绿僵菌系统发育及防治松褐天牛成虫的菌株筛选

doi:10.11707/j.1001-7488.LYKX20240014

摘要/Abstract

摘要：

目的: 研究国内外80株绿僵菌的系统发育关系及其对松褐天牛成虫的毒力，为筛选用于防治松褐天牛成虫的高毒力菌株提供参考。方法: 提取80株绿僵菌的DNA，使用引物ITS1/ITS4和T1/T22分别扩增真菌的ITS区域和β-tubulin基因序列并测序。采用MAFFT比对序列，通过IQ-TREE和Mrbayes 3.2.7软件分别采用最大似然法和贝叶斯法构建系统发育树。使用羽化3天内的健壮松褐天牛成虫测定绿僵菌的毒力。在PDA培养基上培养绿僵菌，将收集获得的孢子用0.05%的吐温-80溶液分别配置成浓度为10⁷ 个·mL^–1的孢子悬浮液，0.05%的吐温80溶液作为空白对照。采用浸渍法处理松褐天牛成虫，以累计校正死亡率为指标评价菌株的致病力。结果: 从国内外菌库中收集的123株菌株中80株为绿僵菌，分为6个主要类群，其中57株属于金龟子绿僵菌复合种，11株属于黄绿绿僵菌复合种，6株属于莱氏绿僵菌，4株属于M. viride，1株属于柱孢绿僵菌，1株属于新西兰绿僵菌。高毒力菌株主要分布在金龟子绿僵菌复合种和黄绿绿僵菌复合种；蝗绿僵菌、柱孢绿僵菌、新西兰绿僵菌、莱氏绿僵菌和M. viride对松褐天牛的成虫未表现出致病力。14株绿僵菌菌株毒力较高（LT₅₀<15天，15天校正死亡率>80%），可作为防治松褐天牛的备选菌株。结论: 基于国内外80株绿僵菌菌株的系统发育和毒力测试结果，筛选出对松褐天牛成虫具有高毒力的菌株，其中14株具有较大应用潜力，建立了绿僵菌的系统发育关系，确认了高毒力菌株的分布。

关键词: 绿僵菌, 松褐天牛, 菌株鉴定筛选, 生物防治

Abstract:

Objective: The phylogenetic relationship of 80 Metarhizium strains isolated from domestic and abroad and their virulence to Monochamus alternatus adult were investigated in order to screen highly virulent strains for the control of M. alternatus adult. Method: The ITS1/ITS4 and T1/T22 primer pairs were used to amplify the ITS region and β-tubulin gene sequences of 80 Metarhizium strains, respectively. MAFFT was used to align the sequences, and the phylogenetic tree was constructed using the maximum likelihood method and the Bayesian method with IQ-TREE and Mrbayes 3.2.7 software. The virulence of Metarhizium strains was determined using M. alternatus adults in 1-3 days after emergence. Metarhizium strains were cultured on PDA medium, and the spores were collected and prepared into a spore suspension with a concentration of 10⁷·mL^?1 using 0.05% Tween 80 solution, and 0.05% Tween 80 solution was used as a blank control. The impregnation method was used to treat M. alternatus adults, and the pathogenicity of the strains was evaluated by using the cumulative corrected mortality rate of M. alternatus adults. Result: Among the 123 strains collected from domestic and international libraries, there are 80 strains of Metarhizium, which are divided into 6 major taxa, including 57 strains belonging to M. anisopliae species complex, 11 strains belonging to M. flavoviride species complex, 6 strains belonging to M. rileyi, 4 strains belonging to M. viride, one strain belonging to M. cylindrosporum, and one strain belonging to M. novozealandicum. The highly virulent strains were mainly found in M. anisopliae species complex and M. flavoviride species complex. M. acridum, M. cylindrosporum, M. novozealandicum, M. rileyi, and M. viride did not show pathogenicity against M. alternatus adults. A total of 14 strains of Metarhizium were highly virulence (LT₅₀< 15 d, 15-day adjusted mortality >80%), which can be used as alternative strains for the control of M. alternatus. Conclusion: Based on the phylogenetic and virulence test results of 80 strains of Metarhizium from domestic and international, strains with high virulence against M. alternatus adult have been screened, among which 14 strains have high application potential. The phylogenetic relationships of Metarhizium are established, and the distribution of highly virulent strains against M. alternatus adult is confirmed.

Key words: Metarhizium, Monochamus alternatus, screening and identification of strains, biocontrol

中图分类号:

S763.3

刘程林,应玥,王瑞珍,刘云朋,王青华,孔德治,曲良建. 绿僵菌系统发育及防治松褐天牛成虫的菌株筛选[J]. 林业科学, 2025, 61(2): 152-162.

Chenglin Liu,Yue Ying,Ruizhen Wang,Yunpeng Liu,Qinghua Wang,Dezhi Kong,Liangjian Qu. Phylogenesis of Metarhizium Strains and Their Screening for Biocontrol of Monochamus alternatus Adults (Coleoptera: Cerambycidae)[J]. Scientia Silvae Sinicae, 2025, 61(2): 152-162.

图/表 5

表1

表2

鉴定为绿僵菌的菌株"

序号 No.	菌株编号 Strain number	采集区 Geographic location	种名 Species
1	CBS115995	奥地利 Austria	棕色绿僵菌 M. brunneum
2	CBS125.65	德国 Germany	黄绿绿僵菌 M. flavoviride
3	CBS130.22	未知 Unknown	平沙绿僵菌 M. pinghaense
4	CBS130.71	乌克兰 Ukraine	鳞腮绿僵菌 M. lepidiotae
5	CBS130.92	巴西 Brazil	鳞腮绿僵菌 M. lepidiotae
6	CBS158.84	巴西 Brazil	金龟子绿僵菌 M. anisopliae
7	CBS160.96	巴布亚新几内亚 Papua New Guinea	鳞腮绿僵菌 M. lepidiotae
8	CBS194.65	科特迪瓦 Ivory Coast	莱氏绿僵菌 M. rileyi
9	CBS218.29	捷克共和国 Czech Republic	平沙绿僵菌 M. pinghaense
10	CBS218.56	捷克共和国 Czech Republic	黄绿绿僵菌 M. flavoviride
11	CBS229.52	荷兰 Netherlands	瘿绵蚜绿僵菌 M. pemphigi
12	CBS247.64	法国 France	罗伯茨绿僵菌 M. robertsiie
13	CBS248.64	法国 France	大孢绿僵菌 M. majus
14	CBS258.90	中国 China	贵州绿僵菌 M. guizhouense
15	CBS285.59	德国 Germany	M. indigoticum
16	CBS289.67	厄立特里亚 Eritrea	罗伯茨绿僵菌 M. robertsii
17	CBS316.51	俄勒冈州 Oregon	棕色绿僵菌 M. brunneum
18	CBS339.35	阿根廷 Argentina	蝗绿僵菌 M. acridum
19	CBS348.65	马达加斯加 Madagascar	M. viride
20	CBS380.73A	德国 Germany	黄绿绿僵菌 M. flavoviride
21	CBS380.73B	德国 Germany	黄绿绿僵菌 M. flavoviride
22	CBS407.96	未知 Unknown	M. viride
23	CBS454.78	佛罗里达 Florida	罗伯茨绿僵菌 M. robertsii
24	CBS459.75	密歇根州 Michigan	平沙绿僵菌 M. pinghaense
25	CBS464.70	以色列 Isreal	平沙绿僵菌 M. pinghaense
26	CBS473.73	荷兰 Netherlands	黄绿绿僵菌 M. flavoviride
27	CBS544.81	厄瓜多尔 Ecuador	蝗绿僵菌 M. acridum
28	CBS614.85	所罗门群岛 Solomon Islands	黄绿绿僵菌小孢变种 M. minus
29	CBS615.84	菲律宾 Philippines	柱孢绿僵菌 M. cylindrosporum
30	CBS643.67	萨摩亚 Samoa	大孢绿僵菌 M. majus
31	CBS648.67	法国 France	M. bibonidarum
32	CBS650.67	伊朗 Iran	罗伯茨绿僵菌 M. robertsii
33	CBS655.67	法国 France	棕色绿僵菌 M. brunneum
34	CBS656.67	科特迪瓦 Ivory Coast	罗伯茨绿僵菌 M. robertsii
35	CBS657.67	新咯里多尼亚 New Caledonia	平沙绿僵菌 M. pinghaense
36	CBS658.67	法国 France	罗伯茨绿僵菌 M. robertsii
37	CBS659.71	未知 Unknown	M. viride
38	CBS660.67	新西兰 New Zealand	棕色绿僵菌 M. brunneum
39	CBS660.71	未知 Unknown	M. viride
40	CBS661.67	新西兰 New Zealand	新西兰绿僵菌 M. novozealandicum
41	CBS662.67	荷兰 Netherlands	棕色绿僵菌 M. brunneum
42	CBS663.67	荷兰 Netherlands	黄绿绿僵菌 M. flavoviride
43	CBS700.74	法国 France	黄绿绿僵菌 M. flavoviride
44	CBS732.73A	巴西 Brazil	莱氏绿僵菌 M. rileyi
45	CBS732.73B	佛罗里达 Florida	莱氏绿僵菌 M. rileyi
46	CBS733.73	以色列 Isreal	莱氏绿僵菌 M. rileyi
47	CBS806.71	美国 USA	莱氏绿僵菌 M. rileyi
48	CFCC80689	广东 Guangdong	鳞腮绿僵菌 M. lepidiotae
49	CFCC80785	北海道虻天郡 Hokkaido	平沙绿僵菌 M. pinghaense
50	CFCC80787	日本茨城 Ibaraki	平沙绿僵菌 M. pinghaense
51	CFCC80788	宁夏 Ningxia	棕色绿僵菌 M. brunneum
52	CFCC80789	中国台北 Taipei , China	金龟子绿僵菌 M. anisopliae
53	CFCC80790	日本茨城 Ibaraki	鳞腮绿僵菌 M. lepidiotae
54	CFCC80793	日本冲绳 Okinawa	金龟子绿僵菌 M. anisopliae
55	CFCC80794	日本冲绳 Okinawa	瘿绵蚜绿僵菌 M. pemphigi
56	CFCC80795	福建永定 Yongding, Fujian	金龟子绿僵菌 M. anisopliae
57	CFCC80799	福建上杭 Shanghang, Fujian	金龟子绿僵菌 M. anisopliaee
58	CFCC80800	福建上杭 Shanghang, Fujian	金龟子绿僵菌 M. anisopliaee
59	CFCC80801	福建上杭 Shanghang, Fujian	金龟子绿僵菌 M. anisopliae
60	CFCC80802	福建惠安 Hui'an, Fujian	平沙绿僵菌 M. pinghaense
61	CFCC80804	福建永泰 Yongtai, Fujian	金龟子绿僵菌 M. anisopliae
62	CFCC80805	福建南安 Nan'an, Fujian	M. indigoticum
63	CFCC80806	福建漳浦 Zhangpu, Fujian	平沙绿僵菌 M. pinghaense
64	CFCC80807	福建漳浦 Zhangpu, Fujian	平沙绿僵菌 M. pinghaense
65	CFCC80808	福建福州 Fuzhou, Fujian	平沙绿僵菌 M. pinghaense
66	CFCC80809	福建福州 Fuzhou, Fujian	平沙绿僵菌 M. pinghaense
67	CFCC80810	福建东山 Dongshan, Fujian	平沙绿僵菌 M. pinghaense
68	CFCC80811	福建云霄 Yunxiao, Fujian	平沙绿僵菌 M. pinghaense
69	CFCC80812	福建武夷山 Wuyi Mountain, Fujian	鳞腮绿僵菌 M. lepidiotae
70	CFCC82105	日本茨城 Ibaraki	鳞腮绿僵菌 M. lepidiotae
71	CFCC82107	福建三明 Sanming，Fujian	大孢绿僵菌 M. majus
72	CFCC86175	河北 Hebei	平沙绿僵菌 M. pinghaense
73	CFCC87360	湖北武汉 Wuhan, Hubei	平沙绿僵菌 M. pinghaense
74	CFCC87867	安徽 Anhui	鳞腮绿僵菌 M. lepidiotae
75	CFCC88444	河北保定 Baoding, Hebei	平沙绿僵菌 M. pinghaense
76	CFCC88953	未知 Unknown	鳞腮绿僵菌 M. lepidiotae
77	CFCC88954	未知 Unknown	罗伯茨绿僵菌 M. robertsii
78	CFCC88956	未知 Unknown	平沙绿僵菌 M. pinghaense
79	CFCC88957	广东 Guangdong	平沙绿僵菌 M. pinghaense
80	CFCC88236	河南信阳 Xinyang, Henan	莱氏绿僵菌 M. rileyi

表2

图1

图2

表3

80株绿僵菌对松褐天牛成虫毒力测试结果①"

种名 Species	菌株编号 Strain number	LT₅₀（95%置信区间） LT₅₀（95% Confidence interval）/d	15天校正死亡率 Adjusted mortality after 15 days(%)	采集区 Geographic location
棕色绿僵菌 M. brunneum	CBS115995	21.15(20.33~21.96)	11.53	奥地利Austria
	CBS316.51	18.73(16.69~21.03)	35.75	俄勒冈州Oregon
	CBS655.67	21.95(19.05~24.77)	34.12	法国France
	CBS660.67	19.94(18.70~21.14)	19.54	新西兰New Zealand
	CBS662.67	15.35(10.45~60.37)	49.25	荷兰Netherlands
	CFCC80788	8.21(6.78~9.72)	82.16	宁夏Ningxia
平沙绿僵菌 M. pinghaense	CBS130.22	15.69(14.86~16.49)	45.22	未知Unknown
	CBS218.29	23.46(22.00~24.85)	11.52	捷克共和国Czech Republic
	CBS459.75	18.98(16.83~21.19)	32.13	密歇根州Michigan
	CBS464.70	21.55(18.32~24.86)	23.40	以色列Isreal
	CBS657.67	16.64(15.25~17.94)	41.96	新喀里多尼亚New Caledonia
	CFCC80785	6.91(6.35~7.42)	99.69	北海道虻天郡Hokkaido
	CFCC80787	8.56(7.33~9.60)	93.09	日本茨城Ibaraki
	CFCC80802	6.37(5.33~7.35)	94.71	福建Fujian
	CFCC80806	6.55(5.30~7.78)	94.76	福建Fujian
	CFCC80807	9.65(9.05~10.23)	95.62	福建Fujian
	CFCC80808	6.81(6.07~7.50)	95.26	福建Fujian
	CFCC80809	31.53(27.30~36.11)	21.39	福建Fujian
	CFCC80810	27.06(24.94~28.91)	5.69	福建Fujian
	CFCC80811	22.53(21.69~23.35)	7.42	福建Fujian
	CFCC86175	16.56(15.09~18.02)	43.54	河北Hebei
	CFCC87360	11.35(9.69~13.04)	65.56	湖北Hubei
	CFCC88444	11.67(11.07~12.25)	78.24	河北Hebei
	CFCC88956	11.23(8.77~13.53)	63.78	未知Unknown
	CFCC88957	4.91(4.18~5.54)	99.95	广东Guangdong
金龟子绿僵菌 M. anisopliae	CBS158.84	2.09(0.96~3.14)	96.77	巴西Brazil
	CFCC80789	12.11(9.95~14.57)	64.69	中国台北Tapei , China
	CFCC80793	11.40(10.60~12.16)	73.75	日本冲绳Okinawa
	CFCC80795	7.71(2.60~12.60)	68.20	福建Fujian
	CFCC80799	19.52(18.61~20.41)	27.29	福建Fujian
	CFCC80800	13.71(12.51~14.79)	57.87	福建Fujian
	CFCC80801	17.27(14.54~20.96)	43.80	福建Fujian
	CFCC80804	4.25(3.29~5.01)	98.34	福建Fujian
罗伯茨绿僵菌 M. robertsii	CBS247.64	21.76(20.47~23.08)	7.35	法国France
	CBS289.67	17.83(16.17~19.45)	37.19	厄立特里亚Eritrea
	CBS454.78	15.06(14.11~15.95)	49.58	佛罗里达Florida
	CBS650.67	20.21(19.39~21.04)	23.95	伊朗Iran
	CBS656.67	20.01(19.04~20.95)	21.09	科特迪瓦Ivory Coast
	CBS658.67	9.65(8.77~10.41)	86.70	法国France
	CFCC88954	15.53(14.26~16.78)	46.97	未知Unknown
大孢绿僵菌 M. majus	CBS248.64	39.80(37.29~42.52)	1.93	法国France
	CBS643.67	42.29(37.47~47.49)	17.05	萨摩亚Samoa
	CFCC82107	54.35(51.27~58.06)	0.19	福建Fujian
贵州绿僵菌 M. guizhouense	CBS258.90	14.87(12.74~16.53)	50.49	中国China
M. indigoticum	CBS285.59	29.92(23.40~37.06)	1.00	德国Germany
M. indigoticum	CFCC80805	37.02(32.19~45.26)	16.37	福建Fujian
鳞腮绿僵菌 M. lepidiotae	CBS130.71	9.07(8.15~9.92)	85.98	乌克兰Ukraine
	CBS130.92	18.30(16.96~19.68)	34.36	巴西Brazil
	CBS160.96	22.56(20.93~24.14)	27.11	巴布亚新几内亚Papua New Guinea
	CFCC80689	21.20(17.83~24.35)	33.98	广东Guangdong
	CFCC80790	11.78(10.85~12.65)	71.62	日本茨城Ibaraki
	CFCC80812	11.14(8.78~13.54)	67.58	福建Fujian
	CFCC82105	11.86(10.65~13.08)	68.98	日本茨城Ibaraki
	CFCC87867	10.69（8.75~12.51）	67.22	安徽Anhui
	CFCC88953	22.22（20.00~24.87）	26.70	未知Unknown
蝗绿僵菌 M. acridum	CBS544.81	－	1.15	厄瓜多尔Ecuador
蝗绿僵菌 M. acridum	CBS339.35	65.61（54.03~89.69）	0.11	阿根廷Argentina
黄绿绿僵菌 M. flavoviride	CBS125.65	19.69（17.18~22.03）	30.29	德国Germany
	CBS218.56	19.00（17.27~21.30）	31.02	捷克共和国Czech Republic
	CBS380.73A	21.76（20.47~23.08）	13.94	德国Germany
	CBS380.73B	23.58（21.73~25.30）	9.97	德国Germany
	CBS473.73	25.23（22.70~27.79）	27.75	荷兰Netherlands
	CBS663.67	6.35（5.74~6.89）	99.04	荷兰Netherlands
	CBS700.74	21.12（15.47~43.04）	37.21	法国France
瘿绵蚜绿僵菌 M. pemphigi	CBS229.52	14.41（13.38~15.37）	54.67	荷兰Netherlands
瘿绵蚜绿僵菌 M. pemphigi	CFCC80794	14.06（11.27~16.78）	53.39	日本冲绳Okinawa
M. bibonidarum	CBS648.67	7.42（5.80~8.90）	83.64	法国France
黄绿绿僵菌小孢变种 M. minus	CBS614.85	22.85（21.27~24.43）	21.39	所罗门群岛Solomon Islands
M. viride	CBS348.65	－	5.36	马达加斯加Madagascar
	CBS407.96	79.12（69.06~93.45）	2.64	未知Unknown
	CBS659.71	－	10.97	未知Unknown
	CBS660.71	88.65（77.58~105.43 ）	0.76	未知Unknown
莱氏绿僵菌 M. rileyi	CBS194.65	－	9.23	科特迪瓦Ivory Coast
	CBS732.73A	－	9.52	巴西Brazil
	CBS732.73B	－	10.54	佛罗里达Florida
	CBS733.73	－	7.51	以色列Isreal
	CBS806.71	－	4.47	美国USA
	CFCC88236	－	12.10	河南Henan
柱孢绿僵菌 M. cylindrosporum	CBS615.84	63.65（59.33~68.87）	2.74	菲律宾Philippines
新西兰绿僵菌 M. novozealandicum	CBS661.67	99.34（86.88~120.72）	0.41	新西兰New Zealand

表3

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基因 Genes	引物 Primers	序列 Sequence	退火温度 Annealing temperature/℃	参考文献 Reference
ITS	ITS1	5’-TCCGTAGGTGAACCTGCGG-3’	53	郭鹏豪等，2013
ITS	ITS4	5’-TCCTCCGCTTATTGATATGC-3’	53	郭鹏豪等，2013
TUB	Tub-F	5’-CCTCCAAACCGGTCAATG-3’	55	Shimomoto et al.，2011
TUB	Tub-R	5’-CTGGGTCAACTCGGGGAC-3’	55	Shimomoto et al.，2011

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