罗汉果土传病害拮抗细菌的筛选及鉴定

王瑞昊; 邓业成; 陈广桂; 张泽; 张燕玲; 邓志勇; 蓝福生; 郭丽霞; 张川梅; 梁保明; 骆海玉; 张明良

doi:10.19303/j.issn.1008-0384.2021.08.009

罗汉果土传病害拮抗细菌的筛选及鉴定

doi: 10.19303/j.issn.1008-0384.2021.08.009

1.
广西师范大学生命科学学院，广西　桂林　541004
2.
桂林吉福思罗汉果有限公司，广西　桂林　541006

基金项目: 广西重点研发计划项目（桂科AB1850025）；桂林市重点研发计划项目（20190211-19）

详细信息

作者简介:
王瑞昊（1995−），男，硕士生，主要从事天然产物研究（E-mail：429567099@qq.com）

通讯作者:
邓业成（1965−），男，博士，教授，主要从事天然产物及植物保护研究（E-mail：dyecheng@163.com）

中图分类号: S 435
计量
- 文章访问数: 918
- HTML全文浏览量: 313
- PDF下载量: 64
- 被引次数: 0
出版历程
- 收稿日期: 2021-02-01
- 修回日期: 2021-05-03
- 网络出版日期: 2021-05-17
- 刊出日期: 2021-08-28

Antagonistic Bacteria against Soil-borne Diseases on Siraitia grosvenorii

1.
College of Life Sciences, Guangxi Normal University, Guilin, Guangxi　541004, China
2.
Guilin GFS Monk Fruit Crop., Guilin, Guangxi　541006, China

摘要

摘要: 目的从罗汉果根际土壤中筛选分离出对白绢病(southern blight)与根腐病(root rot)有抑制作用的拮抗细菌，为罗汉果土传病害生物防治提供合适菌株。方法采用稀释涂布法和划线法从罗汉果果园土壤中分离纯化出拮抗细菌114株。随后对分离出的拮抗细菌采用平板对峙法进行抑菌活性筛选。通过16S rDNA序列分析，结合菌落形态特征，生理生化特征对菌株进行鉴定。结果 22株细菌对至少1种罗汉果土传病原真菌表现出不同程度的抑制作用。其中5株细菌即TYX-2、TYX-3、TYX-4、TYX-7和TYX-8对根腐病与白绢病抑制作用最为明显，对罗汉果根腐病菌的抑制率分别为67.53%、57.50%、64.17%、60.00%和66.67%。对罗汉果白绢病菌的抑制率分别为83.03%、87.58%、53.31%、82.27%和86.67%。经过形态学、生理生化特征以及16S rDNA系统发育树的分析，确定TYX-2、TYX-7和TYX-8为贝莱斯芽孢杆菌(Bacillus velezia)，TYX-3为枯草芽孢杆菌(Bacillus subtilis)，TYX-4为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。贝莱斯芽孢杆菌TYX-2、枯草芽孢杆菌TYX-3和解淀粉芽孢杆菌TYX-4发酵液对2种罗汉果病原真菌也有较好的抑制作用，对罗汉果根腐病菌的抑菌率分别为47.83%、48.67%和45.83%，对罗汉果白绢病菌的抑菌率分别为52.00%、50.42%和55.83%。结论分离鉴定出的TYX-2、TYX-3和TYX-4分别为贝莱斯芽孢杆菌、枯草芽孢杆菌和解淀粉芽孢杆菌，具有较高的生防应用价值，为进一步研制复合生防菌剂，防治罗汉果土传病害奠定理论基础。
- 罗汉果 /
- 根腐病菌 /
- 白绢病菌 /
- 分离鉴定 /
- 拮抗细菌
Abstract: Object Microbes with potential of being used as biological control agents against the southern blight and root rot diseases on Siraitia grosvenorii were screened and identified from the rhizosphere soil in the field. Method A total of 114 strains of presumably antagonistic microorganisms were isolated from the soil at a S. grosvenorii orchard by means of dilution coating and streaking. The isolates were screened for their antibacterial activity using the plate confrontation method, and subsequently, identified by 16S rDNA sequence analysis along with observations on the colony morphology and microbial physiochemical characteristics. Result Twenty-two isolated strains displayed varying degrees of inhibition effect on one or both root rot and southern blight pathogens. They included the bacteria under the coded names of TYX-2, TYX-3, TYX-4, TYX-7, and TYX-8 with inhibition rates on the root rot of 67.53%, 57.50%, 64.17%, 60.00%, and 66.67%, respectively, and on the southern blight with the rates of 83.03%, 87.58%, 53.31%, 82.27%, and 86.67%, respectively. TYX-2, TYX-7, and TYX-8 were identified to be Bacillus velezia, TYX-3 B. subtilis, and TYX-4 B. amyloliquefaciens. The fermentation broths of TYX-2, TYX-3, and TYX-4 also showed significant inhibition rates on the root rot pathogen at 47.83%, 48.67%, and 45.83%, respectively, and 52.00%, 50.42%, and 55.83% on the southern blight. Conclusion The isolated B. velez, B. subtilis, and B. amyloliquefaciens strains displayed significant inhibitory effects on the root rot and southern blight of S. grosvenorii. They could be studied further for a potential application as agents for biocontrol on the diseases.
- Siraitia grosvenorii /
- root rot /
- southern blight /
- isolation and identification /
- biocontrol bacteria

HTML全文

图 1 土壤细菌对罗汉果土壤病原菌的抑制作用

注：A～E分别为TYX-4、TYX-2、TYX-3、TYX-7、TYX-8对根腐病的抑制效果。G～K分别为TYX-4、TYX-2、TYX-3、TYX-7、TYX-8对白绢病的抑制效果。F为根腐病对照，L为白绢病对照。

Figure 1. Inhibition of bacteria strains on pathogens of S. grosvenorii

Note: A～E respectively TYX-4, TYX-2, TYX-3, TYX-7, TYX-8 inhibit effect on root rot.G～K respectively TYX-4, TYX-2, TYX-3, TYX-7, TYX-8 inhibitory effect on Southern blight.F is the root rot control，L is the Southern blight control.

下载: 全尺寸图片幻灯片

图 2 生防细菌的菌落形态

Figure 2. Colony morphology of bacteria strains with biocontrol potential

下载: 全尺寸图片幻灯片

图 3 菌株TYX-2的16S rDNA序列系统发育树

Figure 3. Phylogenetic tree based on 16S rDNA sequence of TYX-2

下载: 全尺寸图片幻灯片

图 4 菌株TYX-3的16S rDNA序列系统发育树

Figure 4. Phylogenetic tree based on 16S rDNA sequence of TYX-3

下载: 全尺寸图片幻灯片

图 5 菌株TYX-4的16S rDNA序列系统发育树

Figure 5. Phylogenetic tree based on 16S rDNA sequence of TYX-4

下载: 全尺寸图片幻灯片

图 6 菌株TYX-7的16S rDNA序列系统发育树

Figure 6. Phylogenetic tree based on 16S rDNA sequence of TYX-7

下载: 全尺寸图片幻灯片

图 7 菌株TYX-8的16S rDNA序列系统发育树

Figure 7. Phylogenetic tree based on 16S rDNA sequence of TYX-8

下载: 全尺寸图片幻灯片

图 8 拮抗菌株发酵产物对病原菌的抑菌效果

注：A、B、C依次为TYX-2、TYX-3、TYX-4对根腐病的抑菌效果。D、E、F依次为TYX-2、TYX-3、TYX-4对根腐病的抑菌效果,G、H为根腐病菌和白绢病菌对照组。

Figure 8. Antibacterial effect of fermentation broths of antagonistic strains on pathogens

Note: A, B, C are the antibacterial effects of TYX-2, TYX-3, and TYX-4 on root rot. D, E, F are the antibacterial effects of TYX-2, TYX-3, and TYX-4 on root rot in turn. G and H are the control group of root rot and. Southern blight.

下载: 全尺寸图片幻灯片

表 1 22株土壤细菌对罗汉果2种土传病原真菌的拮抗活性

Table 1. Antagonistic activities of 22 strains of bacteria against pathogens of two soil-borne diseases on S. grosvenorii

菌株 Strain	抑菌率（± SD）Inhibition rate/%		菌株 Strain	抑菌率（± SD）Inhibition rate/%
菌株 Strain	根腐病菌 Root rot	白绢病菌 Southern blight	菌株 Strain	根腐病菌 Root rot	白绢病菌 Southern blight
TYX-1	31.66 ± 3.57	14.81 ± 2.58	TYX-13	—	7.36 ± 1.54
TYX-2	67.53 ± 2.89	83.03 ± 0.86	TYX-14	38.85 ± 1.33	—
TYX-3	57.50 ± 2.04	87.58 ± 1.67	TYX-15	5.26 ± 0.56	5.89 ± 0.49
TYX-4	64.17 ± 1.18	53.31 ± 0.86	TYX-16	13.82 ± 5.89	—
TYX-5	27.43 ± 2.63	—	TYX-17	27.54 ± 2.36	25.90 ± 0.38
TYX-6	16.63 ± 5.52	20.56 ± 0.84	TYX-18	35.63 ± 5.27	14.45 ± 2.23
TYX-7	60.00 ± 2.04	82.27 ± 2.97	TYX-19	23.13 ± 2.45	—
TYX-8	66.67 ± 1.16	86.68 ± 0.59	TYX-20	15.84 ± 0.33	—
TYX-9	36.67 ± 11.79	—	TYX-21	34.19 ± 1.44	15.43 ± 0.00
TYX-10	35.86 ± 5.48	16.22 ± 0.56	TYX-22	—	31.29 ± 2.51
TYX-11	8.33 ± 1.22	—	CK	—	—
TYX-12	25.31 ± 1.89	45.72 ± 2.47

下载: 导出CSV

表 2 LB琼脂平板上各菌株形态

Table 2. Colony morphology of various strains on LB agar

菌株 Strain	菌落生长速度 Colony growth	菌落颜色 Colony color	菌落形态 Colony morphology	表面形态 Surface morphology	菌落表面 Colony surface
TYX-2	快	白色	呈圆形菌落	平整	黏稠湿润
TYX-3	快	白色	呈圆形菌落	平整	干燥
TYX-4	中	淡黄	呈椭圆菌落	粗糙	光滑湿润
TYX-7	快	白色	呈椭圆菌落	平整	湿润黏稠
TYX-8	快	白色	呈椭圆菌落	平整	湿润黏稠

下载: 导出CSV

表 3 拮抗细菌的生理生化特征

Table 3. Physiochemical properties of bacteria strains with biocontrol potential

测定指标Test index	菌株 Strain
测定指标Test index	TYX-4	TYX-2	TYX-7	TYX-3	TYX-8
革兰氏染色Gram stain	+	+	+	+	+
过氧化氢接触酶Oxidize activities	+	+	+	+	+
吲哚产生Indole test	−	+	−	−	+
明胶水解Gelatin hydrolysis	−	+	+	+	+
甲基红Methl red test	−	−	−	−	−
淀粉水解Starch hydrolusis	+	+	+	+	+
乳糖Lactose	−	−	−	−	−
甘露醇Mannitol	+	−	−	+	+
葡萄糖Glucose	+	+	+	+	+
D-木糖D-Xylose	−	−	−	+	−
D-果糖D-Fructose	−	−	+	−	−
接触酶Catalase	+	+	+	+	+
柠檬酸盐利用Citrate utilization	+	−	−	+	−
V-P实验Voges-Proskauer	+	+	+	+	+
注：+代表反应为阳性，−代表反应为阴性。 Note: + indicates that the reaction is positive，and − indicates that the reaction is negative.

下载: 导出CSV

表 4 拮抗菌株发酵产物的抑菌效果

Table 4. Antibacterial effect of fermentation broths of antagonistic strains

菌株 Strain	抑菌率（± SD） Inhibition rate/%
菌株 Strain	根腐病菌 Root rot	白绢病菌 Southern blight
TYX-2	47.83 ± 2.53	52.00 ± 2.23
TYX-3	48.67 ± 2.27	50.42 ± 3.93
TYX-4	45.83 ± 2.76	55.83 ± 2.35

下载: 导出CSV

参考文献(16)

[1]	赵二劳, 赵丽婷, 李满秀. 罗汉果的保健功能及产品开发 [J]. 食品研究与开发, 2006, 27(3):125−126. doi: 10.3969/j.issn.1005-6521.2006.03.049 ZHAO E L, ZHAO L T, LI M X. Health-care function and products development of siraitiagrosvenorii [J]. Food Research and Development, 2006, 27(3): 125−126.(in Chinese) doi: 10.3969/j.issn.1005-6521.2006.03.049
[2]	黄志江, 黄捷, 孙滢川. 罗汉果的药用研究 [J]. 广西师范大学学报(自然科学版), 1998, 16(4):75−79. HUANG Z J, HUANG J, SUN Y C, et al. A Survey in the pharmaceutical studies on siraitiagrosvenorii [J]. Journal of Guangxi Normal University (Natural Science Edition), 1998, 16(4): 75−79.(in Chinese)
[3]	MURILLO A C, MULLENS B A. A review of the biology, ecology, and control of the northern fowl mite, Ornithonyssus sylviarum (Acari: Macronyssidae) [J]. Veterinary parasitology, 2017, 246: 30−37. doi: 10.1016/j.vetpar.2017.09.002
[4]	何斐, 张忠良, 崔鸣, 等. 生防放线菌剂对魔芋根域微生物区系的影响 [J]. 应用与环境生物学报, 2015, 21(2):221−227. HE F, ZHANG Z L, CUI M, et al. Effect of biocontrol actinomycetes agents on microflora in the root-zone of Amorphophallus konjacorphophallus konjac K. Koch ex N. E. Br K. Koch ex N. E. Br [J]. Chinese Journal of Applied and Environmental Biology, 2015, 21(2): 221−227.(in Chinese)
[5]	陈志杰, 张锋, 张淑莲, 等. 温室黄瓜土传病害流行因素及环境友好型防治技术对策 [J]. 农业环境科学学报, 2006(B09):697−700. CHEN Z J, ZHANG F, ZHANG S L, et al. Epidemic factors of soil-borne diseases of cucumber and environment-friendly control technique under sunlight greenhouse in loess hilly region [J]. Journal of Agro-Environment Science, 2006(B09): 697−700.(in Chinese)
[6]	高菲, 卜春亚, 靳永胜, 等. 草莓根腐病拮抗细菌的分离筛选与鉴定 [J]. 广东农业科学, 2012, 39(3):4−8. doi: 10.3969/j.issn.1004-874X.2012.03.002 GAO F, BU C Y, JIN Y S, et al. Screening and identification of antagonistic bacteria against Colletotrichumgloeosporioides [J]. Guangdong Agricultural Sciences, 2012, 39(3): 4−8.(in Chinese) doi: 10.3969/j.issn.1004-874X.2012.03.002
[7]	AHMED A S, EZZIYYANI M, PÉREZ SÁNCHEZ C, et al. Effect of chitin on biological control activity of Bacillus spp. and Trichoderma harzianum against root rot disease in pepper (Capsicumannuum) plants [J]. European Journal of Plant Pathology, 2003, 109(6): 692−711.
[8]	季倩茹, 陈静, 胡远亮, 等. 3种芽孢杆菌菌剂对黄瓜枯萎病的防效及其作用机制初探 [J]. 华中农业大学学报, 2020, 39(5):101−107. JI Q R, CHEN J, HU Y L, et al. Control effect of three Bacillus strains on cucumber fusarium wilt and its mechanism [J]. Journal of Huazhong Agricultural University, 2020, 39(5): 101−107.(in Chinese)
[9]	柳慧丽. 枯草芽孢杆菌KC-5的分离鉴定及发酵工艺优化[D]. 南京: 南京农业大学, 2014. LIU H L. Isolation and Identification of Bacillicus Subtilis KC-5 and Its Fermentation Optimization[D]. Nanjing: Nanjing Agricultural University, 2014. (in Chinese).
[10]	何亚登. 2种生防菌的发酵、土壤定殖及防治烟草土传病害的研究[D]. 福州: 福建农林大学, 2019. HE Y D. Study on Fermentation and Soil Colonization and Control of Tobacco Soil-Borne Diseases of Two Biocontrol Agents[D]. Fuzhou: Fujian Agriculture and Forestry University, 2019. (in Chinese).
[11]	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.
[12]	任凤山, 高亮, 张博. 5种杀菌剂防治番茄茎腐病的室内筛选和田间使用效果 [J]. 农学学报, 2016, 6(5):18−22. doi: 10.11923/j.issn.2095-4050.cjas15090002 REN F S, GAO L, ZHANG B. Laboratory Screening and Field Control Effect of Five Kinds of Fungicides on Tomato Basal Stem Rot [J]. Journal of Agriculture, 2016, 6(5): 18−22.(in Chinese) doi: 10.11923/j.issn.2095-4050.cjas15090002
[13]	高琳娜, 曹克强, 段英姿, 等. 拮抗细菌Bs-0728对板蓝根根腐病的防治作用 [J]. 植物保护, 2011, 37(5):97−100. doi: 10.3969/j.issn.0529-1542.2011.05.017 GAO L N, CAO K Q, DUAN Y Z, et al. Biocontrol of Isatisindigotica root rot by the bacterial strain Bs-0728 [J]. Plant protection,, 2011, 37(5): 97−100.(in Chinese) doi: 10.3969/j.issn.0529-1542.2011.05.017
[14]	CHAURASIA B, PANDEY A, PALNI L M S, et al. Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformations in pathogenic fungi in vitro [J]. Microbiological Research, 2005, 160(1): 75−81. doi: 10.1016/j.micres.2004.09.013
[15]	BOWERS J H, LOCKE J C. Effect of formulated plant extracts and oils on population density of Phytophthora nicotianae in soil and control of Phytophthora blight in the greenhouse [J]. Plant Disease, 2004, 88(1): 11−16. doi: 10.1094/PDIS.2004.88.1.11
[16]	闫博巍. 土地类芽孢杆菌对玉米促生作用及其对大斑病生防机理研究[D]. 大庆: 黑龙江八一农垦大学, 2020. YAN B W. Research on the growth-promoting effect of Paenibacillus terrae on maize and its biological control mechanism against NCLB[D]. Daqing: Heilongjiang Bayi Agricultural University, 2020. (in Chinese).