威海地区西洋参根腐病病原菌鉴定及生防木霉筛选

doi:10.3976/j.issn.1002-4026.20240080

摘要/Abstract

摘要：

为探寻西洋参绿色栽培的有效生物防治资源,针对威海地区栽培西洋参根腐病,开展病原菌种类鉴定与高效生防木霉筛选工作。研究运用组织分离法,结合形态学观察及双基因(ITS/TEF1-α)系统发育分析,对西洋参根腐病病原真菌进行分离鉴定;借助柯赫氏法则测定其致病性;利用qPCR技术定量分析发病与健康西洋参根际土壤中主要病原菌的丰度差异;通过平板拮抗实验与盆栽实验筛选生防木霉菌株。结果表明,从西洋参根腐病病根组织共分离出125株真菌,其中镰孢菌属占比达70.91%,为优势属。经鉴定,腐皮镰孢(XYS-1)、尖孢镰孢(XYS-2)、层出镰孢(XYS-33)和交链格孢(XYS-44)这4株真菌体外接种可引发西洋参根腐病。qPCR分析表明,发病根际土壤中腐皮镰孢、尖孢镰孢和交链格孢的丰度分别比健康植株高出42.35%、13.80%和33.44%。此外,筛选出的木霉菌株HB20111、KZ23651、QT20747对相应病原菌抑制效果良好,抑制率分别为66.94%、76.00%和65.20%。温室盆栽实验进一步证实,接种木霉能提升西洋参株高、根鲜重、叶绿素含量和根系活力,降低根腐病发生率。该研究明确了威海西洋参根腐病病原菌种类,筛选出具有生防活性的木霉,为该地区西洋参根腐病的绿色防治奠定了重要基础。

关键词: 西洋参, 根腐病, 病原菌鉴定, 病原菌丰度, 木霉菌, 生物防治

Abstract:

In order to explore effective biological control resources for the cultivation of American ginseng, identification of pathogen species and screening of highly effective biocontrol Trichoderma were carried out for the root rot of American ginseng in Weihai. Through tissue isolation, morphological analysis, and dual-gene (ITS/TEF1-α) phylogenetic analysis, the pathogens responsible for root rot were isolated and identified. The pathogenicity was confirmed using Koch's postulates, and the abundance of key pathogens in the rhizosphere soils of diseased and healthy plants was analyzed through quantitative polymerase chain reaction (qPCR). Finally, biocontrol Trichoderma strains were screened through plate antagonism assays and pot experiments. The results showed that 125 fungal strains were isolated from the rotten roots of American ginseng, with Fusarium being the dominant genus, accounting for 70.91%. Four pathogenic strains were identified: F. solani (XYS-1), F. oxysporum (XYS-2), F. proliferatum (XYS-33), and Alternaria alternata (XYS-44). qPCR analysis revealed that the abundance of F. solani, F. oxysporum, and A. alternata in the rhizosphere soils of diseased plants was 42.35%, 13.80%, and 33.44% higher, respectively, than healthy plants. Three Trichoderma strains showed significant inhibitory effects against these pathogens. Specifically, strain HB20111 inhibited F. solani by 66.94%, strain KZ23651 inhibited F. oxysporum by 76.00%, and strain QT20747 inhibited A. alternata by 65.20%. Greenhouse pot experiments showed that Trichoderma inoculation increased plant height, root fresh weight, chlorophyll content in the leaf, and root activity of American ginseng while reducing the incidence of root rot. In this study, we identified the pathogens causing the root rot of American ginseng in Weihai and screened biocontrol Trichoderma strains, which provided a foundation for sustainable control of the root rot of American ginseng in this region.

Key words: American ginseng, root rot, pathogen identification, pathogen abundance, Trichoderma, biocontrol

中图分类号:

章嘉会, 李红梅, 陈冬梅, 杨翰, 扈进冬, 李纪顺, 魏艳丽. 威海地区西洋参根腐病病原菌鉴定及生防木霉筛选[J]. 山东科学, 2025, 38(4): 67-77.

ZHANG Jiahui, LI Hongmei, CHEN Dongmei, YANG Han, HU Jindong, LI Jishun, WEI Yanli. Identification of pathogens causing root rot of American ginseng in Weihai and screening of biocontrol Trichoderma strains[J]. Shandong Science, 2025, 38(4): 67-77.

图/表 11

表1

表2

表3

图1

图2

图3

图4

图5

图6

图7

图8

参考文献 42

[1]	ZOU L Q, KUANG X J, SUN C. Advances in transcriptomic studies and ginsenoside biosynthesis of American ginseng[J]. Chinese Herbal Medicines, 2015, 7(2): 116-122. DOI: 10.1016/s1674-6384(15)60028-4.
[2]	BI Y M, ZHANG X M, JIAO X L, et al. The relationship between shifts in the rhizosphere microbial community and root rot disease in a continuous cropping American ginseng system[J]. Frontiers in Microbiology, 2023, 14: 1097742. DOI: 10.3389/fmicb.2023.1097742.
[3]	赵璟琛, 伏松平, 胡加怡, 等. 西洋参枯萎病病原分离与鉴定[J]. 西北农业学报, 2022, 31(9): 1222-1227. DOI: 10.7606/j.issn.1004-1389.2022.09.015.
[4]	任绪明, 蒋景龙, 余妙, 等. 西洋参根腐病与三七素积累之间关系[J]. 中国实验方剂学杂志, 2018, 24(6): 42-46. DOI: 10.13422/j.cnki.syfjx.20180691.
[5]	DURAIRAJ K, VELMURUGAN P, VEDHANAYAKISRI K A, et al. Reprint of “Molecular and phenotypic characterization of pathogenic fungal strains isolated from ginseng root rot”[J]. Physiological and Molecular Plant Pathology, 2019, 105: 28-33. DOI: 10.1016/j.pmpp.2018.11.005.
[6]	杨姗姗, 王仪, 刘紫祺, 等. 西洋参根腐病抗性相关基因PqDELLA1的克隆及表达分析[J]. 中国现代中药, 2022, 24(5): 776-783. DOI: 10.13313/j.issn.1673-4890.20210507001.
[7]	JIAO X L, LU X H, CHEN A J, et al. Effects of Fusarium solani and F. oxysporum infection on the metabolism of ginsenosides in American ginseng roots[J]. Molecules, 2015, 20(6): 10535-10552. DOI: 10.3390/molecules200610535.
[8]	张天宇, 李恭民, 陈伟群, 等. 西洋参锈腐病病原研究[J]. 西北农林科技大学学报(自然科学版), 1991, 4(1): 43-48.
[9]	毕武, 陈娟, 焦晓林, 等. 北京地区西洋参根腐病病原鉴定及其致病性[J]. 植物保护, 2011, 37(5):135-138.
[10]	李阔, 王红阳, 郭秀芝, 等. 木霉属真菌诱导根及根茎类中药材抗根腐病的研究及应用进展[J]. 中国中药杂志, 2023, 48(18): 4942-4949. DOI: 10.19540/j.cnki.cjcmm.20230515.101.
[11]	黄钦. 西洋参根腐病生防菌剂研发[D]. 大理: 大理大学, 2021: 1-29.
[12]	杨蕊, 肖月, 李萍, 等. 澳洲坚果叶部病害病原菌鉴定及其生防菌筛选[J]. 经济林研究, 2023, 41(1): 282-291. DOI: 10.14067/j.cnki.1003-8981.2023.01.029.
[13]	李青, 谢昶琰, 张苗, 等. 防控甜瓜枯萎病病菌的生防菌筛选及其根际定殖[J]. 江苏农业学报, 2023, 39(2): 336-343. DOI: 10.3969/j.issn.1000-4440.2023.02.005.
[14]	张子恒. Burkholderia cepacia JA38菌株发酵工艺优化及对人参锈腐病菌生防作用研究[D]. 长春: 吉林农业大学, 2020: 41-57.
[15]	陈国忠, 冯志彬, 张兴晓, 等. 一种用于防治西洋参根腐病的多粘类芽孢杆菌及其应用: CN107937326B[P]. 2020-11-24.
[16]	牛兆颖, 王凤娇, 张淑凤, 等. 一株巨大芽孢杆菌及其在防治西洋参根腐病中的应用: CN109207392A[P]. 2019-01-15.
[17]	张悦丽, 王宪昌, 张博, 等. 一种预防西洋参根腐病的种植方法: CN114258830B[P]. 2023-04-25.
[18]	侯丽娟. 威海地区西洋参种植及高产栽培技术[J]. 农业科技通讯, 2017(7):283-286.
[19]	郭淑涵. 西洋参根腐病和炭疽病病原菌鉴定、生防菌筛选及效果评价[D]. 泰安: 山东农业大学, 2024.
[20]	MATHERON M E, PORCHAS M. Comparative ability of six fungicides to inhibit development of Phytophthora gummosis on Citrus[J]. Plant Disease, 2002, 86(6): 687-690. DOI: 10.1094/PDIS.2002.86.6.687.
[21]	方中达. 植病研究方法[M]. 3版. 北京: 中国农业出版社, 1998.
[22]	魏景超遗. 真菌鉴定手册[M]. 上海: 上海科学技术出版社, 1979.
[23]	张天宇. 中国真菌志(第十六卷)链格孢属[M]. 北京: 科学出版社, 2003.
[24]	董雪田, 朱恺丽, 曲音波, 等. 一种快速提取三孢布拉霉基因组的方法[J]. 食品与生物技术学报, 2021, 40(10): 63-71.
[25]	WHITE T J, BRUNS T, LEE S, et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[M]// PCR Protocols. Amsterdam: Elsevier, 1990: 315-322. DOI: 10.1016/b978-0-12-372180-8.50042-1.
[26]	O'DONNELL K, KISTLER H C, CIGELNIK E, et al. Multiple evolutionary origins of the fungus causing Panama disease of banana: Concordant evidence from nuclear and mitochondrial gene genealogies[J]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(5): 2044-2049. DOI: 10.1073/pnas.95.5.2044.
[27]	O'DONNELL K, NIRENBERG H I, AOKI T, et al. A multigene phylogeny of the Gibberella fujikuroi species complex: Detection of additional phylogenetically distinct species[J]. Mycoscience, 2000, 41(1): 61-78. DOI: 10.1007/BF02464387.
[28]	GARDES M, BRUNS T D. ITS primers with enhanced specificity for basidiomycetes:application to the identification of mycorrhizae and rusts[J]. Molecular Ecology, 1993, 2(2): 113-118. DOI: 10.1111/j.1365-294x.1993.tb00005.x.
[29]	LIEVENS B, BROUWER M, VANACHTER A C R C, et al. Quantitative assessment of phytopathogenic fungi in various substrates using a DNA macroarray[J]. Environmental Microbiology, 2005, 7(11): 1698-1710. DOI: 10.1111/j.1462-2920.2005.00816.x.
[30]	秦召, 李巧云, 段宗彪, 等. 一种快速、特异性检测小麦中链格孢菌(Alternaria alternata)的巢式PCR方法[J]. 河南农业科学, 2014, 43(10): 67-73. DOI: 10.3969/j.issn.1004-3268.2014.10.016.
[31]	张宪政. 植物叶绿素含量测定:丙酮乙醇混合液法[J]. 辽宁农业科学, 1986, 6(3): 26-28.
[32]	王学奎. 植物生理生化实验原理和技术[M]. 北京: 高等教育出版社, 2006.
[33]	LI J H, PHILP J, LI J S, et al. Trichoderma harzianum inoculation reduces the incidence of clubroot disease in Chinese cabbage by regulating the rhizosphere microbial community[J]. Microorganisms, 2020, 8(9): 1325. DOI: 10.3390/microorganisms8091325.
[34]	李丽, 蒋景龙, 董艳鑫, 等. 连作西洋参根际土壤化感物质筛选及化感效应分析[J]. 西北农业学报, 2022, 31(8): 1046-1057. DOI: 10.7606/j.issn.1004-1389.2022.08.012.
[35]	胡加怡, 赵璟琛, 胡红艳, 等. 秦巴山区西洋参病害种类调查[J]. 陕西农业科学, 2021, 67(12): 81-84. DOI: 10.3969/j.issn.0488-5368.2021.12.022.
[36]	文增叶, 李定华, 代梦瑶, 等. 三七根腐病病原菌尖孢镰刀菌的生物学特性分析[J]. 中药材, 2019, 42(9): 1978-1984. DOI: 10.13863/j.issn1001-4454.2019.09.003.
[37]	刘坤, 孙文松, 沈宝宇, 等. 辽宁新宾人参根腐病病原真菌的分离与鉴定[J]. 中国农学通报, 2022, 38(32): 86-91. doi: 10.11924/j.issn.1000-6850.casb2021-1117
[38]	汪静, 梁宗锁, 康冰, 等. 文山三七根腐病病原真菌的鉴定与药剂防治[J]. 西北林学院学报, 2015, 30(1): 158-163. DOI: 10.3969/j.issn.1001-7461.2015.01.26.
[39]	杨婷, 杨宽, 何迟, 等. 三七黑斑病病原菌Alternaria alternata Keissl.生物学特性研究[J]. 中药材, 2018, 41(4): 763-770.
[40]	何洁, 梁霜, 张国俊, 等. 太子参叶斑病病原鉴定及室内药剂筛选[J]. 南方农业学报, 2021, 52(8): 2124-2132.
[41]	燕嗣皇, 吴石平, 陆德清, 等. 木霉生防菌对根际微生物的影响与互作[J]. 西南农业学报, 2005, 3(1): 40-46.
[42]	HARMAN G E, HOWELL C R, VITERBO A, et al. Trichoderma species-opportunistic, avirulent plant symbionts[J]. Nature Reviews Microbiology, 2004, 2(1): 43-56.

引物		序列(5'-3')	扩增程序
ITS	ITS1	TCCGTAGGTGAACCTGCGG	94 ℃预变性4 min;94 ℃变性10 s,53 ℃退火30 s,72 ℃延伸30 s,30个循环后,72 ℃终延伸5 min
ITS	ITS4	TCCTCCGCTTATTGATATGC
TEF1-α	EF-1H	ATGGGTAAGGAAGACAAGAC
TEF1-α	EF-2T	GGAAGTACCAGTGATCATGTT

病原菌	引物	序列(5'-3')	扩增程序
F. solani	ITS1F-F	CTTGGTCATTTAGAGGAAGTAA	Gardes et al^[28]; Lievens et al^[29]
F. solani	AFP346-R	GGTATGTTCACAGGGTTG ATG
F. oxysporum	ITS1F-F	CTTGGTCATTTAGAGGAAGTAA
F. oxysporum	AFP308-R	CGAATTAACGCGAGTCCC AAC
A. alternata	Aa1-F	TATGAAGGCGGGCTGGAA	秦召等^[30]
A. alternata	Aa1-R	GTCGCACTCTCTATCAGCAAA	秦召等^[30]

菌株编号	拉丁名	来源
C61	Trichoderma harzianum	黑龙江省小兴安岭林地
LTR-2	Trichoderma harzianum	北京市农贸市场灵芝
LK20397	Trichoderma harzianum	山西省运城盐湖
TW21990	Trichoderma harzianum	湖南省洞庭湖
KZ23651	Trichoderma harzianum	吉林省抚松县森林
KZ23646	Trichoderma harzianum	吉林省清宇县森林
HB20111	Trichoderma atroviride	黄河三角洲湿地
QT20747	Trichoderma longibrachiatum	内蒙古自治区临河区