棉花内生细菌YUPP-10及其分泌蛋白CGTase对棉花枯萎病的防治作用及机理

doi:10.3864/j.issn.0578-1752.2021.17.011

摘要/Abstract

摘要：

【目的】棉花枯萎病（Fusarium wilt）是棉花种植中较为严重的土传病害之一,主要采用化学方法进行防治,但对环境和人畜安全造成一定影响。生物防治因其专一性强、安全性高的特点,成为防治棉花枯萎病的重要途径。本研究旨在获得一株高效拮抗细菌,并明确其防治机理,为棉花枯萎病生物防治提供技术支持。【方法】前期获得一株能够破坏β-1,4糖苷键的棉花内生蜡状芽孢杆菌（Bacillus cereus）YUPP-10,通过平板对峙培养、共培养法和悬滴法分别测试其对棉花枯萎病菌（尖镰孢,Fusarium oxysporum）菌丝生长、产孢和孢子萌发的影响;利用YUPP-10菌液浸种处理,研究其对棉花生物量的影响;以尖镰孢菌土为基质种植棉花,生长1周后,用LB液体培养基培养的YUPP-10制成不同浓度（分别为1×10⁸、1×10⁷和1×10⁶ cfu/mL）的生防菌剂灌根处理棉苗,于温室中进行棉花枯萎病的防治效果研究;通过Fosmid文库筛选到了YUPP-10关键抑菌物质为环糊精糖基转移酶（CGTase,EC 2.4.1.19）,研究其对尖镰孢菌丝生长、产孢和孢子萌发的影响;利用拟南芥花序侵染法将其转化拟南芥,获得能够稳定表达CGTase的转基因株系,研究转基因拟南芥对枯萎病的抗性,以及在病原胁迫下部分防御基因的转录。【结果】YUPP-10菌株发酵液对尖镰孢菌丝生长、产孢和孢子萌发具有显著的抑制作用,随着发酵液浓度的升高,对尖镰孢产孢量和孢子萌发的抑制率越高,最高抑制率分别为98.41%和51.65%。低浓度的YUPP-10菌液能促进棉花种子发芽、出苗和地上部分的生长。YUPP-10发酵液灌根处理后,棉苗的病情指数和病株率显著降低,其中,1×10⁷ cfu/mL的YUPP-10发酵液对棉花枯萎病的防治效果最高,达到45.11%。YUPP-10菌株的关键抑菌物质CGTase能够水解羧甲基纤维素和葡甘聚糖,表明其具有破坏β-1,4糖苷键的能力,进而检测了其对尖镰孢的抑制效果,结果表明CGTase对尖镰孢的菌丝生长、产孢和孢子萌发具有显著的抑制作用,对产孢和孢子萌发的最高抑制率分别达到62.63%和30.83%。转CGTase的拟南芥提高了对枯萎病的抗性,过表达CGTase的拟南芥在病原胁迫下部分防御基因的转录水平升高。【结论】蜡状芽孢杆菌YUPP-10菌株能抑制尖镰孢的生长、促进棉花部分生物量指标的增长并控制枯萎病的危害,CGTase能够抑制尖镰孢的生长,转CGTase拟南芥对枯萎病的抗性增强。

关键词: 蜡状芽孢杆菌, 棉花枯萎病, 尖镰孢萎蔫专化型病菌, 环糊精糖基转移酶, 防治效果

Abstract:

【Objective】 Fusarium wilt is one of the most important soil-borne diseases in cotton planting. Chemical methods are mainly used to control this disease, however, it has certain impact on the environment, human and animal safety. Biological control has become an important way to control cotton Fusarium wilt because of its high specificity and safety. The objective of this study is to screen an efficient antagonistic bacteria, and characterize the biocontrol mechanism of bacteria against Fusarium wilt, thus providing a technical basis for cotton Fusarium wilt control with biocontrol bacteria. 【Method】 In a previous study, an endophytic bacterium Bacillus cereus YUPP-10 was isolated from vascular of cotton, which can hydrolyze polysaccharides with β-1,4 linkage. The effects of YUPP-10 on hyphal growth, sporulation, and spore germination of F. oxysporum were tested using the confront culture method, enclosed chamber test and hanging drop method, respectively. The seeds were soaked by YUPP-10, and then, the seeding germination and the biomass of cotton were detected. The cotton were cultivated in substrate with F. oxysporum, and after a week of growth, YUPP-10 cultured with LB liquid medium was treated at different concentrations (1×10 ⁸, 1×10⁷and 1×10⁶ cfu/mL, respectively), and the control efficacy against Fusarium wilt was studied in the greenhouse. The key antibacterial substances of YUPP-10 were obtained by Fosmid library, and the direct effects of recombinant cyclodextrin glycosyltransferase (CGTase) on F. oxysporum hyphal growth, sporulation, and spore germination were studied. The overexpression vector was transformed into Arabidopsis thaliana Col-0 via the floral dip method. The resistance of CGTase-overexpressing transgenic plants against Fusarium wilt was assessed with an in vitro technique. The transcriptional levels of some defense genes were analyzed under pathogen challenge. 【Result】 YUPP-10 significantly inhibited the hyphal growth, sporulation, and spore germination of F. oxysporum, the most inhibition rate of spore yield and germination was 98.41% and 51.65%, respectively. Low concentration of YUPP-10 could promote the germination rate, emergence rate and stem length of cotton seeds. After the treatment of YUPP-10, the diseased plant rate and disease index were significantly lower than those of the control group. The control efficacy was 45.11% at the concentration of 1×10 ⁷ cfu/mL. CGTase was the key antimicrobial substance of YUPP-10, the effects of added CGTase on the transparent circles of carboxymethyl cellulose and glucomannan were measured, the results showed that CGTase could hydrolyze polysaccharides with β-1,4 linkage. CGTase also had significant inhibitory effects on the growth, sporulation and spore germination of the pathogen, the most inhibition rate of spore yield and germination was 62.63% and 30.83%, respectively. The CGTase-overexpressing A. thaliana enhanced disease resistance by enhancing the expression of defense genes. 【Conclusion】 YUPP-10 is an efficient biocontrol agent that inhibits the F. oxysporum growth, promots germination rate, emergence rate and stem length of cotton seeds, and protects cotton plant from F. oxysporum infection. CGTase can inhibit the growth of F. oxysporum, and its transgenic A. thaliana enhances the resistance to Fusarium wilt.

Key words: Bacillus cereus, cotton Fusarium wilt, Fusarium oxysporum f. sp. vasinfectum, cyclodextrin glycosyltransferase (CGTase), control efficacy

周京龙,冯自力,魏锋,赵丽红,张亚林,周燚,冯鸿杰,朱荷琴. 棉花内生细菌YUPP-10及其分泌蛋白CGTase对棉花枯萎病的防治作用及机理[J]. 中国农业科学, 2021, 54(17): 3691-3701.

ZHOU JingLong,FENG ZiLi,WEI Feng,ZHAO LiHong,ZHANG YaLin,ZHOU Yi,FENG HongJie,ZHU HeQin. Biocontrol Effect and Mechanism of Cotton Endophytic Bacterium YUPP-10 and Its Secretory Protein CGTase Against Fusarium Wilt in Cotton[J]. Scientia Agricultura Sinica, 2021, 54(17): 3691-3701.

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.0578-1752.2021.17.011

https://www.chinaagrisci.com/CN/Y2021/V54/I17/3691

图/表 11

图1

表1

表2

表3

表4

图2

图3

图4

表5

图5

图6

参考文献 36

[1]	SANOGO S, ZHANG J. Resistance sources, resistance screening techniques and disease management for Fusarium wilt in cotton. Euphytica, 2016, 207(2): 255-271. doi: 10.1007/s10681-015-1532-y
[2]	ZHANG Z, DIAO H, WANG H, WANG K, ZHAO M. Use of Ganoderma lucidum polysaccharide to control cotton fusarium wilt, and the mechanism involved. Pesticide Biochemistry and Physiology, 2019, 158: 149-155. doi: 10.1016/j.pestbp.2019.05.003
[3]	LANG J, HU J, RAN W, XU Y, SHEN Q. Control of cotton Verticillium wilt and fungal diversity of rhizosphere soils by bio-organic fertilizer. Biology and Fertility of Soils, 2012, 48(2): 191-203. doi: 10.1007/s00374-011-0617-6
[4]	张海军, 李泽方. 绿色木霉GY20对棉花枯萎病菌的抑菌作用. 西北农业学报, 2012, 21(3): 193-197.
	ZHANG H J, LI Z F. Biocontrol mechanism of Trichoderma viride GY20 against Fusarium oxysporum f. sp. vasinfectum F12. Acta Agriculturae Boreali-Occidentalis Sinica, 2012, 21(3): 193-197. (in Chinese)
[5]	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)
[6]	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.
[7]	PEREG L, MCMILLAN M. Scoping the potential uses of beneficial microorganisms for increasing productivity in cotton cropping systems. Soil Biology and Biochemistry, 2015, 80: 349-358. doi: 10.1016/j.soilbio.2014.10.020
[8]	李海薇, 韩万里, 王梦瑶, 罗明, 韩剑, 顾爱星. 棉花枯萎病拮抗短小芽胞杆菌筛选鉴定及生防研究. 中国生物防治学报, 2018, 34(3): 440-448.
	LI H W, HAN W L, WANG M Y, LUO M, HAN J, GU A X. Screening and identification of antagonistic bacteria Bacillus pumilus against cotton Fusarium wilt and biocontrol research. Chinese Journal of Biological Control, 2018, 34(3): 440-448. (in Chinese)
[9]	柳自清, 张博然, 刘叶, 郭楠楠, 顾爱星. 短小芽孢杆菌KX-33做种衣剂对棉花枯萎病的盆栽防效. 新疆农业大学学报, 2020, 43(5): 323-329.
	LIU Z Q, ZHANG B R, LIU Y, GUO N N, GU A X. Effect of Bacillus pumilus KX-33 seed coating agent on Verticillium wilt of cotton. Journal of Xinjiang Agricultural University, 2020, 43(5): 323-329. (in Chinese)
[10]	戴蓬博, 蓝星杰, 张伟卫, 甘良, 王阳, 宗兆锋. 生防菌株SC11的鉴定、定殖及对棉花枯萎病防治效果研究. 植物病理学报, 2016, 46(2): 273-279.
	DAI P B, LAN X J, ZHANG W W, GAN L, WANG Y, ZONG Z F. Identification, colonization and disease suppressive effect of strain SC11 against cotton Fusarium wilt. Acta Phytopathologica Sinica, 2016, 46(2): 273-279. (in Chinese)
[11]	PLEBAN S, CHERNIN L, CHET I. Chitinolytic activity of an endophytic strain of Bacillus cereus. Letters in Applied Microbiology, 1997, 25(4): 284-288. doi: 10.1046/j.1472-765X.1997.00224.x
[12]	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. doi: 10.1007/s10526-007-9144-7
[13]	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. doi: 10.1111/lam.2007.44.issue-1
[14]	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. doi: 10.1111/jam.12242
[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]	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 and Food Chemistry, 2010, 58(5): 2668-2672. doi: 10.1021/jf903855y
[17]	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. 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, 37(12): 15-17. (in Chinese)
[18]	CLOUGH S J, BENT A F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 1998, 16(6): 735-743. doi: 10.1046/j.1365-313x.1998.00343.x
[19]	MARASCO R, ROLLI E, ETTOUMI B, VIGANI G, MAPELLI F, BORIN S, ABOU-HADID A F, EL-BEHAIRY U A, SORLINI C, CHERIF A, ZOCCHI G, DAFFONCHIO D. A drought resistance- promoting microbiome is selected by root system under desert farming. PLoS ONE, 2012, 7(10): e48479. doi: 10.1371/journal.pone.0048479
[20]	RASHID S, CHARLES T C, GLICK B R. Isolation and characterization of new plant growth-promoting bacterial endophytes. Applied Soil Ecology, 2011, 61(5): 217-224. doi: 10.1016/j.apsoil.2011.09.011
[21]	HARDOIM P R, VAN OVERBEEK L S, VAN ELSAS J D. Properties of bacterial endophytes and their proposed role in plant growth. Trends in Microbiology, 2008, 16(10): 463-471. doi: 10.1016/j.tim.2008.07.008
[22]	ALI S, CHARLES T C, GLICK B R. Delay of flower senescence by bacterial endophytes expressing 1-aminocyclopropane-1-carboxylate deaminase. Journal of Applied Microbiology, 2012, 113(5): 1139-1144. doi: 10.1111/jam.2012.113.issue-5
[23]	COUTINHO B G, LICASTRO D, MENDONÇA-PREVIATO L, CÁMARA M, VENTURI V. Plant-influenced gene expression in the rice endophyte Burkholderia kururiensis M130. Molecular Plant- Microbe Interactions, 2015, 28(1): 10-21. doi: 10.1094/MPMI-07-14-0225-R
[24]	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. doi: 10.1016/S0144-8617(03)00039-0
[25]	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. doi: 10.1016/S0268-005X(03)00044-4
[26]	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. doi: 10.1016/j.foodres.2014.07.008
[27]	PANGBURN S H, TRESCONY P V, HELLER J. Lysozyme degradation of partially deacetylated chitin, its films and hydrogels. Biomaterials, 1982, 3(2): 105-108. doi: 10.1016/0142-9612(82)90043-6
[28]	CHEN J, PENG H, WANG X, SHAO F, YUAN Z, HAN H. Graphene oxide exhibits broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal conidia by intertwining and membrane perturbation. Nanoscale, 2014, 6(3): 1879-1889. doi: 10.1039/C3NR04941H
[29]	NEJAD P, JOHNSON P A. Endophytic bacteria induce growth promotion and wilt disease suppression in oilseed rape and tomato. Biological Control, 2000, 18(3): 208-215. doi: 10.1006/bcon.2000.0837
[30]	ABDALLAH R A B, MOKNI-TLILI S, NEFZI A, JABNOUN- KHIAREDDINE H, DAAMI- REMADI M. Biocontrol of Fusarium wilt and growth promotion of tomato plants using endophytic bacteria isolated from Nicotiana glauca organs. Biological Control, 2016, 97(10): 80-88. doi: 10.1016/j.biocontrol.2016.03.005
[31]	AJILOGBA C F. RAPD delineation of native Bacillus spp. and analyses of their biocontrol effect on tomato Fusarium wilt[D]. Mafikeng Campus of the North-West University, 2013.
[32]	ABDALLAH R A, NE U O, JABNOUN-KHIAREDDINE H, MOKNI-TLILI S, NEFZI A, MEDIMAGH-SAIDANA S, DAAMI- REMADI M. Endophytic Bacillus spp. from wild Solanaceae and their antifungal potential against Fusarium oxysporum f. sp. lycopersici elucidated using whole cells, filtrate cultures and organic extracts. Journal of Plant Pathology and Microbiology, 2015, 6: 11.
[33]	傅莹, 黄益燕, 肖爱萍, 游春平. 蜡状芽孢杆菌菌株255诱导香蕉产生抗性相关酶初步研究. 广东农业科学, 2011, 38(9): 75-77.
	FU Y, HUANG Y Y, XIAO A Q, YOU C P. Preliminary study on the resistant-related enzymes from banana by the induction of Bacillus cereus strain 255. Guangdong Agricultural Sciences, 2011, 38(9): 75-77. (in Chinese)
[34]	ZHOU J, FENG Z, LIU S, WEI F, SHI Y, ZHAO L, HUANG W, ZHOU Y, FENG H, ZHU H. CGTase, a novel antimicrobial protein from Bacillus cereus YUPP-10, suppresses Verticillium dahliae and mediates plant defence responses. Molecular Plant Pathology, 2021, 22(1): 130-144. doi: 10.1111/mpp.13014
[35]	GUO X, STOTZ H U. Defense against Sclerotinia sclerotiorum in Arabidopsis is dependent on jasmonic acid, salicylic acid, and ethylene signaling. Molecular Plant-Microbe Interactions, 2007, 20(11): 1384-1395. doi: 10.1094/MPMI-20-11-1384
[36]	GLAZEBROOK J, CHEN W, ESTES B, CHANG H S, NAWRATH C, MÉTRAUX J P, ZHU T, KATAGIRI F. Topology of the network integrating salicylate and jasmonate signal transduction derived from global expression phenotyping. The Plant Journal, 2003, 34(2): 217-228. doi: 10.1046/j.1365-313X.2003.01717.x

处理 Treatment	产孢 Sporulation		孢子萌发Spore germination
处理 Treatment	产孢量Spore yield (cfu/mL)	抑制率Inhibition rate (%)	萌发率Germination rate (%)	抑制率Inhibition rate (%)
CK	33.40±1.61a	-	52.87±4.33a	-
1×YUPP-10	0.53±0.19c	98.41	25.56±4.45d	51.65
1/2×YUPP-10	15.20±1.79b	54.49	34.20±3.49c	35.30
1/4×YUPP-10	14.53±0.80b	56.49	39.78±3.42b	27.75

处理 Treatment	发芽率 Germination rate (%)	芽长 Shoot length (cm)
1×YUPP-10	39.08±0.34d	0.23±0.01b
1/2×YUPP-10	81.91±0.88a	0.84±0.32a
1/4×YUPP-10	75.84±0.98b	0.89±0.25a
CK	72.60±1.01c	0.49±0.10ab

处理 Treatment	检测指标 Detection index
处理 Treatment	出苗率Emergence rate (%)	根长Root length (cm)	茎长Stem length (cm)	总鲜重Fresh weight (g)
CK	84.36±0.04b	8.76±0.71a	13.09±0.38b	0.66±0.46a
YUPP-10	94.87±0.04a	10.26±0.66a	14.26±0.39a	0.76±0.49a

处理Treatment	病情指数Disease index	病株率Diseased plant rate (%)	防治效果Control efficacy (%)
1×10⁸	28.23±4.95b	43.04±14.80b	42.79
1×10⁷	27.08±9.54b	42.92±13.54b	45.11
1×10⁶	32.53±3.65ab	56.25±6.05ab	34.06
CK	49.34±9.08a	71.14±13.98a

蛋白浓度 Protein concentration (mg·mL^-1)	产孢 Sporulation		孢子萌发Spore germination
蛋白浓度 Protein concentration (mg·mL^-1)	产孢量 Spore yield (cfu/mL)	抑制率 Inhibition rate (%)	萌发率 Germination rate (%)	抑制率 Inhibition rate (%)
0	35.40±1.51a	-	42.62±2.13a	-
0.12	22.30±0.17b	37.01	38.06±3.35b	10.70
0.24	14.32±1.09c	59.55	32.25±2.19c	24.33
0.48	13.23±0.83c	62.63	29.48±1.62d	30.83