烟云彩螽肠道细菌的分离鉴定及抗菌活性菌株筛选

doi:10.16088/j.issn.1001-6600.2025011603

摘要/Abstract

摘要： 采用稀释涂布法对烟云彩螽肠道细菌进行分离纯化,并结合形态学和分子生物学方法对其进行鉴定;同时,采用平板对峙法测定其对植物病原真菌的拮抗活性,进一步以菌丝生长速率法评价活性肠道细菌发酵产物提取物对植物病原真菌的抗菌效果。结果表明,从烟云彩螽肠道中分离获得76株可培养细菌,其中11株细菌经形态学和分子生物学鉴定归为9个不同属,包括亮杆菌属Leucobacter、微杆菌属Microbacterium、纤维微菌属Cellulosimicrobium、节杆菌属Arthrobacter、赖氨酸芽孢杆菌属Lysinibacillus、葡萄球菌属Staphylococcus、大西洋杆菌属Atlantibacter、克雷伯氏菌属Klebsiella、肠杆菌属Enterobacter。通过平板对峙法测定发现,产酸克雷伯氏菌Klebsiella oxytoca BS.X3-A1对9种供试病原菌均具有拮抗活性。进一步测定该菌株发酵产物对9种供试病原菌的抑制活性,当处理浓度为4 g/L时,其对甘蔗凤梨病菌、茶轮斑病菌、贡柑链格孢菌、水稻胡麻叶斑病菌、甘蓝黑斑病菌的抑制率均为100%;对辣椒炭疽病菌、金橘砂皮病菌、玉米大斑病菌的抑制率均超过80%;对烟草黑胫病菌抑制率相对较弱,为54.68%。接着,测定该菌株对9种植物病原真菌菌丝毒力,发现EC₅₀值在0.394 8~3.908 9 g/L。其中,对甘蔗凤梨病菌抑制效果最佳,EC₅₀值为0.394 8 g/L,小于0.5 g/L;其次是茶轮斑病菌和贡柑链格孢菌,EC₅₀值分别为1.132 0和1.326 8 g/L,均小于1.5 g/L。此外,对玉米大斑病菌、水稻胡麻叶斑病菌、金橘砂皮病菌也表现出较好的抗菌活性,EC₅₀值分别为2.225 6、2.226 3、2.327 2 g/L,均小于3 g/L;而对甘蓝黑斑病菌和烟草黑胫病菌的抗菌活性相对较差,EC₅₀值为3.071 8和3.908 9 g/L。综上,烟云彩螽肠道中具有抗植物病原真菌的细菌,其肠道细菌可产生抗植物病原真菌的次生代谢产物。研究结果为后续从烟云彩螽肠道菌中挖掘抗植物病原真菌活性物质提供参考。

关键词: 烟云彩螽, 肠道细菌, 分离鉴定, 植物病原真菌, 抗菌活性

Abstract: Intestinal bacteria of the Callimenellus fumidus Walker were isolated and purified by the dilution coating method and identified by a combination of morphological and molecular biological methods;at the same time, their antagonistic activity against plant pathogenic fungi was determined by the dual culture method,and the antifungal efficacy of the extracts of the fermentation products of the acvtive intestinal bacteria on plant pathogenic fungi was further evaluated by the mycelial growth rate method. The results showed that 76 culturable bacterial strains were isolated from the intestines of C. fumidus Walker,of which 11 strains were identified through morphological and molecular biological methods as belonging to 9 different genera,including Leucobacter, Microbacterium, Cellulosimicrobium, Arthrobacter, Lysinibacillus, Staphylococcus, Atlantibacter, Klebsiella and Enterobacter. The strain Klebsiella oxytoca BS.X3-A1 was found to have antagonistic activity against all the 9 test pathogens as determined by dual culture method. The inhibitory activity of the fermentation product of the strain against the 9 test pathogens was further determined. At a concentration of 4 g/L,the inhibition rates against Ceratocystis paradoxa,Pestalotiopsis theae,Alternaria citri,Cochliobolus miyabeanus,and Alternaria oleracea all reached 100%. The inhibition rates against anthracnose caused by Colletotrichum capsici,Diaporthe citri and Exserohilum turcicum exceeded 80%, while the inhibition rate against Phytophthora parasitica var. Nicotianae was relatively weaker at 54.68%. The virulence of the strain BS.X3-A1 against 9 phytopathogenic fungi was determined and found to have EC₅₀ values ranging from 0.394 8 to 3.908 9 g/L. Among them,it exhibited the strongest antifungal activity against Ceratocystis paradoxa with EC₅₀ values of 0.394 8 g/L,which was less than 0.5 g/L, followed by Pestalotiopsis theae and Alternaria citri with EC₅₀values of 1.132 0 and 1.326 8 g/L,respectively both below 1.5 g/L. In addition,it also showed good antifungal activity against Exserohilum turcicum,Cochliobolus miyabeanus,and Diaporthe citri with EC₅₀ values of 2.225 6,2.226 3,and 2.327 2 g/L,respectively,which were all less than 3 g/L;while the antifungal activities against Alternaria oleracea and Phytophthora parasitica var. Nicotianae were relatively weaker,with EC₅₀ values of 3.071 8 and 3.908 9 g/L,respectively. The results showed that the intestine of C. fumidus Walker harbors bacteria with antifungal activity against plant pathogenic fungi,and these intestinal bacteria produce secondary metabolites effective against plant pathogenic fungi. The results of the study provide a reference for further exploration of antifungal compounds derived from the intestinal bacteria of the C. fumidus Walker.

Key words: Callimenellus fumidus Walker, intestinal bacteria, isolation and identification, plant pathogenic fungi, antifungal activity

中图分类号: S481;Q939.9

陈雪玉, 施慧露, 黄秋菊, 边迅, 骆海玉. 烟云彩螽肠道细菌的分离鉴定及抗菌活性菌株筛选[J]. 广西师范大学学报（自然科学版）, 2025, 43(6): 204-214.

CHEN Xueyu, SHI Huilu, HUANG Qiuju, BIAN Xun, LUO Haiyu. Isolation and Identification of Intestinal Bacteria from Callimenellus fumidus Walker and Screening of Antifung Active Strains[J]. Journal of Guangxi Normal University(Natural Science Edition), 2025, 43(6): 204-214.

参考文献

[1] 叶佳琴, 郅军锐, 黄宸. 肠道细菌对西花蓟马生长发育和繁殖的影响[J]. 山地农业生物学报, 2020, 39(5): 45-49. DOI: 10.15958/j.cnki.sdnyswxb.2020.05.007.
[2] CALDERÓN-CORTÉS N, QUESADA M, WATANABE H, et al. Endogenous plant cell wall digestion: a key mechanism in insect evolution[J]. Annual Review of Ecology, Evolution, and Systematics, 2012, 43: 45-71. DOI: 10.1146/annurevecolsys110411160312.
[3] ITOH H, HORI T, SATO Y, et al. Infection dynamics of insecticidedegrading symbionts from soil to insects in response to insecticide spraying[J]. The ISME Journal, 2018, 12(3): 909-920. DOI: 10.1038/s4139601700219.
[4] 郑龙玉. 卵携带的微生物对亮斑扁角水虻产卵行为和生长发育的影响[D]. 武汉: 华中农业大学, 2012.
[5] BEHAR A, YUVAL B, JURKEVITCH E. Gut bacterial communities in the Mediterranean fruit fly (Ceratitis capitata) and their impact on host longevity[J]. Journal of Insect Physiology, 2008, 54(9): 1377-1383. DOI: 10.1016/j.jinsphys.2008.07.011.
[6] 徐红星, 郑许松, 刘淑平, 等. 昆虫内共生菌在昆虫防御中的作用[J]. 昆虫知识, 2009, 46(3): 350-354. DOI: 10.3969./j.issn.0452-8255.2009.03.004.
[7] 杜甜甜, 张晨宁, 诸卫平, 等. 中药材提取物抑制植物病原菌研究进展[J]. 生态科学, 2024, 43(1): 265-272. DOI: 10.14108/j.cnki.10088873.2024.01.031.
[8] 黄世文, 刘连盟, 赵可菡, 等. 三唑类杀菌剂对水稻药害机理及解决方案[J]. 植物保护, 2022, 48(4): 107-113.DOI: 10.16688/j.zwbh.2021288.
[9] 宋佳露, 程星凯, 刘鹏飞, 等. 植物病原菌对杀菌剂多药抗性的发生现状[J]. 植物保护, 2021, 47(6): 28-33, 65. DOI: 10.16688/j.zwbh.2020413.
[10] BAI Y B, GAO Y Q, NIE X D, et al. Antifungal activity of griseofulvin derivatives against phytopathogenic fungi in vitro and in vivo and three-dimensional quantitative structure-activity relationship analysis[J]. Journal of Agricultural and Food Chemistry, 2019, 67(22): 6125-6132. DOI: 10.1021/acs.jafc.9b00606.
[11] 邹修为, 熊有明, 谢剑波, 等. 一株辣椒疫霉拮抗菌Y439分离鉴定及生防作用研究[J]. 湖南农业科学, 2022(6): 1-5. DOI: 10.16498/j.cnki.hnnykx.2022.006.001.
[12] 杨德伟, 施春兰, 解紫薇, 等. 铜绿假单胞菌HZ15的生物活性[J]. 微生物学通报, 2025, 52(4): 1537-1550. DOI: 10.13344/j.microbiol.china.240612.
[13] 周定中, 曹露, 王茂淋, 等. 黑水虻肠道细菌抗菌筛选及其活性物质分子鉴定[J]. 微生物学通报, 2012, 39(11): 1614-1621. DOI: 10.13344/j.microbiol.china.2012.11.011.
[14] CHANWORAWIT K, WANGSOONTHORN P, DEEVONG P. Characterization of chitinolytic bacteria newly isolated from the termite Microcerotermes sp. and their biocontrol potential against plant pathogenic fungi[J]. Bioscience, Biotechnology, and Biochemistry, 2023, 87(9): 1077-1091. DOI: 10.1093/bbb/zbad080.
[15] WANG Y N, YANG D, YU Z G. New lactones produced by Streptomyces sp. SN5431 and their antifungal activity against Bipolaris maydis[J]. Microorganisms, 2023, 11(3): 616. DOI: 10.3390/microorganisms11030616.
[16] 白锦荣. 广西螽斯科区系研究[D]. 保定: 河北大学, 2014.
[17] 武永霞. 中国拟叶螽亚科分类(直翅目:螽斯科)[D]. 保定: 河北大学, 2019.
[18] 付东冉, 许小霞, 冯淑杰, 等. 小菜蛾肠道成团泛菌PxG45的分离鉴定及其抗真菌活性[J]. 昆虫学报, 2023, 66(10): 1343-1353. DOI: 10.16380/j.kcxb.2023.10.008.
[19] PAN Y Y, BIAN X. Four new species of the genus Phlugiolopsis (Orthoptera: Tettigoniidae: meconematinae) from Guangxi, China[J]. Zootaxa, 2024, 5443(2): 287-297. DOI: 10.11646/zootaxa.5443.2.11.
[20] RAN Y Q, BIAN X. A new species of Xiphidiopsis (Euxiphidiopsis)(Orthoptera: Tettigoniidae: meconematinae) from Guangxi, China[J]. Zootaxa, 2023, 5383(3): 398-400. DOI: 10.11646/zootaxa.5383.3.9.
[21] 陈春. 湖南省螽斯科区系研究[D]. 保定: 河北大学, 2024.
[22] 谢慧聪. 中国拟叶螽亚科系统发育研究[D]. 上海: 华东师范大学, 2022. DOI: 10.27149/d.cnki.ghdsu.2022.001087.
[23] 马兰. 中国蝈螽属物种界定、遗传多样性及系统进化关系研究[D]. 保定: 河北大学, 2021.
[24] 黄慧敏. 优雅蝈螽肠道可培养细菌组研究[D]. 保定: 河北大学, 2023.
[25] ZHENG X, ZHU Q D, QIN M, et al. The role of feeding characteristics in shaping gut microbiota composition and function of Ensifera (Orthoptera)[J]. Insects, 2022, 13(8): 719. DOI: 10.3390/insects13080719.
[26] 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.
[27] 田书鑫, 刘南南, 王桂清. 对峙培养法在生防菌抑制效果研究中的应用[J]. 河南农业科学, 2019, 48(8): 1-6. DOI: 10.15933/j.cnki.10043268.2019.08.001.
[28] LUO H Y, ZHOU Q Y, DENG Y C, et al. Antifungal activity of the extract and the active substances of endophytic Nigrospora sp. from the traditional Chinese medicinal plant Stephania kwangsiensis[J]. Natural Product Communications, 2017, 12(12): 1889-1892. DOI: 10.1177/1934578X1701201219.
[29] 李奇聪, 陈洁萍, 欧艳绍, 等. 柑橘内生真菌LJZY11次生代谢产物抑菌活性研究[J]. 广西师范大学学报(自然科学版), 2023, 41(1): 155-163. DOI: 10.16088/j.issn.10016600.2021102801.
[30] 车旭婷. 优雅蝈螽肠道微生物多样性研究[D]. 保定: 河北大学, 2019.
[31] 郑翔. 螽亚目食性与肠道菌群结构和功能的关系及肠道功能微生物的筛选鉴定[D]. 保定: 河北大学, 2023.
[32] 何明川, 施春兰, 魏聪聪, 等. 烟草黑胫病拮抗细菌的分离、鉴定及发酵条件优化[J]. 南方农业学报, 2022, 53(6): 1604-1615. DOI: 10.3969/j.issn.20951191.2022.06.014.
[33] 叶敏, 毛雪, 田小卫. 黄瓜枯萎病拮抗昆虫共生菌的筛选及抑菌机制初步研究[J]. 江苏农业科学, 2024, 52(19): 143-150. DOI: 10.15889/j.issn.10021302.2024.19.020.
[34] YUAN Y, ZHANG S J, TAN X S, et al. Intestinal bacterium Bacillus siamensis M54 from Allomyrina dichotoma is a potential biocontrol agent against maize stalk rot[J]. Biological Control, 2024, 199: 105660. DOI: 10.1016/j.biocontrol.2024.105660.
[35] 杨丹丹, 张心青, 杨传伦, 等. 一株产酸克雷伯氏菌发酵培养基优化[J]. 微生物学杂志, 2020, 40(6): 75-83. DOI: 10.3969/j.issn.10057021.2020.06.011.
[36] MAWAD A M M, HASSANEIN M, ALDABY E S, et al. Desulphurisation kinetics of thiophenic compound by sulphur oxidizing Klebsiella oxytoca SOB1[J]. Journal of Applied Microbiology, 2021, 130(4): 1181-1191. DOI: 10.1111/jam.14829.
[37] HUSSENEDER C, PARK J S, HOWELLS A, et al. Bacteria associated with Piezodorus guildinii (Hemiptera: Pentatomidae), with special reference to those transmitted by feeding[J]. Environmental Entomology, 2017, 46(1): 159-166. DOI: 10.1093/ee/nvw112.
[38] 高欢欢, 覃冬云, 代晓彦, 等. 肠道细菌弗氏柠檬酸杆菌和产酸克雷伯氏菌对斑翅果蝇生长发育和物质代谢的影响[J]. 昆虫学报, 2020, 63(4): 462-469. DOI: 10.16380/j.kcxb.2020.04.009.
[39] 林伟, 骆海玉, 邓业成, 等. 血散薯块根内生真菌Periconia igniaria Stdif10发酵产物的生物活性研究[J]. 河南农业科学, 2020, 49(1): 75-81. DOI: 10.15933/j.cnki.10043268.2020.01.010.
[40] 蒙思妤, 邓业成, 廖金梅, 等. 拟可可毛球二孢菌等7种草珊瑚内生真菌发酵产物的抑菌活性研究[J]. 福建农业学报, 2022, 37(8): 1056-1066. DOI: 10.19303/j.issn.10080384.2022.008.011.
[41] 韦坚芬, 邓志勇, 邓业成, 等. 草珊瑚内生真菌SgG4抗植物病原真菌的活性物质研究[J]. 广西师范大学学报(自然科学版), 2024, 42(2): 175-182. DOI: 10.16088/j.issn.10016600.2023041801.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed