铁皮石斛镰刀菌根腐病病原菌的鉴定及其对链霉菌发酵液的敏感性分析

doi:10.16409/j.cnki.2095-039x.2022.02.004

摘要/Abstract

摘要： 根腐病是危害浙江省铁皮石斛的重要病害之一，鉴定根腐病的病原菌是有效防治该病害的前提条件。为进一步明确前期经柯赫法则验证为病原菌的镰刀菌（编号为GF-14）分类地位，本研究通过形态学鉴定，并结合内源转录间隔区（rDNA-ITS）、翻译延伸因子（TEF-1α）和RNA聚合酶基因（RPB1和RPB2）等序列的同源性比对及系统发育分析，确认该致病菌属于变红-木贼镰刀菌复合种群Fusarium incarnatum-equiseti species complex。进一步采用菌丝生长速率法，测定了该病原菌对多种杀菌剂的敏感性，并选取对菌丝生长抑制效果最佳的杀菌剂进行田间防效的测定。结果表明：本实验室分离获得的生防菌淀粉酶产色链霉菌Streptomyces diastatochramogenes 1628发酵液对该致病菌的抑制效果最佳，发酵液稀释10倍后抑制率高达98.22%；而宁南霉素、井冈霉素则对该致病菌无显著抑制作用。菌株对峙试验发现淀粉酶产色链霉菌1628对菌株GF-14的菌丝生长抑制率为32.65%。田间防效试验表明其发酵液对铁皮石斛根腐病病株具有防治效果，为34.90%。本研究明确了铁皮石斛镰刀菌根腐病病原菌的分类地位，并筛选到能有效抑制该病原菌的微生物杀菌剂，为后续铁皮石斛根腐病的防治提供依据。

关键词: 铁皮石斛, 根腐病, 镰刀菌, 病原菌鉴定, 淀粉酶产色链霉菌发酵液

Abstract: Root rot is one of the main diseases that have been harming Dendrobium officinale in Zhejiang Province. Pathogen identification is crucial to prevent and control root rot effectively. Fusarium sp. (No. GF-14) has been verified by Koch's rule, which is an important pathogen of D. officinale root rot. First, homology alignment and phylogenetic tree of sequences was analyzed, which included the internally transcribed spacer (rDNA-ITS), translation elongation factor (TEF-1α) and RNA polymerase genes (RPB1 and RPB2). At the same time, the morphological identification was also performed. It was confirmed that the pathogen GF-14 was F. incarnatum-equiseti species complex isolate. Afterwards, the susceptibility of GF-14 to a variety of fungicides was determined by mycelial growth rate method. The fungicide with best inhibitory effect on mycelial growth was selected to determine the field control effect. The results showed that the fermentation broth of Streptomyces diatatochromogenes 1628 diluted 10 times had the best biocontrol effect on the pathogenic fungus GF-14 (98.22%), while Ningnanmycin and Jinggangmycin had no effect on GF-14. Furthermore, the inhibitory rate of 1628 on the mycelial growth of GF-14 was 32.65% in the confrontation experiment, and the fermentation production of 1628 had a great biocontrol effect on root rot in the field (34.90%). This study clarified the pathogen classification of D. officinale root rot and proved the metabolites of 1628 could be used for biocontrol the disease in the future.

Key words: Dendrobium officinale, root rot, Fusarium, identification of pathogen, fermentation broth of Streptomyces diastatochramogenes

中图分类号:

S476

张祥丽, 曹瑱艳, 杨怡华, 宋阳, 申屠旭萍, 俞晓平. 铁皮石斛镰刀菌根腐病病原菌的鉴定及其对链霉菌发酵液的敏感性分析[J]. 中国生物防治学报, 2022, 38(1): 258-266.

ZHANG Xiangli, CAO Zhenyan, YANG Yihua, SONG Yang, SHENTU Xuping, YU Xiaoping. Identification of the Pathogen Causing Root Rot of Dendrobium officinale and Sensitivity to the Fermentation Broth of Streptomyces[J]. Chinese Journal of Biological Control, 2022, 38(1): 258-266.

参考文献

[1] 中国药典2010年版(一部)[S]. 北京:中国医药科技出版社, 2010, 265-266.
[2] 肖开心, 朱滢嘉, 陈瑞, 等. 铁皮石斛药理作用研究进展[J]. 河南中医, 2020, 40(5):788-792.
[3] 戴德江, 沈颖, 沈瑶, 等. 浙产特色中药材病虫害化学防治的研究进展[J]. 农药学学报, 2019, 21(s1):759-771.
[4] 曹星星. 铁皮石斛病原真菌分离与鉴定及印度梨形孢促生作用研究[D]. 杭州:浙江大学, 2015.
[5] 李梦娇. 铁皮石斛常见真菌病害的鉴定及复合菌剂的应用研究[D]. 昆明:云南农业大学, 2017.
[6] Wu X L, Wang Y, Liu F, et al. Identification of Coptis chinensis root rot disease pathogenic Fusarium spp. fungi[J]. China Journal of Chinese Materia Medica, 2020, 45(6):1323-1328.
[7] Scherm B, Balmas V, Spanu F, et al. Fusarium culmorum:causal agent of foot and root rot and head blight on wheat[J]. Molecular Plant Pathology, 2013, 14(4):323-341.
[8] Latiffah Z, Hayati M, Baharuddin S, et al. Identification and pathogenicity of Fusarium species associated with root rot and stem rot of Dendrobium[J]. Asian Journal of Plant Pathology, 2010, 3(1):14-21.
[9] Chittem K, Mathew F M, Gregoire M, et al. Identification and characterization of Fusarium spp. associated with root rots of field pea in North Dakota[J]. European Journal of Plant Pathology, 2015, 143(4):641-649.
[10] 孔琼, 袁盛勇, 郭建伟, 等. 铁皮石斛根腐病病原菌鉴定及生物学特性研究[J]. 中药材, 2018, 41(7):1566-1570.
[11] 毛文龙, 陈玲菱, 刘涛, 等. 杭州地区设施大棚铁皮石斛主要病虫害绿色防控技术[J]. 现代园艺, 2020, 43(16):54-55.
[12] 曹瑱艳, 杨怡华, 申屠旭萍, 等. 浙江省铁皮石斛根腐病病原真菌的鉴定[J]. 植物保护学报, 2020, 47(1):178-186.11.
[13] O'Donnell K, Sutton D A, Rinaldi M G, et al. Internet-accessible DNA sequence database for identifying fusaria from human and animal infections[J]. Journal of Clinical Microbiology, 2010, 48(10):3708-3718.
[14] 申屠旭萍, 于冰, 李丹婷, 等. HPLC法测定发酵液中丰加霉素的研究[J]. 中国计量大学学报, 2013, 24(3):215-218.
[15] Shentu X P, Liu N, Tang G, et al. Improved antibiotic production and silent gene activation in Streptomyces diastatochromogenes by ribosome engineering[J]. Journal of Antibiotics, 2016, 69(5):406-410.
[16] 朴怡情, 申屠旭萍, 叶子弘. 木霉素标准样品的研制[J]. 中国计量大学学报, 2019, 30(4):484-489.
[17] 申屠旭萍, 石一珺, 俞晓平. GC法测定研究发酵液中木霉素[J]. 中国药学杂志, 2008, (22):1755-1756.
[18] 易润华, 朱西儒, 周而勋. 简化CTAB法快速微量提取丝状真菌DNA[J]. 湛江海洋大学学报, 2003(6):72-73.
[19] 魏景超. 真菌鉴定手册[M]. 北京:科学出版社, 1979.
[20] 布斯. 镰刀菌属[M]. 北京:农业出版社, 1988.
[21] 张匀华. 小麦赤霉病发病率与病情指数关系初步研究[J]. 黑龙江农业科学, 2000(3):3-5.
[22] 周艳丽, 李薇, 薛超, 等. 30%壬菌铜微乳剂防治水稻白叶枯病药效试验[J]. 农药, 2014, 53(5):375-376.
[23] Avila C F, Moreira G M, Nicolli C P, et al. Fusarium incarnatum-equiseti species complex associated with Brazilian rice:Phylogeny, morphology and toxigenic potential[J]. International Journal of Food Microbiology, 2019, 2:306.
[24] Xia J W, Sandoval-Denis M, Crous P W, et al. Numbers to names-restyling the Fusarium incarnatum-equiseti species complex[J]. Persoonia, 2019, 43:186-221.
[25] Kim B, Choi Y J. Fusarium fujikuroi causing Fusarium wilt of Lactuca serriola in Korea[J]. Plant Disease, 2020, 105(2):502.
[26] Torbati M, Arzanlou M, da Silva Santos A C. Fungicolous Fusarium species:ecology, diversity, isolation, and identification[J]. Current Microbiology, 2021, 78(8):2850-2859.
[27] 朱将伟. 生物农药的应用与研究进展——生防菌及其相关的生物农药[J]. 绿色科技, 2019(22):203-205.
[28] Kwon M K, Hong J R, Kim Y H, et al. Soil-environmental factors involved in the development of root rot/vine on Cucurbits caused by Monosporascus cannonballus[J]. The Plant Pathology Journal, 2001, 17(1):45-51.
[29] El-Sayed S A, Abdel-Monaim M F. Integrated control management of root rot disease in lupine plants by using some bio-agents, chemical inducers and fungicides[J]. Plant Science, 2017, 1:1.
[30] Saad M M G, Kandil M, Mohammed Y M M. Isolation and identification of plant growth-promoting bacteria highly effective in suppressing root rot in Fava Beans[J]. Current Microbiology. 2020, 77(9):2155-2165.
[31] 杨瑞先, 刘萍, 王祖华, 等. 牡丹根腐病原菌拮抗细菌抑菌活性物质分析[J]. 生物技术通报, 2021, 38(2):1-11.
[32] 卢志军, 云晓敏, 刘西莉. 生防菌株HL29的鉴定及其抑菌活性研究[J]. 植物病理学报, 2005(S1):79-85.
[33] 姚晓明, 施德, 何春龙, 等. 浙江省主要中药材病虫发生现状与防治对策[J]. 浙江农业科学, 2014(12):1785-1788.
[34] 曹瑱艳, 申屠旭萍, 俞晓平. 杭白菊枯萎病的病原菌分离鉴定及淀粉酶产色链霉菌对其防治效果[J]. 中国生物防治学报, 2019, 35(2):265-271.
[35] 申屠旭萍, 于冰, 边亚琳, 等. 拮抗链霉菌B28的分类鉴定及其生防作用研究[J]. 植物病理学报, 2012, 42(1):105-109.
[36] 于冰, 申屠旭萍, 俞晓平. 丰加霉素对黄瓜立枯丝核菌的拮抗作用[J]. 中国生物防治学报, 2011, 27(3):373-377.