.0578-1752.2017.14.008

摘要/Abstract

摘要： 【目的】棉花黄萎病(Verticillium wilt)是世界性难以防治的病害，筛选有效的生防微生物资源成为解决棉花黄萎病的重要途径之一，本研究旨在分离一株高效拮抗细菌，并明确其防治机理，为生物防治棉花黄萎病提供技术支持。【方法】用葡甘聚糖作为碳源筛选一株能够降解β-1,4糖苷键的棉花内生细菌YUPP-10，通过平板对峙培养、平板对扣培养和悬滴法等测定YUPP-10对大丽轮枝菌(Verticillium dahliae)菌丝生长和孢子萌发的抑制效果；用YUPP-10无菌滤液培养大丽轮枝菌微菌核，检测其对微菌核萌发的影响；利用基质接种法进行盆栽试验探究其对棉花黄萎病的防治效果；通过胚根、叶片接种病原菌，木质化和活性氧爆发研究YUPP-10诱导抗病能力；采用实时荧光定量PCR（qRT-PCR）方法检测防御相关基因的表达。【结果】成功获取一株蜡状芽孢杆菌（Bacillus cereus）YUPP-10，其整体代谢产物在7 d时对大丽轮枝菌的抑菌带宽为0.73 cm，挥发性代谢产物在10 d时对大丽轮枝菌菌落的抑制率为77.03%；YUPP-10无菌培养液对大丽轮枝菌孢子和菌核萌发的抑制率分别为17.22%—71.25%和10.69%—26.62%，且抑制率与无菌滤液的浓度呈正相关。在用YUPP-10玉米蛭石培养物拌土处理的盆栽试验中，其对棉花黄萎病的最高防效达到80.60%。诱导抗性试验发现YUPP-10诱导了胚根和叶片抗大丽轮枝菌的侵染，诱导了细胞木质化和活性氧的爆发。qRT-PCR检测结果显示YUPP-10成功激活了苯丙氨酸解氨酶（PAL）、过氧化物酶（POD）、多酚氧化酶（PPO）、几丁质酶（CHI）和病程相关基因（PR10）的表达，对大丽轮枝菌在棉花体内的扩散和生长起到了抑制作用。【结论】YUPP-10通过直接抑制病原菌的生长，同时诱导植物防御反应来抵抗病原菌的侵染和扩展，其具有良好的应用前景。

关键词: 内生细菌, 棉花黄萎病, 诱导抗性, 防御机理

Abstract: 【Objective】Control of Verticillium wilt is a worldwide problem in agriculture and horticulture, screening of effective biocontrol microbial resources has become one of the important ways to control Verticillium wilt. The use of microbial antagonists to control these pathologies fits modern sustainable agriculture criteria. The objective of this study is to screen an efficient antagonistic bacteria, and characterize the biocontrol mechanism of bacteria against Verticillium wilt, thus providing a technical basis for control of Verticillium wilt of cotton with biocontrol bacteria. 【Method】An endophytic bacterium, which can hydrolyze polysaccharides with β-1,4 linkage, was isolated by enrichment medium, which was made of konjac glucomannan as carbon source. The inhibition and prevention of YUPP-10 against V. dahliae and Verticillium wilt were tested. The inhibition rate of YUPP-10 against V. dahliae was assessed using the confront culture method, enclosed chamber test and hanging drop method, cultured microsclerotia with aseptic culture filtrate, and the control effect of cottonon Verticillium wilt was detected by substrate inoculation method in a pot experiment. Induced disease resistance of YUPP-10 in cotton were analysed by embryo and leaves inoculated with V. dahliae, sedimentation of lignin and reactive oxygen species (ROS). In addition, the expression level of defense genes in G. hirsutum leaves were detected by real-time quantitative PCR (qRT-PCR). 【Result】A Bacillus cereus isolate YUPP-10 was screened. The results showed that YUPP-10 and its volatile organic compounds significantly inhibited the colony growth of V. dahliae, with the width of the inhibition zone of YUPP-10 was 0.73 cm after 7 days, and the inhibition rate of volatile organic compounds of YUPP-10 was 77.03% after 10 days. B. cereus YUPP-10 culture filtrate (CF) suppressed V. dahliae spore and microsclerotia germination in a dose-dependent manner. The inhibition rate of CF on V. dahliae spore and microsclerotia germination ranged from 17.22% to 71.25% and 10.69% to 26.62%, respectively. The inhibition rate was 80.60%, when cotton seedlings were pro-inoculated with substrate containing YUPP-10 in a pot experiment. YUPP-10 induced disease resistance, including immature embryo and leaves against V. dahliae, ROS, sedimentation of lignin and some defense genes, such as PAL, POD, PPO, CHI and PR10.【Conclusion】It was concluded that YUPP-10 is an efficient biocontrol agent that protects cotton plant from V. dahliae infection. Our data demonstrate that V. dahliae growth is restricted and the additional signals from YUPP-10 must participate in the regulation of the immune response against V. dahliae. Therefore, the YUPP-10 has a great potential in controlling Verticillium wilt.

Key words: endophytic bacterium, Verticillium wilt, induced resistance, defense mechanism

周京龙，冯自力，冯鸿杰，李云卿，袁媛，李志芳，魏锋，师勇强，赵丽红，孙正祥，朱荷琴，周燚. 棉花内生蜡状芽孢杆菌YUPP-10对棉花黄萎病的防治作用及机制[J]. 中国农业科学, 2017, 50(14): 2717-2727.

ZHOU JingLong, FENG ZiLi, FENG HongJie, LI YunQing, YUAN Yuan, LI ZhiFang, WEI Feng, SHI YongQiang, ZHAO LiHong, SUN ZhengXiang, ZHU HeQin, ZHOU Yi. Biocontrol Effect and Mechanism of Cotton Endophytic Bacterium Bacillus cereus YUPP-10 Against Verticillium Wilt in Gossypium hirsutum[J]. Scientia Agricultura Sinica, 2017, 50(14): 2717-2727.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.14.008

https://www.chinaagrisci.com/CN/Y2017/V50/I14/2717

参考文献

[1] Zhang Z, Zhao J, Ding L, Zou L, Li Y, Chen G, Zhang T. Constitutive expression of a novel antimicrobial protein, Hcm1, confers resistance to both Verticillium and Fusarium wilts in cotton. Scientific Reports, 2016, 6: 20773.

[2] Holliday P, Holliday P. A dictionary of plant pathology. New York: Cambridge University Press, 2001.

[3] Schulz B, Boyle C. What are Endophytes? //Microbial Root Endophytes. Berlin: Springer, 2006.

[4] Ryan R P, Germaine K, Franks A, Ryan D J, Dowling D N. Bacterial endophytes: recent developments and applications. FEMS Microbiology Letters, 2008, 278(1): 1-9.

[5] Berg G, Eberl L, Hartmann A. The rhizosphere as a reservoir for opportunistic human pathogenic bacteria. Environmental Microbiology, 2005, 7(11): 1673-1685.

[6] 李社增, 鹿秀云, 马平, 高胜国, 刘杏忠, 刘干. 防治棉花黄萎病的生防细菌NCD-2的田间效果评价及其鉴定. 植物病理学报, 2005, 35(5): 451-455.

Li S Z, Lu X Y, Ma P, Gao S G, Liu X Z, Liu G. Evaluation of biocontrol potential of a bacterial strain NCD-2 against cotton verticillium wilt in field trials. Acta Phytopathologica Sinica, 2005, 35(5): 451-455. (in Chinese)

[7] Li B, Li Q, Xu Z, Zhang N, Shen Q, Zhang R. Responses of beneficial Bacillus amyloliquefaciens SQR9 to different soilborne fungal pathogens through the alteration of antifungal compounds production. Frontiers in Microbiology, 2014, 5: 636.

[8] Pereg L, Mcmillan M. Scoping the potential uses of beneficial microorganisms for increasing productivity in cotton cropping systems. Soil Biology & Biochemistry, 2015, 80: 349-358.

[9] Pleban S, Chernin L, Chet I. Chitinolytic activity of an endophytic strain of Bacillus cereus. Letters in Applied Microbiology, 1997, 25(4): 284-288.

[10] Li J G, Jiang Z Q, Xu L P, Sun F F, Guo J H. Characterization of chitinase secreted by Bacillus cereus strain CH2 and evaluation of its efficacy against Verticillium wilt of eggplant. BioControl, 2008, 53(6): 931-944.

[11] Kishore G K, Pande S. Chitin-supplemented foliar application of chitinolytic Bacillus cereus reduces severity of Botrytis gray mold disease in chickpea under controlled conditions. Letters in Applied Microbiology, 2007, 44(1): 98-105.

[12] Hammami I, Siala R, Jridi M, Ktari N, Nasri M, Triki M A. Partial purification and characterization of chiIO8, a novel antifungal chitinase produced by Bacillus cereus IO8. Journal of Applied Microbiology, 2013, 115(2): 358-366.

[13] Wang J, Zhang L, Teng K, Sun S, Sun Z, Zhong J. Cerecidins, novel lantibiotics from Bacillus cereus with potent antimicrobial activity. Applied & Environmental Microbiology, 2014, 80(8): 2633-2643.

[14] Niu D D, Liu H X, Jiang C H, Wang Y P, Wang Q Y, Jin H L, Guo J H. The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces systemic resistance in Arabidopsis thaliana by simultaneously activating salicylate- and jasmonate/ethylene-dependent signaling pathways. Molecular Plant-Microbe Interactions, 2011, 24(5): 533-542.

[15] Gong C, Liu Y, Liu S Y, Cheng M Z, Zhang Y, Wang R H, Chen H Y, Li J F, Chen X L, Wang A X. Analysis of Clonostachys rosea-induced resistance to grey mould disease and identification of the key proteins induced in tomato fruit. Postharvest Biology & Technology, 2017, 123: 83-93.

[16] Huang C J, Tsay J F, Chang S Y, Yang H P, Wu W S, Chen C Y. Dimethyl disulfide is an induced systemic resistance elicitor produced by Bacillus cereus C1L. Pest Management Science, 2012, 68(9): 1306-1310.

[17] Katsuraya K, Okuyama K, Hatanaka K, Oshima R, Sato T, Matsuzaki K. Constitution of konjac glucomannan: chemical analysis and ¹³C NMR spectroscopy. Carbohydrate Polymers, 2003, 53(2): 183-189.

[18] Fang W, Wu P. Variations of konjac glucomannan (KGM) from Amorphophallus konjac and its refined powder in China. Food Hydrocolloids, 2004, 18(1): 167-170.

[19] Lafarge C, Cayot N, Hory C, Goncalves L, Chassemont C, Le Bail P. Effect of konjac glucomannan addition on aroma release in gels containing potato starch. Food Research International, 2014, 64: 412-419.

[20] Pangburn S H, Trescony P V, Heller J. Lysozyme degradation of partially deacetylated chitin, its films and hydrogels. Biomaterials, 1982, 3(2): 105-108.

[21] Whipps J M, Magan N. Effects of nutrient status and water potential of media on fungal growth and antagonist-pathogen interactions1. Bulletin Oepp/eppo Bulletin, 1987, 17: 581-591.

[22] Li S K, Ji Z Q, Zhang J W, Guo Z Y, Wu W J. Synthesis of 1-acyl-3-isopropenylbenzimidazolone derivatives and their activity against Botrytis cinerea. Journal of Agricultural & Food Chemistry, 2010, 58(5): 2668-2672.

[23] Hu X, Bai Y, Chen T, Hu D, Yang J, Xu X. An optimized method for in vitro production of Verticillium dahliae microsclerotia. European Journal of Plant Pathology, 2013, 136(2): 225-229.

[24] López-Escudero F J, Mwanza C, Blanco-López M A. Reduction of Verticillium dahliae microsclerotia viability in soil by dried plant residues. Crop Protection, 2007, 26(2): 127-133.

[25] 朱荷琴, 冯自力, 李志芳, 赵丽红, 师勇强. 蛭石沙土无底纸钵定量蘸菌液法鉴定棉花品种(系)的抗黄萎病性. 中国棉花, 2010(12): 15-17.

Zhu H Q, Feng Z L, Li Z F, Zhao L H, Shi Y Q. Dip quantitative fungal solution method by using vermiculite sand without bottom paper bowl to identify cotton varieties (lines) of resistance to Verticillium wilt. China Cotton, 2010(12): 15-17. (in Chinese)

[26] 赵丽红, 冯自力, 李志芳, 冯鸿杰, 师勇强, 张芸, 朱荷琴. 棉花抗黄萎病鉴定与评价标准的商榷. 棉花学报, 2017, 29(1): 50-58.

Zhao L H, Feng Z L, Li Z F, Feng H J, Shi Y Q, Zhang Y, Zhu H Q. Development of an improved standard for identifying and evaluating verticillium wilt resistance in cotton. Cotton Science, 2017, 29(1): 50-58. (in Chinese)

[27] 张芸, 冯自力, 冯鸿杰, 李志芳, 师勇强, 赵丽红, 朱荷琴, 杨家荣. 内生球毛壳属真菌CEF-082对棉花黄萎病的控制作用. 植物病理学报, 2016, 46(5): 697-706.

Zhang Y, Feng Z L, Feng H J, Li Z F, Shi Y Q, Zhao L H, Zhu H Q, Yang J R. Control effect of endophytic fungus Chaetomium globosum CEF-082 against Verticillium wilt in Gossypium hirsutum. Acta Phytopathologica Sinica, 2016, 46(5): 697-706. (in Chinese)

[28] Wubben M J, Callahan F E, Hayes R W, Jenkins J N. Molecular characterization and temporal expression analyses indicate that the MIC (Meloidogyne Induced Cotton) gene family represents a novel group of root-specific defense-related genes in upland cotton (Gossypium hirsutum L.). Planta, 2008, 228(1): 111-123.

[29] Han Q, Wu F, Wang X, Qi H, Shi L, Ren A, Liu Q, Zhao M, Tang C. The bacterial lipopeptide iturins induce Verticillium dahliae cell death by affecting fungal signalling pathways and mediate plant defence responses involved in pathogen-associated molecular pattern-triggered immunity. Environmental Microbiology, 2015, 17(4): 1166-1188.

[30] Kuiper I, Lagendijk E L, Bloemberg G V, Lugtenberg B J. Rhizoremediation: a beneficial plant-microbe interaction. Molecular plant-microbe interactions, 2004, 17(1): 6-15.

[31] Roderick G K, Hufbauer R, Navajas A M. Evolution and biological control. Evolutionary Applications, 2012, 5(5): 419-423.

[32] Nakamura N, Nakano K, Sugiura N, Matsumura M. A novel cyanobacteriolytic bacterium, Bacillus cereus, isolated from a Eutrophic Lake. Journal of Bioscience & Bioengineering, 2003, 95(2): 179-184.

[33] Kai M, Effmert U, Berg G, Piechulla B. Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani. Archives of Microbiology, 2007, 187(5): 351-360.

[34] Vespermann A, Kai M, Piechulla A B. Rhizobacterial volatiles affect the growth of fungi and Arabidopsis thaliana. Applied & Environmental Microbiology, 2007, 73(17): 5639-5641.

[35] Sharifi R, Ryu C M. Are bacterial volatile compounds poisonous odors to a fungal pathogen Botrytis cinerea, alarm signals to Arabidopsis seedlings for eliciting induced resistance, or both? Frontiers in Microbiology, 2016, 7: 196.

[36] Song G C, Ryu C M. Two volatile organic compounds trigger plant self-defense against a bacterial pathogen and a sucking insect in cucumber under open field conditions. International Journal of Molecular Sciences, 2013, 14(5): 9803-9819.

[37] Thimmaraju R, Meredith L B, Kunjeti S G, Donofrio N M, Czymmek K J, Paré P W, Bais H P. The rhizobacterial elicitor acetoin induces systemic resistance in Arabidopsis thaliana. Communicative & Integrative Biology, 2010, 3(2): 130-138.

[38] Johri B N, Sharma A, Virdi J S. Rhizobacterial diversity in India and its influence on soil and plant health. Advances in Biochemical Engineering/biotechnology, 2003, 84: 49-89.

[39] González-Sánchez M Á, Vicente A D, Pérez-García A, Pérez-Jiménez R, Romero D, Cazorla F M. Evaluation of the effectiveness of biocontrol bacteria against avocado white root rot occurring under commercial greenhouse plant production conditions. Biological Control, 2013, 67(2): 94-100.

[40] Pieterse C M J, Zamioudis C, Berendsen R L, Weller D M, Van Wees S C, Bakker P A. Induced systemic resistance by beneficial microbes. Annual Review of Phytopathology, 2014, 52: 347-375.

[41] Boller T, Felix G. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annual Review of Plant Biology, 2009, 60: 379-406.

[42] Borges A A, Jiménezarias D, Sandalio L M, Pérez J A. Priming crops against biotic and abiotic stresses: MSB as a tool for studying mechanisms. Frontiers in Plant Science, 2014, 5: 642.

[43] Bhuiyan N H, Selvaraj G, Wei Y, King J. Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion. Journal of Experimental Botany, 2009, 60(2): 509-521.

[44] Wang N, Liu M, Guo L, Yang X, Qiu D. A novel protein elicitor (PeBA1) from Bacillus amyloliquefaciens NC6 induces systemic resistance in tobacco. International Journal of Biological Sciences, 2016, 12(6): 757-767.