大丽轮枝菌弱致病力菌株Vd171对棉花黄萎病的诱导免疫作用及机制

doi:10.3864/j.issn.0578-1752.2018.06.006

摘要/Abstract

摘要： 【目的】明确大丽轮枝菌（Verticillium dahliae）弱致病力菌株Vd171对棉花黄萎病的诱导免疫效果及作用机制, 为探索棉花黄萎病生物防治新途径提供依据。【方法】以大丽轮枝菌弱致病力菌株Vd171为研究对象，温室苗期评估不同接种方法、接种时期、接种次数及接种体类型的诱导免疫效果；不同接种方法包括蘸根、灌根、茎部针刺、叶面喷雾和浸种，均采用Vd171分生孢子悬浮液（1×10⁷个分生孢子/mL），蘸根、灌根和叶面喷雾为每钵10 mL，茎部针刺为每株0.2 mL，浸种时间为12 h；不同接种时期试验设置接种棉花黄萎病菌强致病力菌株Vd080前2、4、6、8 d及之后的1、2、4 d接种Vd171共7个处理；接种次数试验设置接种Vd080前接种Vd171 一次和两次；接种体类型采用Vd171的分生孢子悬浮液和查氏培养滤液。采用水培方法培育棉苗，先接种Vd171，4 d后接种Vd080GFP，不同时间取根组织，振荡冲洗Vd080GFP分生孢子，显微镜下计数，测定弱致病力菌株免疫后病原菌在棉苗根部的定殖量；接种Vd080GFP后第7天，PDA平板上分离棉苗下胚轴切段，检测病原菌在植株体内的扩展速度。取接种Vd171后不同时段的棉苗，采用qPCR方法检测植株内防御相关酶基因4CL、CHI、POD、PPO和PAL的表达量；测定POD、PPO、PAL和CHI的活性变化。将棉苗茎部做切片，用间苯三酚染色，显微镜下观察茎部维管束木质化情况；将棉苗叶片用DAB染色，观察叶片活性氧爆发情况。【结果】在接种大丽轮枝菌强致病力菌株Vd080前4 d接种Vd171，棉株的免疫效果最好，防治效果达到89.4%；几种接种方法相比较，以蘸根防治效果最好，为70.0%，其次是叶面喷雾，为54.3%，浸种和灌根分别为45.0%和39.0%，而针刺效果最差，为2.2%；接种Vd171一次和两次对棉株均具有较好的免疫效果，其防治效果分别为85.6%和81.4%；先接种分生孢子、培养滤液对棉株均具有较好的免疫效果，其防治效果分别为85.6%和81.1%；先接种Vd171能阻止Vd080在棉花根部的定殖和在植株体内的扩展；Vd171能显著诱导棉苗防御相关基因PAL、4CL和CHI的表达，其中，GhPAL、Gh4CL和GhCHI变化最大，分别为对照处理的2.2、8.5和2.6倍，均显著或极显著高于对照；经Vd171诱导的棉苗下胚轴中PPO、PAL、POD和CHI酶活性均极显著高于对照，分别较对照提高31.9%、131.0%、57.1%和102.1%，子叶中PAL、CHI和POD酶活性也显著高于对照，分别较对照提高22.1%、39.6%和7.1%，PPO酶活性与对照无显著差异；先接种Vd171诱导了细胞木质化和活性氧的爆发。【结论】大丽轮枝菌弱致病力菌株Vd171可以有效地诱导棉花对黄萎病产生抗性，在棉花黄萎病防治上具有较好的应用前景。

关键词: 棉花, 黄萎病, 大丽轮枝菌, 弱致病力菌株, 诱导免疫

Abstract: 【Objective】 The objective of this study is to clarify the induced immunity effect and mechanism of the weak pathogenicity isolate of Verticillium dahliae Vd171 against Verticillium wilt in cotton, and to provide a basis for exploring new ways of biological control of Verticillium wilt in cotton. 【Method】 The weak pathogenicity isolate of V. dahlia Vd171 was used to evaluate the induced immune effects of different inoculation methods, inoculation periods, inoculation times and inoculum types in greenhouse. Different inoculation methods included root-dipping, root-irrigation, stem-injection, foliar-spraying and seed-soaking by Vd171 spore suspension with 1×10⁷ conidia/mL. Ten mL spore suspension for each pot was used in root-dipping, root-irrigation and foliar-spraying and 0.2 mL for each plant in stem-injection. The seeds were soaked for 12 h with spore suspension. Seven treatments were set to confirm the inoculation periods of Vd171 which including 2, 4, 6, 8 days before inoculated Vd080 (a high pathogenicity isolate of V. dahliae) and 1, 2, 4 days after inoculated Vd080. The inoculation times of Vd171 was one or two times before inoculated Vd080. The inoculum types included spore suspension and filtrate of Czapek of Vd171. The cotton seedlings were cultivated by hydroponics and inoculated by Vd080_GFP 4 days after Vd171. Then the roots were collected in different times and the spores of Vd080_GFP were washed in sterile water. The colonization of Vd080_GFPon cotton rootwas analyzed by counting the spores of Vd080_GFPunder microscope. Furthermore, in order to analyze the extension of Vd080 in cotton stem, the small hypocotyl pieces was cultivated on PDA plates at the 7th day after inoculated by Vd080_GFP. The transcript level of 4CL, CHI, POD, PPO and PAL was analyzed by qPCR. The enzyme activity of POD, PPO, PAL and CHI was measured. The sedimentation of lignin was observed by phloroglucinol staining and the reactive oxygen species (ROS) of leaves was observed by DAB staining. 【Result】 The highest control efficacy was obtained when Vd171 was inoculated 4 days prior to inoculation by Vd080 (control efficacy of 89.4%). The control efficacy had significant differences among the five inoculation methods. Root-dipping treatment provided a control efficacy of 70.0%, followed by foliar-spraying (54.3%), seed-soaking (45.0%), root-irrigation (39.0%), and stem-injection (2.2%). There was no statistical significant difference between the control efficacy against Vd080 of pre-inoculated by Vd171 for one and two times (85.6% and 81.4%). Particularly, both cotton pre-inoculated with conidiospore and culture filtrate of Vd171 treatments could reduce disease indices of Verticillium wilt, with the control efficacy of 85.6% and 81.1%. There was no significant difference between the two treatments. Cotton plants pre-inoculated with Vd171 could reduce the colonization of Vd080 in cotton root and prevent its expansion in cotton stem. In addition, the transcript level of GhPAL, Gh4CL and GhCHI increased significantly by induction of Vd171, which was 2.2, 8.5 and 2.6 times of the control treatment, respectively. The activity of PPO, PAL, POD and CHI in hypocotyl increased by 31.9%, 131.0%, 57.1% and 102.1% than that of control treatment, respectively, and activity of PAL, CHI and POD in cotyledon also remarkably increased by 22.1%, 39.6% and 7.1% than that of control treatment, respectively. Meanwhile, sedimentation of lignin and ROS were induced by Vd171.【Conclusion】 The weak virulent isolate Vd171 can effectively induce cotton resistant to Verticillium wilt and has a good application prospect in the control of Verticillium wilt in cotton.

Key words: cotton, Verticillium wilt, Verticillium dahliae, weak pathogenicity isolate,

')">induced immunity

张一豪，冯鸿杰，袁媛，靳羽莹，师勇强，张朝军，李付广. 大丽轮枝菌弱致病力菌株Vd171对棉花黄萎病的诱导免疫作用及机制[J]. 中国农业科学, 2018, 51(6): 1067-1078.

ZHANG YiHao, FENG HongJie, YUAN Yuan, JIN YuYing, SHI YongQiang, ZHANG ChaoJun, LI FuGuang. Induced Immunity Effect and Mechanism of the Weak Pathogenicity Isolate of Verticillium dahliae Vd171 Against Verticillium Wilt in Cotton[J]. Scientia Agricultura Sinica, 2018, 51(6): 1067-1078.

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.0578-1752.2018.06.006

https://www.chinaagrisci.com/CN/Y2018/V51/I6/1067

参考文献

[1] 朱荷琴, 简桂良, 宋晓轩. 棉田黄萎病菌致病型群落结构研究. 棉花学报, 2004, 16(3): 147-151.

ZHU H Q, JIAN G L, SONG X X. Community structure of pathogenic type of Verticillium dahliae Kleb. in cotton field. Cotton science, 2004, 16(3): 147-151. (in Chinese)

[2] 简桂良, 卢美光, 朱荷琴. 棉田黄萎病菌致病型结构初步研究. 植物保护学报, 2005, 32(1): 109-110.

JIAN G L, LU M G, ZHU H Q. A preliminary study on the virulence of pathogenicity of Verticillium dahliae in cotton field. Acta Phytophylacica Sinica, 2005, 32(1): 109-110. (in Chinese)

[3] 贾涛, 裴国亮, 杨家荣, 徐进, 简桂良. 棉田土壤中棉花黄萎病菌的致病力分化. 植物保护学报, 2007, 34(5): 519-523.

JIA T, PEI G L, YANG J R, XU J, JIAN G L. Pathogenicity differentiation of Verticillium dahliae of cotton in naturally infested soil. Acta Phytophylacica Sinica, 2007, 34(5): 519-523. (in Chinese)

[4] 赵蕾, 滕安娜. 木霉对植物的促生及诱导抗性研究进展. 植物保护, 2010, 36(3): 43-46.

ZHAO L, TENG A N. Research progress in Trichoderma-stimulated plant growth and induced plant resistance. Plant protection, 2010, 36(3): 43-46. (in Chinese)

[5] MACE M E. Contribution of tyloses and terpenoid aldehyde phytoalexins to Verticillium wilt resistance in cotton. Physiological Plant Pathology, 1978, 12(1): 1-11.

[6] 吴洵耻, 刘波. 棉花黄萎病菌菌株间交互保护作用的研究(1)两菌株有效间隔天数的试验. 植物病理学报, 1987, 17(4): 215-218.

WU X C, LIU B. The interprotection between strains of Verticillium dahlia Kleb (1) Experiment of effective intervals between strains. Acta Phytopathologica Sinica, 1987, 17(4): 215-218. (in Chinese)

[7] ZHU H Q, FENG Z L, Li Z F, SHI Y Q, ZHAO L H, YANG J R. Characterization of two fungal isolates from cotton and evaluation of their potential for biocontrol of Verticillium wilt of cotton. Journal of Phytopathology, 2013, 161(2): 70-77.

[8] 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, 37(12): 15-17.

ZHU H Q, FENG Z L, LI Z F, ZHAO L H, SHI Y Q. A new method for identification of cotton resistance to Verticillium wilt with vermiculate and sand in bottomless paper pot dipping in quantitative spores. China Cotton, 2010, 37(12): 15-17. (in Chinese)

[9] 姚耀文, 傅翠真, 王文录, 李庆基, 张元恩, 陈璧, 张中义, 穆罕默德·依明·艾沙, 邓先明, 张树琴, 郜树德, 李腾友. 棉花黄萎病菌生理型鉴定的初步研究. 植物保护学报, 1982, 9(3): 145-148.

YAO Y W, FU C Z, WANG W L, LI Q J, ZHANG Y N, CHEN B, ZHANG Z Y, Muhammed Amin Eysa , DENG X M, ZHANG S Q, GAO S D, LI T W. Preliminary studies on physiological forms of cotton Verticillium wilt fugus. Acta Phytophylacica Sinica, 1982, 9(3): 145-148. (in Chinese)

[10] 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)

[11] FERRER M A, CALDERÓN A A, MUÑOZ R, BARCELÓ A R. 4-Methoxy-α-naphthol as a specific substrate for kinetic, zymographic and cytochemical studies on plant peroxidase activities. Phytochemical Analysis, 1990, 1(2): 63-69.

[12] ROBISON S P, DRY I B. Broad bean leaf polyphenol oxidase is a 60-kilodalton protein susceptible to proteolytic cleavage. Plant Physiology, 1992, 99(1): 317-323.

[13] PODILE A R, LAXMI V D V. Seed bacterization with Bacillus subtilis AF1 increases phenylalanine ammonia-lyase and reduces the incidence of Fusarial wilt in pigeonpea. Journal of Phytopathology, 1998, 146(5/6): 255-259.

[14] REISSIG J L, STORMINGER J L, LELOIR L F. A modified colorimetric method for the estimation of n-acetylamino sugars. Journal of Biological Chemistry, 1955, 217(2): 959-966.

[15] 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. 中国农业科学, 2017, 50(14): 2717-2727.

ZHOU J L, FENG Z L, FENG H J, LI Y Q, YUAN Y, LI Z F, WEI F, SHI Y Q, ZHAO L H, SUN Z X, ZHU H Q, ZHOU Y. Biocontrol effect and mechanism of cotton endophytic bacterium Bacillus cereus YUPP-10 against Verticillium wilt in Gossypium hirsutum. Scientia Agricultura Sinica, 2017, 50(14): 2717-2727. (in Chinese)

[16] DODDS J A. Cross-protection and systemic acquired resistance for control of plant diseases//Bellows T S, Fisher T W, Caltagirone L E, Dahlsten D L, Gordh G, Huffaker C B. Handbook of Biological Control. Academic Press, 1999: 549-556.

[17] 俞振明, 李家玉, 林志华, 张奇, 何海斌. 植物抗性诱导防御病虫草害的研究进展. 农业科学研究, 2013, 34(2): 69-76.

YU Z M, LI J Y, LIN Z H, ZHANG Q, HE HＢ. Research advances on induced-defense of plant resistance. Journal of Agricultural Science, 2013, 34(2): 69-76. (in Chinese)

[18] 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.

[19] 吴蔼民, 顾本康, 傅正擎, 蒋正峰. 内生菌73a防治棉花黄萎病机理. 江苏农业学报, 2002, 18(1): 48-51.

WU A M, GU B K, FU Z Q, JIANG Z F. Mechanism of controlling cotton Verticillium wilt with entophytic bacterium 73a. Jiangsu Journal of Agricultural Sciences, 2002, 18(1): 48-51. (in Chinese)

[20] 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)

[21] 田绍仁, 聂太礼, 王梦亮, 杨军. 拮抗细菌C-02防治棉花黄萎病的机理研究. 棉花学报, 2012, 24(5): 420-426.

TIAN S R, NIE T L, WANG M L, YANG J. Study on antagonistic mechanism of bacterium strain C-02 against Verticillium wilt of cotton. Cotton Science, 2012, 24(5): 420-426. (in Chinese)

[22] Schnathorst W C, MATHRE D E. Cross protection in cotton with strains of Verticillium albo-atrum. Phytopathology, 1996, 56: 1204?1209.

[23] VAGELAS I, LEONTOPOULOS S. Cross-protection of cotton against Verticillium wilt by Verticillium nigrescens. Emirates Journal of Food and Agriculture, 2015, 27(9): 687-691.

[24] Borges A A, Jiménez-arias D, Expósito-Rodríguez M, 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.

[25] 薛鑫, 张芊, 吴金霞. 植物体内活性氧的研究及其在植物抗逆方面的应用. 生物技术通报, 2013(10): 6-11.

XUE X, ZHANG Q, WU J X. Research of reactive oxygen species in plants and its application on stress tolerance. Biotechnology Bulletin, 2013(10): 6-11. (in Chinese)

[26] 李莉, 赵越, 马君兰. 苯丙氨酸代谢途径关键酶: PAL、C4H、4CL研究新进展. 生物信息学, 2005, 4(5): 187-189.

LI L, ZHAO Y, MA J L. Recent progress on key enzymes: PAL, C4H, 4CL of phenylalanine metabolism pathway. China Journal of Bioinformatics, 2005, 4(5): 187-189. (in Chinese)

[27] Tuan P A, Li X, Park N I, Lee S Y, Kim H H, Park S U. Molecular cloning of 4-coumarate: CoA ligase and total phenolic content in garlic (Allium sativum). Plant Omics Journal, 2011, 4(1): 25-28.

[28] 欧阳光察, 薛应龙. 植物苯丙烷类代谢的生理意义及其调控. 植物生理学通讯, 1988(3): 9-16.

OUYANG G C, XUE Y L. Physiological role and regulation of phenylpropanoid in plant. Plant Physiology Communications, 1988(3): 9-16. (in Chinese)

[29] 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.

[30] MOHAMMADI M, ROOHPARVAR R, TORABI M. Induced chitinase activity in resistant wheat leaves inoculated with an incompatible race of puccinia striiformis f. sp. tritici, the causal agent of yellow rust disease. Mycopathologia, 2002, 154(3): 119-126.