4株绿僵菌的鉴定及对松褐天牛成虫致病效果比较

doi:10.11707/j.1001-7488.20200110

摘要/Abstract

摘要：

目的: 为筛选对松褐天牛毒力更高的微生物资源，开展生物防治通过培养性状、分生孢子形态及系统发育树鉴定4个绿僵菌菌株，比较测定各自对松褐天牛的毒力。方法: 对4株绿僵菌的培养性状和分生孢子形态特征进行描述，并对4株绿僵菌的EF-1α基因进行克隆和测序，构建系统进化发育树，综合鉴定4株绿僵菌的分类地位。测定了不同孢子浓度及接种方法（浸虫、浸枝和无纺布）对松褐天牛成虫的致病效果，构建4个菌株对松褐天牛的毒力方程。结果: 1）菌株WTKH和WP08鉴定为平沙绿僵菌；菌株LV2鉴定为金龟子绿僵菌；菌株qc1401鉴定为瘿绵蚜绿僵菌。2）平沙绿僵菌WP08菌株和金龟子绿僵菌LV2菌株对松褐天牛校正死亡率较高，瘿绵蚜绿僵菌qc1401菌株次之，平沙绿僵菌WTKH菌株最低。4个菌株对松褐天牛的致死速度依次为WP08 > LV2 > qc1401 > WTKH，LT₅₀分别为5.52、10.38、16.93和20.80天。3）以平沙绿僵菌WP08为供试菌株，在1.0×10⁸、1.0×10⁷、1.0×10⁶个·mL^-1共3个浓度下，孢子浓度越高则松褐天牛校正死亡率越高、半数死亡时间越短，孢子浓度为1.0×10⁸、1.0×10⁷时，试验期内校正死亡率均达到100%；半数死亡时间LT₅₀分别为11.38、16.44和24.22天。4）以平沙绿僵菌WP08为供试菌株，3种接种方法40天内松褐天牛死亡率均为100%，LT₅₀浸虫法为5.52天，浸枝法为11.38天，无纺布法为17.21天。结论: 4株绿僵菌均可侵染松褐天牛致其死亡，其中，平沙绿僵菌WP08的致死率和致死速度较快，为防治松褐天牛的优良菌株。

关键词: 虫生真菌, 绿僵菌, 松褐天牛, 毒力测定, EF-1α, LT₅₀

Abstract:

Objective: Monochamus alternates, causing great economic losses every year, is not only an important borer pest in pinewood, but also the main vector of pine nematode disease. To screen for more effective biocontrol agents against M. alternates, four Metarhizium strains were identified by molecular and morphological characteristics, and their virulence against M. alternates adults was analyzed. Method: In this study, cultural characteristics and conidia morphology of four Metarhizium strains were observed respectively. The EF-1α gene of each strain was cloned and sequenced, and a phylogenetic tree was constructed to comprehensively identify the taxonomic status of 4 strains to species level. The virulence of four Metarhizium strains against M. alternatus adult was tested with different conidia concentrations and inoculation methods(dipping, twig dipping, and nonwoven cloth), and the pathogenesis was evaluated. Result: 1) WP08 and WTKH were clusted into the clade of M. pingshaense, and LV2 was into M. anisopliae, and qc1401 was into M. pemphogum. 2) Among the four strains, WP08 and LV2 caused the highest corrected death rate (100%) in the 40-day experimental period, and the corrected death rate of qc1401 and WTKH was 91.9% and 87.9%, respectively. Lethal speed of each strain was in the order of WP08 > LV2 > qc1401 > WTKH with the LT₅₀ of 5.52, 10.38, 16.93 and 20.80 d, respectively. 3) Among the conidium concentration series of 1.0×10⁸, 1.0×10⁷, 1.0×10⁶ conidia·mL^-1 of WP08, the higher the conidia concentration, the higher the corrected death rate and the shorter the lethal time of M. alternatus adults. The corrected death rate was as high as 100% at conidium concentration of 1.0×10⁸ and 1.0×10⁷ conidia·mL^-1 of WP08, and was 83.9% at conidium concentration of 1.0×10⁶ conidia·mL^-1, and the LT₅₀ was 11.38, 16.44 and 24.22 d at the three concentration, separately. 4) For strain of WP08, all the three inoculation method could cause a death rate of 100% in the experimental period of 40 days, and the LT₅₀ of insect dipping, twig dipping, and nonwoven cloth were 5.52, 11.38, and 17.21 d, respectively. Conclusion: Four Metarhizium strains are identified as M. pingshaense, M. anisopliae, and M. pemphogum, and all the strains can infect M. alternatus adults and cause their death. M. pingshaense WP08 strain exhibits higher death rate and faster lethal speed than the other three stains, and shows great potential as biocontrol agent for M. alternatus.

Key words: entomopathogenic fungus, Metarhizium, Monochamus alteratus, virulence test, EF-1α, LT₅₀

中图分类号:

S763.306.4

张亚波,张威,张守科,彭瀚,孟珂,舒金平,王浩杰. 4株绿僵菌的鉴定及对松褐天牛成虫致病效果比较[J]. 林业科学, 2020, 56(1): 103-111.

Yabo Zhang,Wei Zhang,Shouke Zhang,Han Peng,Ke Meng,Jinping Shu,Haojie Wang. Identification of Four Entomopathogenic Metarhizium Strains and Their Virulence against Monochamus alternatus Adults[J]. Scientia Silvae Sinicae, 2020, 56(1): 103-111.

图/表 8

图1

图2

图3

表1

图4

表2

图5

表3

参考文献 0

	郭庆港, 王培培, 鹿秀云, 等. 棉花枯萎病菌新生理型菌株的分子鉴定. 植物病理学报, 2018. 48 (2): 145- 153.
	Guo Q G , Wang P P , Lu X Y , et al. Molecular identification of the new genotype strains of Fusarium oxysporum f. sp. vasinfectum in China. Acta Phytopathologica Sinica, 2018. 48 (2): 145- 153.
	何学友, 陈顺立, 杨希, 等. 金龟子绿僵菌在森林土壤中的分布及对松褐天牛致病性测定. 菌物学报, 2007. 26 (2): 289- 294.
	He X Y , Chen S L , Yang X , et al. The investigation of Metarhizium anisopliae in forest soil in Fujian and Jiangxi provinces and pathogenicity against Monochamus alternatus. Mycosystema, 2007. 26 (2): 289- 294.
	何学友, 蔡守平, 余培旺, 等. 金龟子绿僵菌MaYTTR-04菌株对松褐天牛成虫的致病力. 昆虫学报, 2008. 51 (1): 102- 107. doi: 10.3321/j.issn:0454-6296.2008.01.016
	He X Y , Cai S P , Yu P W , et al. Virulence of a strain of Metarhizium anisopliae MaYTTR-04 against adults of Monochamus alternatus. Acta Entomologica Sinica, 2008. 51 (1): 102- 107. doi: 10.3321/j.issn:0454-6296.2008.01.016
	何学友, 陈顺立, 黄金水. 感染松褐天牛的金龟子绿僵菌菌株的初步筛选. 昆虫学报, 2005. 48 (6): 975- 981. doi: 10.3321/j.issn:0454-6296.2005.06.026
	He X Y , Chen S L , Huang J S . Preliminary screening of virulent strains of Metarhizium anisopliae against Monochamus alternatus. Acta Entomologica Sinica, 2005. 48 (6): 975- 981. doi: 10.3321/j.issn:0454-6296.2005.06.026
	江英成. 绿僵菌和白僵菌侵染马尾松毛虫试验比较. 浙江农林大学学报, 2000. 17 (4): 410- 413. doi: 10.3969/j.issn.2095-0756.2000.04.014
	Jang Y C . Comparison on infection of Metarhizium anisopliae and Beauveria bassiana to Dendrolimus punctatus. Journal of Zhejiang Forestry College, 2000. 17 (4): 410- 413. doi: 10.3969/j.issn.2095-0756.2000.04.014
	蒋平, 吾中良, 柴希民, 等. 松褐天牛传递松材线虫数量的研究. 南京林业大学学报:自然科学版, 2002. 26 (3): 69- 71.
	Jiang P , Wu Z L , Chai X M , et al. Transmission Characteristic of Bursaphelenchus xylophilus Through Monochamus alternatus. Journal of Nanjing Forestry University:Natural Sciences Edition, 2002. 26 (3): 69- 71.
	林长春, 周成枚, 赵锦年, 等. 松褐天牛成虫羽化出孔规律研究. 林业科学研究, 2002. 15 (2): 131- 135. doi: 10.3321/j.issn:1001-1498.2002.02.002
	Lin C C , Zhou C M , Zhao J N , et al. Adult eclosion of Monochamus alternatus. Forest Research, 2002. 15 (2): 131- 135. doi: 10.3321/j.issn:1001-1498.2002.02.002
	刘洪剑, 朴春根, 汪来发, 等. 白僵菌和肿腿蜂对松褐天牛幼虫的作用. 林业科学, 2007. 43 (5): 64- 68.
	Liu H J , Piao C G , Wang L F , et al. Biocontrol of Monochamus alternatus by Beauveria bassiana and Scleroderma guani. Scientia Silvae Sinicae, 2007. 43 (05): 64- 68.
	潘永胜, 徐福元, 韩正敏, 等. 松褐天牛成虫高毒力绿僵菌菌株筛选及液体振荡培养条件. 林业科学研究, 2010. 23 (1): 102- 107.
	Pan Y S , Xu F Y , Han Z M , et al. Studies on the screening and liquid shake culture condition of high virulent Metarhizium anisopliae against Monochamus alternatus Hope Adults. Forest Research, 2010. 23 (1): 102- 107.
	史先慧, 马涛, 陆雪雷, 等. 松褐天牛成虫行为与化学生态学研究进展. 林业科学研究, 2017. 30 (5): 854- 865.
	Shi X H , Ma T , Lu X L , et al. Research progress in adult behavior and chemical ecology of Monochamus alternatus Hope adults. Forest Research, 2017. 30 (5): 854- 865.
	宋漳, 卢凤美, 陈辉. 绿僵菌和白僵菌对刚竹毒蛾的毒力比较. 西北林学院学报, 2003. 18 (3): 43- 46. doi: 10.3969/j.issn.1001-7461.2003.03.013
	Song Z , Lu F M , Chen H . Cimparison on infection of Metarhizium anisopliae and Beauveria bassiana to Pantana phyllostachysae. Journal of Northwest Forestry University, 2003. 18 (3): 43- 46. doi: 10.3969/j.issn.1001-7461.2003.03.013
	王滨, 樊美珍, 李增智. 白僵菌无纺布结合引诱剂防治鞘翅目林业害虫研究初报. 中国生物防治, 2003. 19 (2): 91- 92.
	Wang B , Fan M Z , Li Z Z . Control forest beetles with Beauveria nonwoven fabric sheet in combination with beetle attractants. Chinese Journal of Biological Control, 2003. 19 (2): 91- 92.
	王峰, 刘斌, 农向群, 等. 绿僵菌属4个生防潜力菌株的多基因鉴定. 中国生物防治学报, 2017. 33 (6): 780- 787.
	Wang F , Liu B , Nong X Q , et al. Multigene identification of four potential biocontrol strains in Metarhizium genus. Chinese Journal of Biological Control, 2017. 33 (6): 780- 787.
	王四宝, 黄勇平, 张心团, 等. 松褐天牛成虫高毒力病原真菌筛选及林间感染试验. 中国森林病虫, 2004. 23 (6): 13- 16. doi: 10.3969/j.issn.1671-0886.2004.06.003
	Wang S B , Huang Y P , Zhang X T , et al. Screening and biological control of high virulent strains against Monochamus alternatus adults. Forest pest and disease, 2004. 23 (6): 13- 16. doi: 10.3969/j.issn.1671-0886.2004.06.003
	徐金柱, 何雪香, 秦长生, 等. 绿僵菌无纺布菌条制作工艺. 中国生物防治, 2007. 23 (2): 147- 150.
	Xu J Z , He X J , Qin C S , et al. Making technics of nonwoven fabric strips with Metarhizium anisopliae. Chinese Journal of Biological Control, 2007. 23 (2): 147- 150.
	徐金柱, 秦长生, 李增智, 等. 绿僵菌无纺布菌条的制作:Ⅰ.液体种子培养条件筛选. 生物学杂志, 2006. 23 (3): 22- 24.
	Xu J Z , Qin C S , Li Z Z , et al. The making of nonwoven fabric strips with Metarhizium(Ⅰ) screening the condition for liquid. Journal of Biology, 2006. 23 (3): 22- 24.
	展茂魁, 杨忠岐, 王小艺, 等. 松褐天牛成虫松材线虫病的传播能力. 林业科学, 2014. 50 (7): 74- 81.
	Zhan M K , Yang Z Q , Wang X Y , et al. Capacity of transmitting Bursaphelenchus xylophilus by the vector Monochamus alternatus adults. Scientia Silvae Sinicae, 2014. 50 (7): 74- 81.
	张立钦, 刘军. 松褐天牛优良白僵菌菌株筛选. 南京林业大学学报:自然科学版, 2000. 24 (2): 33- 37.
	Zhang L Q , Liu J . The screening virulent strain of Beauveria bassiana to Monochamus alternatus. Journal of Nanjing Forestry University:Natural Scinece Edition, 2000. 24 (2): 33- 37.
	张亚波, 叶碧欢, 吴小双, 等. 一株虫生真菌的分离、鉴定及其对筛胸梳爪叩甲幼虫致病力测定. 中国生物防治学报, 2015. 31 (6): 868- 875.
	Zhang Y B , Ye B H , Wu X S , et al. Isolation and identification of an entomopathogenic fungal strain and its virulence to larvae of Melanotus cribricollis. Chinese Journal of Biological Control, 2015. 31 (6): 868- 875.
	张亚波, 叶碧欢, 刘振勇, 等. 不同绿僵菌菌株对筛胸梳爪叩甲幼虫的毒力测定. 林业科学研究, 2014. 27 (1): 66- 70.
	Zhang Y B , Ye B H , Liu Z Y , et al. Virulence Bioassay of different Metarhizium strains against Melanotus cribricollis Larvae. Forest Research, 2014. 27 (1): 66- 70.
	赵锦年, 应杰. 松褐天牛取食为害与松树枯死关系的研究. 林业科学, 1989. 25 (5): 432- 438. doi: 10.3321/j.issn:1001-7488.1989.05.010
	Zhao J N , Ying J . A study on relationship between the damage of pine sawyer beetle (Monochamus alternatus) and pine dying. Scientia Silvae Sinicae, 1989. 25 (5): 432- 438. doi: 10.3321/j.issn:1001-7488.1989.05.010
	赵学球, 蔡守平, 王四宝, 等. 球孢白僵菌释放后在森林系统中定殖及持续控虫作用的证据. 菌物学报, 2007. 26 (1): 84- 88.
	Zhao X Q , Cai S P , Wang S B , et al. Evidence of survival and sustainable control on pests of Beauveria bassiana after artificial release in Massion pine plantation. Mycosystema, 2007. 26 (1): 84- 88.
	Bischoff J F , Rehner S A , Humber R A . A multilocus phylogeny of the Metarhizium anisopliae lineage. Mycologia, 2009. 101 (4): 512- 530. doi: 10.3852/07-202
	Donald W R , Raymond J S L . Metarhizium spp., cosmopolitan insect pathogenic fungi:mycological aspects. Advances in Applied Microbiology, 2004. 54, 1- 70. doi: 10.1016/S0065-2164(04)54001-7
	Driver F , Milner R J , Trueman W H . A taxonomic revision of Metarhizium based on a phylogenetic analysis of rDNA sequence data. Mycological Research, 2000. 104 (2): 134- 150. doi: 10.1017/S0953756299001756
	Kepler R M , Humber R A , Bischoff J F , et al. Clarification of generic and species boundaries for Metarhizium and related fungi through multigene phylogenetics. Mycologia, 2014. 106 (4): 811- 829. doi: 10.3852/13-319
	Li H , Shen P , Fu P , et al. Characteristics of the emergence of Monochamus alternatus, the vector of Bursaphelenchus xylophilus (Nematoda:Aphelenchoididae), from Pinus thunbergii logs in Nanjing, China, and of the transmission of the nematodes through feeding wounds. Nematology, 2007. 9 (6): 807- 816. doi: 10.1163/156854107782331234
	Nishi O , Iiyama K , Yasunagaaoki C , et al. Species associations and distributions of soil entomopathogenic fungi Metarhizium spp. in Japan. Mycology, 2017. 8 (4): 308- 317. doi: 10.1080/21501203.2017.1386244
	Okitsu M , Kishi Y , Takagi Y . Control of adults of Monochamus alternatus Hope (Coleoptera:Cerambycidae) by application of non-woven fabric strips containing Beauveria bassiana (Deutyeromycotina:Hyphomycetes) on infested tree trunks. Journal of the Japanese Forestry Society, 2000. 82 (3): 276- 280.
	Rehner S A , Buckley E . A Beauveria phylogeny inferred from nuclear ITS and EF1-α sequences:evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia, 2005. 97 (1): 84- 98.
	Rezende J M , Zanardo A B R , da Silva Lopes M , et al. Phylogenetic diversity of Brazilian Metarhizium associated with sugarcane agriculture. Biological Control, 2015. 60 (4): 495- 505.
	Shimazu M . Effects of temperature on growth of Beauveria bassiana F-263, a strain highly virulent to the Japanese pine sawyer, Monochamus alternatus, especially tolerance to high temperatures. Applied Entomology & Zoology, 2004. 39 (3): 469- 475.
	Stamps W T , Linit M J . Interaction of intrinsic and extrinsic chemical cries in the behaviour of Bursaphelenchus xylophilus(Aphelenchida:Aphelenchoididae)in relation to its beetle vectors. Nematology, 2001. 3 (4): 295- 301. doi: 10.1163/156854101317020213
	Tsutsumi T , Yamanaka M . Infection by entomogenous fungus, Beauveria brongniartii(SACC.)PETCH GSES, of adults of yellow spotted Longicorn beetle, Psacothea hilaris PASCOE by dispersing conidia from non-woven fabric sheet containing fungus. Japanese Journal of Applied Entomology & Zoology, 1997. 41 (1): 45- 49.
	Tulloch M . The genus Metarhizium. Transactions of the British Mycological Society, 1976. 66 (3): 407- 411. doi: 10.1016/S0007-1536(76)80209-4

菌株 Isolates	累计死亡率 Mortality(%)	校正死亡率 Corrected cumulative mortality(%)	回归方程 Regression equation	相关系数 Correlation coefficient(r)	LT₅₀/d
WP08	100	100.00a	Y=3.818X-6.000	0.911	5.52a
LV2	100	100.00a	Y=2.116X-6.542	0.930	10.38ab
qc1401	93.30	91.90a	Y=1.012X-2.028	0.963	16.93bc
WTKH	90.00	87.90a	Y=0.735X-0.046	0.964	20.80c
CK	16.67	0.00b	—	—	—

处理 Spores concentration/(conidia·mL^-1)	累计死亡率 Mortality(%)	校正死亡率 Corrected cumulative mortality (%)	回归方程 Regression equation	相关系数 Correlation coefficient(r)	LT₅₀/d
1.0×10⁶ 1.0×10⁷ 1.0×10⁸ CK	85.88 100.00 100.00 16.67	83.00b 100.00a 100.00a 0.00c	Y=0.754X-2.873 Y=0.874X+0.424 Y=1.866X-5.795 —	0.977 0.904 0.936 —	24.22b 16.44ab 11.38a —

接种方法 Inoculation method	累计死亡率 Mortality(%)	校正死亡率(%) Corrected cumulative mortality (%)	回归方程 Regression equation	相关系数 Correlation coefficient(r)
无纺布法 Nonwoven cloth	100.00	100.00a	Y=1.197X-5.085	0.975	17.21c
浸虫法 Insect dipping	100.00	100.00a	Y=3.818X-6.000	0.911	5.52a
浸枝法 Twig dipping	100.00	100.00a	Y=1.866X-5.795	0.936	11.38b
CK	16.67	0.00b	—	—	—

[1]	叶碧欢, 张亚波, 舒金平, 吴鸿, 王浩杰. 3种绿僵菌对筛胸梳爪叩甲幼虫的毒力及保护酶活性的影响[J]. 林业科学, 2018, 54(6): 100-108.
[2]	温小遂, 廖三腊, 唐艳龙, 杨忠岐. 释放花绒寄甲卵防治松褐天牛技术[J]. 林业科学, 2017, 53(10): 133-138.
[3]	曹庆杰, 迟德富. 感染杨干象幼虫的高致病力虫生真菌菌株筛选[J]. 林业科学, 2017, 53(1): 119-127.
[4]	高尚坤, 张彦龙, 唐艳龙, 杨忠岐, 王小艺, 路纪芳, 王健, 司徒春南, 付甫永. 花绒寄甲松褐天牛生物型的越冬特性及耐寒性[J]. 林业科学, 2016, 52(3): 68-74.
[5]	何学友, 蔡守平, 杜月飞, 陈德兰, 黄金水, 李孔泉. 感染油茶象幼虫的高致病力金龟子绿僵菌菌株筛选[J]. 林业科学, 2015, 51(8): 52-59.
[6]	展茂魁, 杨忠岐, 王小艺, 来燕学, 张毅丰. 松褐天牛成虫松材线虫病的传播能力[J]. 林业科学, 2014, 50(7): 74-81.
[7]	张彦龙, 杨忠岐, 张翌楠, 王小艺, 吴成进, 马生福, 陆之贵. 利用花绒寄甲防治越冬后松褐天牛试验[J]. 林业科学, 2014, 50(3): 92-98.
[8]	张亚波, 吴小双, 叶碧欢, 吴盼盼, 舒金平. 平沙绿僵菌侵染筛胸梳爪叩甲幼虫的扫描电镜观察[J]. 林业科学, 2014, 50(11): 197-201.
[9]	杨远亮;杨忠岐;王小艺;喻锦秀;颜学武. 应用花绒寄甲防治松褐天牛[J]. 林业科学, 2013, 49(3): 103-109.
[10]	徐明, 刘冬梅, 徐福元, 施建华. Bt与灭幼脲混剂对美国白蛾第2,3代幼虫的联合毒力及防治效果[J]. 林业科学, 2013, 49(12): 171-174.
[11]	郝德君;陆佳伟;殷丽. 3种生物农药对悬铃木方翅网蝽的室内毒力及解毒酶活性的影响[J]. 林业科学, 2012, 48(7): 86-91.
[12]	张彦龙;杨忠岐;王小艺;吴成进;马生福;陆之贵. 2种昆虫病原线虫对松褐天牛幼虫的控制力测定[J]. 林业科学, 2012, 48(12): 64-70.
[13]	张亚波;吴盼盼;王鹏;刘剑;徐天森;王浩杰;舒金平. 一株绿僵菌的鉴定及其生物学特性[J]. 林业科学, 2012, 48(12): 134-140.
[14]	徐福元;郑华英;刘云鹏;解春霞;徐明;赵振东. 马尾松种源对松褐天牛成虫取食、松材线虫病的抗性分析[J]. 林业科学, 2011, 47(1): 101-106.
[15]	王达;袁芳芳;黄大庄;刘春延;毕华明. 绿僵菌侵染光肩星天牛幼虫过程的透射电镜观察[J]. 林业科学, 2010, 46(5): 113-115.