林业科学  2018, Vol. 54 Issue (6): 100-108   PDF    
DOI: 10.11707/j.1001-7488.20180612
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文章信息

叶碧欢, 张亚波, 舒金平, 吴鸿, 王浩杰
Ye Bihuan, Zhang Yabo, Shu Jinping, Wu Hong, Wang Haojie
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
Effects of Three Different Metarhizium Strains on Virulence and Protective Enzymes Activities of Melanotus cribricollis larvae
林业科学, 2018, 54(6): 100-108.
Scientia Silvae Sinicae, 2018, 54(6): 100-108.
DOI: 10.11707/j.1001-7488.20180612

文章历史

收稿日期:2016-12-06
修回日期:2017-02-16

作者相关文章

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
叶碧欢1,2, 张亚波1, 舒金平1, 吴鸿1, 王浩杰1     
1. 中国林业科学研究院亚热带林业研究所 杭州 311400;
2. 浙江省林业科学研究院 杭州 310023
摘要:【目的】探索绿僵菌侵染后竹林筛胸梳爪叩甲幼虫保护酶系统的应答反应,为筛选高致病力的绿僵菌菌株提供依据。【方法】生物测定平沙绿僵菌WP08菌株、金龟子绿僵菌WTKH菌株及蝗虫绿僵菌菌株3种菌株在不同浓度条件下对该幼虫的致病力,同时测定不同侵染时期幼虫超氧化物歧化酶(SOD)、过氧化物酶(POD)及过氧化氢酶(CAT)3种保护酶含量及活性变化情况。【结果】3株绿僵菌对筛胸梳爪叩甲幼虫均具有致死性,WP08菌株最优,WTKH菌株次之,蝗虫绿僵菌最低。不同菌株的致病力随着浓度的增加而提高,WP08菌株在108spore·g-1干土浓度条件下的致病效果最好,16天时供试幼虫已经全部死亡,但与107spore·g-1干土浓度条件下的致病效果无显著差异。幼虫的3种保护酶在抗绿僵菌侵染过程中其活性变化趋势不同,SOD活性变化无显著差异,CAT活性变化则表现为先下降后上升再下降的趋势,POD活性波动虽与CAT相似,但其后期(5~7天)差异不显著。【结论】平沙绿僵菌WP08菌株对筛胸梳爪叩甲幼虫的致病性优于其他2种菌株,可作为林间应用的首选菌株。绿僵菌可对竹林金针虫体内的3种保护酶活性产生不同程度的影响,且在本次取样时间内CAT的活性变化较明显。
关键词:筛胸梳爪叩甲幼虫    绿僵菌    致病力    保护酶    
Effects of Three Different Metarhizium Strains on Virulence and Protective Enzymes Activities of Melanotus cribricollis larvae
Ye Bihuan1,2, Zhang Yabo1, Shu Jinping1 , Wu Hong1, Wang Haojie1    
1. Research Institute of Subtropical Forestry, CAF Hangzhou 311400;
2. Zhejiang Academy of Forestry Hangzhou 310023
Abstract: 【Objective】The bamboo wireworm (Melanotus cribricollis larvae) is one of the most rampant forest pests causing serious damage to bamboo shoots in southern China. It is hard to monitor and control this pest till now. Metarhizium spp. can be used for biocontrol of the wireworm. The purpose of this study is to screen high pathogenicity Metarhizium strain and explore the immune response of protective enzyme system in the wireworm after the fungal infection.【Method】The pathogenicity tests by applying three Metarhizium strains against the pest larvae with different spore concentrations were conducted. The dynamic activity changes of three protective enzymes in the host larvae, i.e. superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were also determined during different infect periods.【Result】Results showed that all the three isolates possessed lethal property were able to infest M. cirbricollis larvae, among which M. pingshaense WP08 strain was the best, M. anisopliae WTKH strain was the second and M. acridum strain the last. The pathogenicity was enhanced with the increased spore concentration. With 108 spore·g-1 DW soil, M. pingshaense WP08 strain exhibited the highest toxicity and all of the tested larvae died in 16 d, but M. pingshaense WP08 strain with 107 spore·g-1 DW soil had no significant pathogenicity. There was difference in the dynamic change trends of three protective enzyme activities. There was no significant difference in the SOD activity. However, the CAT activity firstly down-regulated, then up-regulated and declined again subsequently, and the POD activity showed similar fluctuation trend to the CAT activity, but there was no significant difference at the late stage (5-7 d).【Conclusion】The present study indicated that M. pingshaense WP08 strain was the best entomopathogenic fungus to infect M. cribricollis larvae compared with other two strains. Therefore, the strain could be first selected as a biocontrol agent for control of the wireworm in bamboo forests. The fungal infection influenced activities of the three protective enzymes, especially to CAT activity.
Key words: larvae of Melanotus cribricollis    Metarhizium spp.    pathogenicity    protective enzymes    

筛胸梳爪叩甲(Melanotus cribricollis)幼虫是当前竹林金针虫中最为重要的种类,近年来对我国南方早园竹(Phyllostachys propinqua)等竹林地产生了严重的危害,造成鲜笋产量减少、品质降低,同时竹林自然更新受到制约,笋农经济收入下降,更不利于竹林在生态环境中发挥重要作用(徐天森,2004; 舒金平等,2012; 叶碧欢等,2016)。鉴于该虫较长的生活周期(2~3年)以及营地下生活习性,其防治工作一直存在困难。当前的防治主要依赖毒死蜱等各类化学试剂,易造成环境污染、农药残留和抗药性等问题,严重阻碍了竹产业“生态、健康、有机”的可持续发展,因此无公害生物防治至关重要。

高效的昆虫致病真菌可能成为筛胸梳爪叩甲幼虫等地下害虫有效防控的关键途径(王鹏等,2010; 张亚波等,2012)。绿僵菌(Metarhizium spp.)是一种广谱性杀虫真菌,目前已报道绿僵菌对鞘翅目(Meissle et al., 2009; Ansari et al., 2013)、鳞翅目(Asi et al., 2013; Ibrahim et al., 2016)和双翅目(Dimbi et al., 2013; Trevisoli et al., 2015)等200余种农林害虫表现出明显的致死效果,对地下害虫尤为有效。然而,不同致病真菌对同一宿主昆虫的毒力及寄生效果存在显著差异,而这种差异的造成与昆虫自身的防御免疫密切相关(Wright et al., 2004; Pelizza et al., 2015)。昆虫在与病原微生物长期协同进化过程中,形成了一套有效的防御体系。病原菌入侵虫体后,昆虫的免疫防御系统启动应对,其中具有自由基清除作用的保护酶系统最为关键(Lozinskaya et al., 2004)。昆虫保护酶系统主要由过氧化物酶(peroxidase,POD)、过氧化氢酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase,SOD)组成,能抑制活性自由基对细胞的毒害(Fridovich,1977; Genestra,2007)。昆虫超氧化物歧化酶有CuZn-SOD和Mn-SOD 2种,主要存在于细胞质或线粒体基质中(Zelko et al., 2002),在超氧阴离子($ \text{O}_{2}^{{\bar{\centerdot }}} $)歧化反应中起关键作用。CAT主要分布于过氧化物酶体中,能通过2种不同的模式有效分解SOD歧化反应等产生的H2O2,以避免具有更强毒性HO·的生成(Ho et al., 2004; Kabel,2014)。POD酶主要功能是清除H2O2和抗脂质过氧化(张傲,2009)。有研究者认为,保护酶活性变化可作为评价昆虫先天性免疫能力的指标参数(Yaninek et al., 1988; Ding et al., 2001; Wu et al., 2015)。

研究昆虫免疫系统的组成、功能及应答机制对于深入揭示昆虫与病源菌互作机制及高效工程菌株的研发有着重要的价值(Ho et al., 2004; Kabel,2014)。本文以金龟子绿僵菌(M. anisopliae) WTKH菌株、蝗虫绿僵菌(M. acridum)和平沙绿僵菌(M. pingshaense)WP08菌株为研究材料,比较分析了不同菌株对竹林筛胸梳爪叩甲幼虫的致病效果,同时也对3种保护酶含量及活性变化进行了测定,以期为高效高毒力的生防菌株筛选提供数据支撑,为科学高效利用或改造生防菌株提供理论支持,为虫菌互作机制研究提供新的重要信息。

1 材料与方法 1.1 试验材料及仪器

1) 昆虫:于2016年4月中下旬浙江省德清县山民村(120°04′ E,30°32′ N)竹林采集筛胸梳爪叩甲幼虫,带回室内饲养,笋期喂食鲜笋,其余时间喂食玉米和小麦种子。

2) 菌株:平沙绿僵菌WP08菌株由中国林业科学研究院亚热带林业研究所的森林保护实验室保存,金龟子绿僵菌WTKH菌株和蝗虫绿僵菌购于中国普通微生物菌种保藏管理中心。3种绿僵菌具体的孢子和产孢小梗形态见图 1所示。

图 1 绿僵菌分生孢子(左)及产孢小梗的形态(右) Figure 1 Morphology of conidium (left) and conidiophore(right) of different Metarhizium spp. isolates

3) 土壤:40目网筛过筛,干燥箱中高温灭菌(121 ℃、4 h)备用。

4) 仪器:本研究所使用的主要仪器设备详见表 1

表 1 本研究所使用的主要仪器设备 Tab.1 Main apparatuses used in present study

5) 试剂:本研究所使用的主要试剂详见表 2

表 2 主要试剂信息 Tab.2 Information of main reagents used in this study
1.2 试验方法 1.2.1 毒力测定

1) 菌株的培养和悬浮液配置:接种、培养几种绿僵菌菌株(PPDA,温度25 ℃± 1 ℃,RH 80% ± 5%),待各菌株大量产孢后,将孢子轻轻刮到试剂瓶中配置成不同浓度的孢子悬浮液(0.05%吐温80);2)悬浮液浓度设置:WTKH菌株和蝗虫绿僵菌均设置2个浓度(1.3×107、1.3×108 spore·g-1干土),WP08菌株则增设1个浓度梯度(1.3×106spore·g-1干土);3)土壤处理:CK组和处理组的土壤分别用0.05%吐温80或不同浓度的孢子悬浮液处理(10%±1%);4)试验条件和重复设置:试验置于人工气候培养箱中(温度25 ℃± 1 ℃、相对湿度80%±10%、黑暗条件),采用干净的玻璃瓶分装,每组处理3个重复,每组重复至少60头虫体,36天为一周期;5)试验结果观察:定期检查虫体的存活情况并将死亡虫体保湿培养以观察其是否因绿僵菌侵染致死(体壁见白色菌丝或绿色孢子),而非侵染死亡的虫体则将发黑腐烂(图 2)。

图 2 筛胸梳爪叩甲幼虫的死亡特征 Figure 2 Death symptom of M. cribricollis larvae A.平沙绿僵菌WP08菌株M. pingshaense WP08 strain, 侵染死亡Death due to infected; B.金龟子绿僵菌WTKH菌株M. anisopliae WTKH strain, 侵染死亡Death due to infected; C.蝗虫绿僵菌菌株M. acridum strain, 侵染死亡Death due to infected; D.非侵染死亡Death due to uninfected.
1.2.2 取样及酶原的制备

筛胸梳爪叩甲幼虫保护酶在不同时间的活性变化测定以WP08菌株(108 spore·g-1干土)为致病菌。每个节点(CK、1天、3天、5天、7天)取5头大小均一的虫体混样,液氮粉碎后加入适量的预冷PBS,离心后保留上层液体(10 000 r·min-1;30 min;4 ℃),4 ℃保存,每组3个重复。

1.2.3 蛋白质浓度的测定

制作BSA标准曲线(R2 =0.980 3),考马斯亮蓝法测定蛋白酶OD595值,计算蛋白质浓度,具体操作参考王学奎(2006)

1.2.4 保护酶活性测定

SOD和POD采用相应的南京建成试剂盒,分别测定OD550和OD420值,CAT根据紫外线吸收法测定OD240值(曹传旺等,2009)。

1.3 数据处理

对试验数据进行方差分析、建立回归方程以及LT50预测,使用软件为SPSS18.0。

2 结果与分析 2.1 病原菌株对筛胸梳爪叩甲幼虫的致死性

毒力测定结果表明,3种菌株对筛胸梳爪叩甲幼虫均具有致死性,且随着浓度的增加致死效果提高(表 3图 3)。WP08菌株的侵染校正累计死亡率显著高于WTKH菌株,蝗虫绿僵菌菌株最低。不同孢子浓度条件下,WP08菌株的侵染校正累计死亡率差异不显著(98.68%,98.46%,97.40%),而WTKH菌株和蝗虫绿僵菌在较低浓度(107spore·g-1干土)时的侵染校正累计死亡率(25.33%,16.67%)均显著低于较高浓度(108spore·g-1干土)时(79.27%,42.00%)。相比其他2种菌株,WP08的LT50(有效致死中时间)最短,其致死速率随着浓度提高而加快。WP08菌株的LT50(天)在2种浓度条件下(107、108spore·g-1干土)无差异(9.1~10.8天),但与较低浓度(106spore·g-1干土)时存在差异。接种16天后,经WP08菌株(108spore·g-1干土)处理的幼虫全部死亡,同时结果显示,108spore·g-1干土条件下,WTKH菌株的LT50(d)为23.4天(表 3图 3)。毒力测定试验过程中还发现了非侵染死亡虫体,尸体发黑发臭最后腐烂。从非侵染死亡的统计结果发现,除WP08菌株2种浓度条件(107,108 spore·g-1干土)外,其他条件的数据均显著高于CK,尤其是WTKH组的死亡率最高(图 4)。

表 3 不同绿僵菌对筛胸梳爪叩甲幼虫的毒力测定 Tab.3 Toxicity test of different Metarhizium isolates to M.cribricollis larvae
图 3 筛胸梳爪叩甲幼虫侵染校正累计死亡率 Figure 3 Corrected cumulative mortality of M.cribricollis larvae
图 4 筛胸梳爪叩甲幼虫的非侵染校正累计死亡率 Figure 4 Corrected cumulative uninfected mortality of M.cribricollis larvae
2.2 平沙绿僵菌WP08菌株侵染对筛胸梳爪叩甲幼虫保护酶活性的影响

根据测定结果可知,绿僵菌的不同侵染时期,幼虫的蛋白质浓度和SOD活性变化均不显著(图 5ab)。CAT活性变化波动较明显,3天时活性最低,7天次之,CK最高。3天和7天的差异不显著,但与其余节点CAT活性差异显著(图 5c)。POD活性变化趋势与CAT较相似,但7天与其他时间节点的差异不显著(图 5d)。

图 5 筛胸梳爪叩甲幼虫蛋白质和3种酶在不同感病时间的浓度变化 Figure 5 Protein concentration dynamics of M.cribricollis larvae during different infected period
3 讨论

当前,竹林金针虫——筛胸梳爪叩甲在我国南方笋用竹区暴发成灾,造成了重大经济损失(邓顺等,2010)。绿僵菌是一类应用广泛的虫生真菌,其防治害虫的历史已逾百年,对金针虫等地下害虫防治有其独特优势,国内外已报道多起成功案例(Roddam et al., 1997; Roberts et al., 2004; Kabaluk et al., 2007; 2014)。王鹏等(2010)获得一株可寄生筛胸梳爪叩甲幼虫的平沙绿僵菌WP08菌株,为了评价和筛选更为高效的绿僵菌菌株,本研究比较了3种不同菌株对该虫的致死效果。不同菌株的孢子和产孢小梗形态存在一定差异,WP08菌株和WTKH菌株的孢子呈长椭圆形,但WP08菌株的孢子个体较大[(4.89~7.23 μm)×(1.38~2.80 μm)],长度近乎是后者[(2.29~3.16 μm))×(0.76~1.17 μm)]的2倍,蝗虫绿僵菌菌株的孢子呈卵圆形[(2.07~4.12 μm)×(1.39~2.57 μm)]。结果表明,3种菌株均对筛胸梳爪叩甲幼虫有一定致死性,但WP08菌株优于WTKH菌株,蝗虫绿僵菌的杀虫效果最差。同时,毒力测定试验结果还显示不同菌株处理对虫体的非感染死亡率也有显著影响,36天时WTKH菌株处理组的非侵染死亡数显著高于其他2种菌株,推测虫体因绿僵菌产生并释放至土壤中的有毒物质而非侵染死亡。在先前研究工作中,笔者推测绿僵菌可能产生某些有毒物质,因此其液体发酵培养液对该虫具有驱避效果(叶碧欢等,2016)。该驱避现象或许能间接地解释绿僵菌非侵染死亡的原因,但是具体的有效致死因子未被挖掘,其作用机制仍有待进一步探究。

昆虫在长期的进化过程中已经形成完整的自由基清除系统,其中包括SOD、POD和CAT的对自由基的协作调控。正常情况下,昆虫能够避免有毒自由基对自身的损伤,维持其正常的动态平衡状态(李会平等,2007)。当遭受病原物侵染、化学农药毒害和不良环境胁迫等外界刺激时,昆虫体内的自由基生成-消除的原有平衡被打破,激发保护酶的防御应答响应(李周直等,1994; Wu et al., 2015)。近年来,关于昆虫保护酶应对温度、植物次生代谢产物、化学农药、线虫、病原菌等胁迫时的活性变化已有大量的研究报道。热胁迫时,小金蝠蛾(Thitarodes xiaojinensis)幼虫的POD活性显著升高(王梦龙等,2014),在应对低温胁迫时,茶淡黄刺蛾(Darna trima)保护酶也起到重要作用(李品武等,2016)。刘玉坤等(2011)研究发现不同寄主植物会对昆虫保护酶产生差异影响,这可能与植物中某些特殊成分有关。韩永强等(2016)研究表明,稻纵卷叶螟(Cnaphalocrocis medinalis)取食时,植物次生代谢物在昆虫体内的积累导致SOD等保护酶活性发生相应变化,致使幼虫存活率降低。化学农药因试剂类型、作用时间和有效浓度等不同而对保护酶活性变化产生差异影响(鄢杰明等,2010; 刘丹等,2012; 付莉,2014; 王莹,2016; 贾变桃等,2016)。吡虫啉处理12 h时可激活榆紫叶甲(Ambrostoma quadriimpressum)SOD活性,而功夫菊酯却表现为显著的抑制活性(王莹,2016)。一般而言,化学农药处理早期可诱导保护酶活性,但因时间的延长被抑制,药剂的毒性作用从而逐渐显现(贾变桃等,2016)。此外,多数农药试剂只在低浓度时对保护酶活性起激活效应,随着浓度增加而产生抑制作用(王莹,2016)。此外,线虫、细菌和真菌类病原物也因侵染剂量、时间和昆虫种类等对保护酶产生不同影响(朱建兰等,2008; 姬国红等,2009; 张奎花等,2012)。红火蚁(Solenopsis invicta)工蚁感染球孢白僵菌(Beauveria bassiana)后,SOD和CAT活性在接种12~24 h后显著下降,POD活性则在72 h时内呈先上升后下降趋势(王龙江等,2010)。桑天牛幼虫和菜青虫感染丝状真菌后,体内3种保护酶活性呈先上升后下降的趋势,反映出昆虫在真菌侵染初期其防御能力增强随后又下降的趋势(李会平等,2007; 李世广等,2016)。发光杆菌(Photorhabdus luminescens)可激发大蜡螟(Galleria mellonella)SOD活性显著升高,却抑制CAT和POD活性(Wu et al., 2015)。不同保护酶在抗病原物侵染过程中,其作用顺序因昆虫种类存在差异,例如菜青虫SOD和CAT在抗异小杆线虫(Heterorhabditis megidis)侵染时首先发生作用,POD随后(张奎花等,2012),而金龟子绿僵菌侵染菜青虫后,首先发生作用的是POD、SOD随后,CAT作用最迟(李世广等,2016)。

根据毒力测定结果,选择最优致病菌株——平沙绿僵菌WP08菌株为研究材料,探究绿僵菌侵染对幼虫体内保护酶活性的影响。由保护酶活性测定结果可知,平沙绿僵菌接染并未影响筛胸梳爪叩甲幼虫体内的蛋白酶浓度和SOD活性,推测虫体内的超氧阴离子自由基当时与接染7天之前其含量差异不显著,尚未影响SOD的活性,也有可能因为该虫还有其他抑制或降低超氧阴离子自由基危害的途径。此外,CAT和POD对H2O2均有酶解作用,而结果也显示该虫的这2种保护酶的活性变化趋势在侵染初期相似,3天时酶活性均较CK显著降低,推测病原菌此阶段在体内快速增殖,保护酶活性受到了强烈抑制,抗氧化性能因此显著减弱。然而,5~7天时CAT活性波动较POD更为明显,CAT活性先显著升高后下降,POD活性虽然有相似波动,但变化不显著,推测这2种酶虽然均受H2O2水平调控,但其响应度或作用先后顺序不完全一致。根据CAT活性变化推测5天时虫体内H2O2的含量因侵染过程的推进已积累到了一定阈值,因而暂时性地激发了CAT活性,随着体内H2O2水平降低到正常阈值,该酶活性变化将趋向稳定(7天),也可能因为此时虫体受到绿僵菌的严重干扰和破坏,其抵御能力已开始明显衰弱。该现象与毒力测定结果相吻合,相对5天时的死亡率(108,1.32%),7天时幼虫死亡数显著增加(13.16%)。同时根据酶活性测定结果可知,接染后阶段(5~7天)的POD和CAT活性变化趋势与SOD截然相反,可能是SOD对病原菌侵染的迟缓响应,其抗氧化活性在后期才逐渐发挥,亦有可能因为该阶段超氧阴离子的过剩积累导致。

4 结论

本研究表明,3株供试的菌株均对筛胸梳爪叩甲幼虫有致病性,但是WP08菌株的杀虫效果最好,可作为有效防控的生防菌株首选。昆虫保护酶作为自由基清除系统,其活性变化与虫体的抗逆性等存在联系,可作为昆虫先天性免疫力中的一项可参指标。绿僵菌侵染后,该幼虫体内的3种保护酶活性变化趋势存在差异,至少在本次取样时间内,绿僵菌对CAT活性影响最为明显,POD次之,SOD差异不显著。本研究通过探究绿僵菌侵染对筛胸梳爪叩甲幼虫保护酶活性的影响,期望不仅能为虫-菌互作研究提供新的基础信息,为生防菌株的高效利用或基因改造提供可靠的理论依据,也能为其他林业害虫的防控提供有效的作用目标。

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