Isolation and Identification of Antagonistic Bacteria BACYitc8-7 Against Deciduous Disease Pathogen (Corynespora cassiicola) and Analysis of Antibacterial Components of Volatile Organic Compounds from Rubber Tree

doi:10.3969/j.issn.1000-2561.2022.08.013

Abstract

Abstract:

Corynespora cassiicola is an important pathogen causing Corynespora leaf fall disease of rubber trees. Chemical fungicides are widely used to prevent and control the disease at present, which brings severe ecological environmental issues in rubber planting areas. Isolating and screening bacteria strains with inhibitory activity against C. cassiicola from rhizosphere soil of rubber trees would provide theoretical foundation for researches and development of biological control agents on Corynespora leaf fall disease. Bacteria with high antagonistic activity was obtained from the rhizosphere soil of rubber trees with leaf fall disease by using dilution plate coating and plate-confrontation, its bacteriostatic diameter was (57.68±0.66)mm. It was observed that nine important plant pathogenic fungi could be inhibited by the strain as well. Based on morphological, physiological, biochemical characteristics and 16S rRNA gene sequence, the strain was basically consistent with Pseudomonas aeruginos, and was named BACYitc8-7. The activity of the volatile compounds of BACYitc8-7 against C. cassiicola was evaluated when LB, NA, Gause, Kim B and PDA were used as nutrition sources. The strongest antagonistic effects were found in Kim B and LB mediums, and the inhibition rate was 43.47%-48.69%. 35 substances, including amines, alcohols, alkenes, phenols, esters and pyrazines were obtained from the antimicrobial components of BACYitc8-7 collected and detected by headspace solid phase micro extraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS), and the top five contents were amine, borane dimethyl sulfide complex, isoamyl alcohol, 1-triene and 2-methyl-3-methylthiofuran, respectively.

Key words: Corynespora cassiicola, antagonistic bacteria, isolation and identification, volatile substances, antibacterial components

CLC Number:

S794.1

DAI Liming, LI Lanlan, SHI Yuping, LIU Yixian, DENG Yueye, WU Zhonghua, CAI Zhiying. Isolation and Identification of Antagonistic Bacteria BACYitc8-7 Against Deciduous Disease Pathogen (Corynespora cassiicola) and Analysis of Antibacterial Components of Volatile Organic Compounds from Rubber Tree[J]. Chinese Journal of Tropical Crops, 2022, 43(8): 1636-1645.

Figures/Tables 8

References 37

[1]	蔡志英. 橡胶树常见病害诊断及其防治[M]. 北京: 中国农业科学技术出版社, 2017.
	CAI Z Y. Diagnosis and control of common diseases of rubber trees[M]. Beijing: China Agricultural Science and Technology Press, 2017. (in Chinese)
[2]	李博勋, 冯艳丽, 刘先宝, 蔡吉苗, 陆翠梅, 郑肖兰, 黄贵修. 我国热带作物多主棒孢种群多样性及致病力分化分析[J]. 热带作物学报, 2019, 40(12): 2456-2465.
	LI B X, FENG Y L, LIU X B, CAI J M, LU C M, ZHENG X L, HANG G X. Genetic diversity and pathogenic variability among isolates of Corynespora cassiicola from tropical crops in China[J]. Chinese Journal of Tropical Crops, 2019, 40(12): 2456-2465. (in Chinese)
[3]	李宝聚, 高苇, 石延霞, 谢学文. 多主棒孢和棒孢叶斑病的研究进展[J]. 植物保护学报, 2012, 39(2): 171-176.
	LI B J, GAO W, SHI Y X, XIE X W. Progress in researches on Corynespora leaf spot[J]. Acta Phytophylacica Sinica, 2012, 39(2): 171-176. (in Chinese)
[4]	李晗, 冉茂, 陈海涛, 吴杰, 张帅, 徐宸, 杨超, 汪代斌, 孙现超. 植物棒孢霉叶斑病的发生及防治研究进展[J]. 植物医生, 2020, 33(1): 15-20.
	LI H, RAN M, CHEN H T, WU J, ZHANG S, XU C, YANG C, WANG D B, SUN X C. Progress in researches of Corynespora leaf spot[J]. Plant Doctor, 2020, 33(1): 15-20. (in Chinese)
[5]	王进强, 何明霞, 张春霞, 周明. 橡胶树棒孢霉落叶病苗圃防治初报[J]. 中国农学通报, 2011, 27(22): 51-54.
	WANG J Q, HE M X, ZHANG C X, ZHOU M. Preliminary report of chemical control Corynespora leaf fall disease of hevea rubber in rubber nursery[J]. Chinese Agricultural Science Bulletin, 2011, 27(22): 51-54. (in Chinese)
[6]	林运萍, 蒋菊生, 白先权, 易杰祥. 橡胶树棒孢霉落叶病的发生及防治[J]. 中国热带农业, 2008(1): 54-55.
	LIN Y P, JIANG J S, BAI X Q, YI J X. The occurrence and prevention of Corynespora leaf fall disease of hevea rubber[J]. China Tropical Agriculture, 2008(1): 54-55. (in Chinese)
[7]	王静, 郑永华. 拮抗菌在果蔬采后病害生物防治中的应用[J]. 生物技术进展, 2013, 3(6): 393-398.
	WANG J, ZHENG Y H. Application of microbial antagonists as biocontrol agents against postharvest diseases of fruits and vegetables[J]. Current Biotechnology, 2013, 3(6): 393-398. (in Chinese)
[8]	赵昱榕, 李磊, 谢学文, 石延霞, 柴阿丽, 孙广玉, 李宝聚. 贝莱斯芽胞杆菌ZF2对多主棒孢病菌防治效果[J]. 中国生物防治学报, 2019, 35(2): 62-70.
	ZHAO Y R, LI L, XIE X W, SHI Y X, CHAI A L, SUN G Y, LI B J. Biocontrol effect of Bacillus velezensis strain ZF2 against Corynespora cassiicola[J]. Chinese Journal of Biological Control, 2019, 35(2): 62-70. (in Chinese)
[9]	时涛, 彭建华, 刘先宝, 戴英葵, 黄贵修. 橡胶树内生细菌多样性初探及拮抗菌株的筛选[J]. 中国森林病虫, 2011, 3(2): 5-9.
	SHI T, PENG J H, LIU X B, DAI Y K, HUANG G X. Rubber endophytic bacteria population diversity and screening of antagonistic strains[J]. Forest Pest and Disease, 2011, 3(2): 5-9. (in Chinese)
[10]	李岚岚, 戴利铭, 蒋桂芝, 刘一贤, 施玉萍, 蔡志英. 橡胶树炭疽病生防内生菌的分离鉴定及抑菌作用研究[J]. 热带作物学报, 2021, 42(10): 2958-2965.
	LI L L, DAI L M, JIANG G Z, LIU Y X, SHI Y P, CAI Z Y. Isolation, identification and bacteriostasis study of endophytic bacteria to control Colletotrichum leaf disease on rubber tree[J]. Chinese Journal of Tropical Crops, 2021, 42(10): 2958-2965. (in Chinese)
[11]	桑建伟. 香蕉枯萎病拮抗内生芽孢杆菌BEB17的分离鉴定及生防潜力分析[D]. 海口: 海南大学, 2018.
	SANG J W. Isolation, identification and biocontrol potential analysis of endophytic Bacillus BEB17 antagonistic banana fusarium wilt[D]. Haikou: Hainan University, 2018. (in Chinese)
[12]	吴利民, 陆宁海, 高扬帆, 田雪亮, 郎剑锋, 徐瑞富. 哈茨木霉TR-12对多主棒孢霉菌的拮抗作用[J]. 湖北农业科学, 2006, 45(6): 749-750.
	WU L M, LU N H, GAO Y F, TIAN X L, LANG J F, XU R F. Antagonism of T. harianum strain TR-12 against Corynespora cassiicola Wei[J]. Hubei Agricultural Sciences, 2006, 45(6): 749-750. (in Chinese)
[13]	刘一贤, 施玉萍, 戴利铭, 李岚岚, 蔡志英. 橡胶褐根病拮抗放线菌17-7的筛选、鉴定及发酵条件优化[J]. 微生物学通报, 2020, 47(1): 118-129.
	LIU Y X, SHI Y P, DAI L M, LI L L, CAI Z Y. Screening, identification and fermentation optimization of an antimicrobial actinomycete strain 17-7 to Phellinus noxius[J]. Microbiology China, 2020, 47(1): 118-129. (in Chinese)
[14]	陈旭玉, 何其光, 甘炳春. 广藿香棒孢霉叶斑病拮抗菌的分离鉴定及生物农药的筛选[J]. 江西农业学报, 2012, 24(6): 64-66.
	CHEN X Y, HE Q G, GAN B C. Isolation and identification of antagonistic bacterium against leaf spot pathogen (Corynespora cassiicola) in Pogostemon cablin and screening of biological medicaments[J]. Acta Agriculturae Jiangxi, 2012, 24(6): 64-66. (in Chinese)
[15]	张力群, 张俊威. 假单胞菌产生的抗生素[J]. 中国生物防治学报, 2015, 31(5): 750-756.
	ZHANG L Q, ZHANG J W. Antibiotics produced by Pseudomonas spp.[J]. Chinese Journal of Biological Control, 2015, 31(5): 750-756. (in Chinese)
[16]	任小平, 谢关林, 王笑. 铜绿假单胞菌ZJ1999对水稻纹枯病的防治及其在水稻上的定殖[J]. 中国生物防治, 2006, 2(1): 54-57.
	REN X P, XIE G L, WANG X. Application and colonization of Pseudomonas aeruginosa ZJ1999 for biocontrol of Rhizoctonia solani, pathogen of rice sheath blight[J]. Chinese Journal of Biological Control, 2006, 2(1): 54-57.. (in Chinese)
[17]	申顺善, 张莹莹, 张维娜, 吕雅悠, 朱卓琳, 朴凤植. 绿针假单胞菌HL5-4对番茄灰霉菌的抑制活性及其定殖能力[J]. 园艺学报, 2016, 43(6): 1195-1202.
	SHEN S S, ZHANG Y Y, ZHANG W N, LV Y Y, ZHU Z L, PIAO F Z. Antifungal activity of Pseudomonas choloeaphtis HL5-4 against tomato gray mold and its colonization ability[J]. Acta Horticulturae Sinica, 2016, 43(6): 1195-1202. (in Chinese)
[18]	宁爽. 内生假单胞菌BTa14、Bar25促生抗病作用及机理的研究[D]. 烟台: 烟台大学, 2019.
	NING S. Study on the growth-promoting, disease-resistance effects and mechanisms of endophytic Pseudomonas BTa14 and Bar25[D]. Yantai: Yantai University, 2019. (in Chinese)
[19]	张清霞, 张迎, 何玲玲, 陈夕军, 童蕴慧, 纪兆林. 水稻纹枯病拮抗细菌7-5的鉴定及其生防机制初步研究[J]. 中国水稻科学. 2018, 32(3): 277-284. DOI
	ZHANG Q X, ZHANG Y, HE L L, CHEN X J, TONG W H, JI Z L. Identification of strain 7-5, antagonistic to rice sheath blight, and preliminary study of its biocontrol mechanism[J]. Chinese Journal of Rice Science, 2018, 32(3): 277-284. (in Chinese)
[20]	梁艳琼, 唐文, 董文敏, 吴伟怀, 李锐, 习金根, 谭施北, 郑金龙, 黄兴, 陆英, 贺春萍, 易克贤. 枯草芽孢杆菌菌株Czk1挥发性物质的抑菌活性及其组分分析[J]. 南方农业学报, 2019, 50(11): 2465-2474.
	LIANG Y Q, TANG W, DONG W M, WU W H, LI R, XI J G, TAN S B, ZHENG J L, HUANG X, LU Y, HE C P, YI K X. Antifugal effect and components analysis of volatile organic compounds from Bacillus subtilis Czk1[J]. Journal of Southern Agriculture, 2019, 50(11): 2465-2474. (in Chinese)
[21]	GE Y H, HUANG X Q, WANG S L, ZHANG X, XU Y. Phenazine-1-carboxylic acid is negatively regulated and pyoluteorin positively regulated by gacA in Pseudomonas sp. M18[J]. FEMS Microbiology Letters, 2004, 237(1): 41-47. DOI URL
[22]	蒋桂芝, 何双凌, 陶亮, 岳海, 贺熙勇. 澳洲坚果叶枯病病原鉴定及其防治药剂筛选[J]. 中国植保导刊, 2018, 38(7): 20-24.
	JIANG G Z, HE S L, TAO L, YUE H, HE X Y. Identification of pathogen of macadamia leaf blight and selection of controlling pesticide[J]. China Plant Protection, 2018, 38(7): 20-24. (in Chinese)
[23]	柳凤, 杨顺锦, 詹儒林, 赵艳龙. 澳洲坚果叶斑病病原鉴定及其生物学特性[J]. 植物保护学报, 2011, 38(5): 437-442.
	LIU F, YANG S J, ZHAN R L, ZHAO Y L. Identification and biological characteristics of macadamia leaf spot caused by Pestalotiopsis versicolor[J]. Acta Phytophylacica Sinica, 2011, 38(5): 437-442. (in Chinese)
[24]	周莲, 蒋海霞, 金凯明, 孙爽, 张薇, 张雪洪, 何亚文. 高产申嗪霉素和吩嗪-1-酰胺的水稻根际铜绿假单胞菌PA1201分离、鉴定与应用潜力[J]. 微生物学报, 2015(4): 401-411.
	ZHOU L, JIANG H X, JIN K M, SUN S, ZHANG W, ZHANG X H, HE Y W. Isolation, identification and characterization of rice rhizobacterium Pseudomonas aeruginosa pa1201 producing high level of biopesticide “shenqinmycin” and phenazine-1-carboxamide[J]. Acta Microbiologica Sinica, 2015(4): 401-411. (in Chinese)
[25]	蒋海霞, 周莲, 何亚文. 铜绿假单胞菌生防菌株抑菌代谢产物及其生防应用研究进展[J]. 微生物学通报, 2015, 42(7): 1338-1349.
	JIANG H X, ZHOU L, HE Y W. Research progress in biocontrol strain Pseudomonas aeruginosa: antifungal metabolites and their applications in biocontrol[J]. Microbiology China, 2015, 42(7): 1338-1349. (in Chinese)
[26]	胡军华, 周泽扬, 张伏军, 蓝希钳, 林立鹏, 唐婧, 马淑华, 谢洁, 肖杰, 潘国庆. 烟草根际细菌铜绿假单胞菌swu31-2的定殖能力及其对烟草青枯病的防治作用[J]. 植物保护, 2009, 35(5): 89-94.
	HU J H, ZHOU Z Y, ZHANG F J, LAN X Q, LIN L P, TANG J, MA S H, XIE J, XIAO J, PAN G Q. Analysis of the colonization of tabacco rhizosphere bacterium swu31-2 and its control effect on tabacco bacterial wilt[J]. Plant Protection, 2009, 35(5): 89-94. (in Chinese)
[27]	WU D Q, YE J, OU H Y, WEI X, XU Y. Genomic analysis and temperature-dependent transcriptome profiles of the rhizosphere originating strain Pseudomonas aeruginosa M18[J]. BMC Genomics, 2011, 12(1): 438-455. DOI URL
[28]	LEE J Y, MOON S S, HWANG B K. Isolation and in vitro and in vivo activity against Phytophthora capsici and Colletotrichum orbiculare of phenazine-1-carboxylic acid from Pseudomonas aeruginosa strain GC-B26[J]. Pest Management Science, 2003, 59(8): 872-882. DOI URL
[29]	RANE M R, SARODE P D, CHAUDHARI B L, CHINCHOLKAR S B. Exploring antagonistic metabolites of established biocontrol agent of marine origin[J]. Applied Biochemistry and Biotechnology, 2008, 151(2/3): 665-675. DOI URL
[30]	肖咪云, 孙孟龙, 阮楚晋, 陈寿昆, 刘裕华, 陆祖军. 生防细菌2016NX1对病原真菌的抑制及发酵条件优化[J]. 广西师范大学学报(自然科学版), 2019, 37(2): 172-182.
	XIAO M Y, SUN M L, RUAN C J, CHEN S K, LIU Y H, LU Z J. Inhibitory effect of biocontrol bacterium 2016NX1 on plant pathogenic fungi and optimization of fermentation conditions[J]. Journal of Guangxi Normal University (Natural Science Edition), 2019, 37(2): 172-182. (in Chinese)
[31]	LI Q, PING N, LU Z, HUANG J, LI G, HSIANG T. Effects of volatile substances of Streptomyces globisporus JK-1 on control of Botrytis cinerea on tomato fruit[J]. Biological Control, 2012, 61(2): 113-120. DOI URL
[32]	FERNANDO W G D, RAMARATHNAM R, KRISHNAMOORTHY A S, SAVCHUK S C. Identification and use of potential bacterial organic antifungal volatiles in biocontrol[J]. Soil Biology and Biochemistry, 2005, 37(5): 955-964. DOI URL
[33]	GOTOR V A, TEIXID N, FRANCESCO A D, USALL J, UGOLINI L, TORRES R, MARI M. Antifungal effect of volatile organic compounds produced by Bacillus amyloliquefaciens CPA-8 against fruit pathogen decays of cherry[J]. Food Microbiology, 2017, 64: 219-225. DOI URL
[34]	李宝庆, 鹿秀云, 郭庆港, 钱常娣, 李社增, 马平. 枯草芽孢杆菌 BAB-1产脂肽类及挥发性物质的分离和鉴定[J]. 中国农业科学, 2010, 43(17): 3547-3554.
	LI B Q, LU X Y, GUO Q G, QIAN C D, LI S Z, MA P. Isolation and identification of lipopeptides and volatile compounds produced by Bacillus subtilis strain BAB-1[J]. Scientia Agricultura Sinica, 2010, 43(17): 3547-3554. (in Chinese)
[35]	RYU C M, FARAG M A, HU C H, REDDY M S, KLOEPPER J W, PAEE P W. Bacterial volatiles induce systemic resistance in Arabidopsis[J]. Plant Physiology, 2004, 134(3): 1017-1026. DOI URL
[36]	RYU C M, FARAG M A, HU C H, REDDY M S, WEI H X, PARE PW, KLOEPPER J W. Bacterial volatiles promote growth in Arabidopsis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(8): 4927-4932.
[37]	TAHIR H A S, GU Q, WU H J, NIU Y D, HUO R, GAO X W. Bacillus volatiles adversely affect the physiology and ultra-structure of Ralstonia solanacearum and induce systemic resistance in tobacco against bacterial wilt[J]. Scientific Reports, 2017, 7(1): 40481-40496. DOI URL

引物Primer	序列（5°-3°）Sequence (5°-3°)	扩增基因Amplified gene	片段大小Amplified segment/bp
27F	AGAGTTTGATCCTGGCTCAG	16S rDNA	1500
1492R	GGTTACCTTGTTACGACCCTT	16S rDNA	1500
PCA2a	TGCCAAGCCTCGCTCCAAC	phzCD	1400
PCA3b	CCGCGTTGTTCCTCGTTCAT	phzCD	1400

引物Primer	序列（5°-3°）Sequence (5°-3°)	扩增基因Amplified gene	片段大小Amplified segment/bp
27F	AGAGTTTGATCCTGGCTCAG	16S rDNA	1500
1492R	GGTTACCTTGTTACGACCCTT	16S rDNA	1500
PCA2a	TGCCAAGCCTCGCTCCAAC	phzCD	1400
PCA3b	CCGCGTTGTTCCTCGTTCAT	phzCD	1400

培养基 Medium	菌落形态 Colony morphology	色素 Pigment	荧光 Fluorescence
LB	菌落呈圆形,表面光滑、湿润,边缘整齐	淡黄色无金属光泽	紫外灯下有荧光
PDA	菌落呈山脉状、湿润、表面粗糙边缘光滑、	淡黄色无金属光泽	紫外灯下有荧光
高氏	菌落呈圆形、湿润、表面与边缘均光滑、无色透明状	无	无
NA	菌落呈圆形、湿润、表面粗糙有小点状突起	早期呈黄绿色,后期呈红褐色	紫外灯下有荧光
金氏B	菌落呈山脉状、湿润、表面粗糙有小点状突起、边缘光滑	铜色金属光泽	紫外灯下有荧光

培养基 Medium	菌落形态 Colony morphology	色素 Pigment	荧光 Fluorescence
LB	菌落呈圆形,表面光滑、湿润,边缘整齐	淡黄色无金属光泽	紫外灯下有荧光
PDA	菌落呈山脉状、湿润、表面粗糙边缘光滑、	淡黄色无金属光泽	紫外灯下有荧光
高氏	菌落呈圆形、湿润、表面与边缘均光滑、无色透明状	无	无
NA	菌落呈圆形、湿润、表面粗糙有小点状突起	早期呈黄绿色,后期呈红褐色	紫外灯下有荧光
金氏B	菌落呈山脉状、湿润、表面粗糙有小点状突起、边缘光滑	铜色金属光泽	紫外灯下有荧光

供试菌株 Plant pathogenes	抑菌带宽 Diameter of inhibition zone/mm	抑菌率 Inhibition rate/%
胶孢炭疽菌	37.95±0.66	54.00±1^cd
五隔大无性孢丽赤壳菌	44.73±0.42	66.67±1^b
橡胶树褐根菌	54.25±1.91	66.33±2^b
尖孢炭疽菌DFMP1	29.50±2.02	46.33±3^e
多主棒孢菌	57.68±0.66	79.33±1^a
茄类镰刀菌	32.43±1.41	52.33±2^d
橡胶疫霉菌	39.14±0.63	57.33±1^c
小孢拟盘多毛孢	66.25±2.03	79.67±3^a
尖孢炭疽菌MLZZP3	26.30±0.93	47.67±2^e