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落叶松八齿小蠹(Ips subelongatus Motschulsky)为鞘翅目、小蠹亚科、齿小蠹属昆虫,在东亚广泛分布,是中国北方3种落叶松(兴安落叶松、华北落叶松和长白落叶松)的次期性严重为害的蛀干害虫[1],在种群暴发期可为害健康树木。作为影响本土针叶树并对非本土针叶树有威胁的主要害虫,该害虫于2003年被中国列入“全国林业危险性有害生物名单”(国家林业局,2003年),于2005年被欧洲地中海植物保护组织列入A2预警名单(EPPO,2005)。此外,小蠹虫携带的多种病原微生物,特别是长喙壳真菌,在小蠹虫为害过程中会加重对树木的危害[2]。
长喙壳真菌是一类包括蛇口壳目(Ophiostomatales)真菌及相似类群真菌的总称,其中一些种类是引起林木病害的重要病原菌,如引起榆树荷兰病的Ophiostoma ulmi和Ophiostoma novo-ulmi、引起月桂枯萎病的Raffaelea lauricola及引起针叶树黑根病的Leptographium wageneri[2-4]。已有研究证实,齿小蠹伴生真菌群落中的先锋种Endoconidiophora spp.,如Endoconidiophora fujiensis,对日本地区的落叶松毒力较强,可引发寄主坏死,或诱导寄主落叶松产生强烈的抗性反应[5]。Endoconidiophora polonica高密度接种在挪威云杉上,能导致其死亡[6]。
小蠹虫和长喙壳真菌在长期的协同进化过程中形成了复杂的伴生关系,二者之间的联系在小蠹虫定殖寄主过程中起着重要作用[7-8]。研究表明,长喙壳真菌可以合成小蠹虫生长发育所必需的甾醇[9]、解毒寄主树木的防御物质[10-12]、释放化学信息素吸引或排斥同种小蠹虫[13-14]等。然而,一些寄主萜类化合物对小蠹虫及其伴生真菌具有毒性,如针叶树在受到小蠹虫攻击时会增加单萜类化合物的合成,以抵抗小蠹虫的入侵,当萜类物质浓度高于小蠹虫的生理耐受阈值时,抑制小蠹虫的攻击[15];离体试验也表明,在培养基中添加α-pinene和limonene等单萜类物质,可抑制长喙壳真菌的生长[14, 16]。
研究明确小蠹虫传播的真菌是否会引起植物病害以及植物是否会对小蠹虫及其伴生真菌产生抗性,是关于小蠹虫传播病菌的2个关键科学问题。本研究将2010—2018年采集分离的12种落叶松八齿小蠹伴生长喙壳真菌代表性菌株接种到5年生落叶松苗上[17],观察不同真菌或菌株之间的致病力是否存在差异,并分析寄主防御相关代谢物质的变化,以探究落叶松八齿小蠹及其伴生长喙壳真菌复合物对落叶松的潜在风险。
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本试验选用的12种长喙壳真菌为由2010—2018年采集的落叶松八齿小蠹虫体和坑道中分离纯化得到的菌株[17],分别为E. fujiensis、E. laricicola、Ophiostoma hongxingense、Ophiostoma peniculi、Ophiostoma pseudobicolor、Ophiostoma subelongati、Ophiostoma genhense、Ophiostoma lotiforme、Ophiostoma rufum、Ophiostoma minus、Ophiostoma multisynnematum和Ophiostoma xinganense,每种真菌各选用2个菌株用于致病力试验(表1)。
表 1 供试菌株信息
Table 1. The information of tested strains
种名
Species菌株号
Strain No.寄主
Host采集地
LocationEndoconidiophora fujiensis MJG55 日本落叶松 Larix kaempferi 辽宁抚顺 Fushun, Liaoning MJG29 日本落叶松 L. kaempferi 辽宁抚顺 Fushun, Liaoning E. laricicola TRL63 西伯利亚落叶松 L. sibirica 新疆阿勒泰 Altay, Xinjiang KBLK9 西伯利亚落叶松 L. sibirica 新疆阿勒泰 Altay, Xinjiang Ophiostoma hongxingense HXS70 兴安落叶松 L. gmelinii 黑龙江哈尔滨 Harbin, Heilongjiang HXS72 兴安落叶松 L. gmelinii 黑龙江哈尔滨 Harbin, Heilongjiang O. peniculi CFCC52688 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia GH77 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia O. pseudobicolor CFCC52683 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia CFCC52684 兴安落叶松 L. gmelinii 内蒙古赤峰 Chifeng, Inner Mongolia O. subelongati CFCC52693 兴安落叶松 L. gmelinii 黑龙江哈尔滨 Harbin, Heilongjiang ZHS37 兴安落叶松 L. gmelinii 黑龙江哈尔滨 Harbin, Heilongjiang O. genhense CFCC52675 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia CFCC52676 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia O. lotiforme CFCC52691 樟子松 Pinus sylvestris var. mongholica 内蒙古海拉尔 Hailar, Inner Mongolia CFCC52692 樟子松 P. sylvestris var. mongholica 内蒙古海拉尔 Hailar, Inner Mongolia O. rufum CFCC52681 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia CFCC52682 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia O. minus CFCC52697 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia CFCC52698 樟子松 P. sylvestris var. mongholica 内蒙古海拉尔 Hailar, Inner Mongolia O. multisynnematum CFCC52677 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia CFCC52678 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia O. xinganense CFCC52679 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia CFCC52680 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia -
选择移栽于中国林科院内的5年生长白落叶松(Larix olgensis A. Henry)作为试验材料。这些落叶松高2.0~3.0 m,平均2.49 m,地径2.05~4.77 cm,平均3.05 cm。
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菌株在2% MEA培养基上25 ℃黑暗条件下培养7 d,用经灭菌的直径6 mm软木塞打孔器在新培养菌落边缘打取菌苔。于2019年7月9日随机选择60棵落叶松,在离地30 cm处的树干上,用经灭菌的直径6 mm的软木塞打孔器打孔至木质部,用无菌牙签将菌苔接入接种孔,然后盖上树皮,用封口膜缠绕,并用胶带固定。以2% MEA培养基作为接种对照。每棵树上设1个接种点,每个处理各设5次重复。
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接种60 d后,撕开接种点的胶带和封口膜,用壁纸刀刮去接种点周围病斑区域的外树皮,测量病斑的长和宽,并记录相关数据。
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测量病斑大小后,每个处理随机选择3个重复,在接种植株病健交界处取约1 cm2样品放至15 mL无菌离心管并带回实验室,于−4 ℃冰箱保存。次日,样品表面经2%次氯酸钠消毒1 min,然后用无菌水冲洗3次,经无菌滤纸吸去表面水分后取约1 mm2组织碎片放置在2% MEA培养基上进行培养,根据菌落形态学和核酸β-微管蛋白(BT)基因片段序列比较鉴定是否为原始接种的长喙壳菌株。
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对MJG55(E. fujiensis)和TRL63(E. laricicola)菌株和对照接种1、7、14、30和60 d后的5棵落叶松进行单萜类物质测定,以评估寄主防御反应强弱。用聚乙烯薄膜(48 cm × 60 cm,雷诺公司,美国)包裹接种点周围树干,用特氟龙管将含有100 mg Porapak-Q 吸附剂(默克公司,德国)的玻璃管连接在小型真空泵上形成闭环动态顶空采样系统,以500 mL·min−1的流速持续1 h收集寄主挥发性物质。将含有挥发物的玻璃管两端用锡纸包裹并缠绕封口膜,放入装有干冰的保温箱并带回实验室。用2 mL色谱级正己烷洗脱寄主挥发物,用温和的氮气将样品浓缩至50 μL,置于−20 ℃冰箱保存直至气相色谱检测。气相色谱分析方法同Liu等人描述[18]。将样品放置在配有火焰离子检测器和自动进样器的气相色谱仪(GC-FID,安捷伦,美国)上,使用口径0.25 mm × 0.2 μm,30 m长的HP-5色谱柱进行分析,每次进样1 μL。
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根据Rajtar等[19]的方法,计算接种点病斑面积(长 × 宽)。单萜类化合物释放速率根据处理组和健康组的差值(Δ)进行分析。使用IBM SPSS Statistics19针对不同菌株产生的病斑、MJG55和TRL63菌株处理的落叶松在同一时间产生的单萜类化合物释放速率、MJG55或TRL63菌株处理在不同时间产生的单萜类化合物释放速率,进行单因素方差分析(LSD test,α=0.05)。
落叶松八齿小蠹伴生长喙壳真菌致病性研究
Pathogenicity of Ophiostomatoid Fungi Associated with Ips subelongatus
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摘要:
目的 通过分析落叶松八齿小蠹伴生长喙壳真菌对寄主的致病能力,探究其对落叶松的危害程度与潜在风险。 方法 利用人工接种的方法,将从落叶松八齿小蠹虫体及坑道中分离到的12种长喙壳真菌接种到长白落叶松上,60 d后观察和测量病斑大小。并对2种Endoconidiophora真菌(E. fujiensis和E. laricicola)诱导寄主抗性的能力进行测定。 结果 结果表明,5种长喙壳真菌E. laricicola、E. fujiensis、Ophiostoma hongxingense、O. peniculi和O. xinganense接种长白落叶松产生的病斑面积大于对照处理,其余7种Ophiostoma真菌产生的病斑面积与对照无显著差异。E. laricicola产生的平均病斑面积最大,为3.51 cm2,且诱导寄主萜类物质含量呈现先升高后降低的趋势,表现出对长白落叶松的较强致病力。而E. fujiensis产生的平均病斑面积为1.70 cm2,诱导寄主萜类物质含量呈现下降趋势,表现出较弱致病力。 结论 E. laricicola对长白落叶松具有强致病性,E. fujiensis、O. hongxingense、O. peniculi和O. xinganense对长白落叶松具有弱致病性,而另外7种Ophiostoma真菌对落叶松无明显致病性。 -
关键词:
- 落叶松八齿小蠹
- / Endoconidiophora
- / Ophiostoma
- / 单萜
Abstract:Objective The paper aims to investigate the harm degree and the potential risk of the ophiostomatoid fungi associated with Ips subelongatus by analyzing their pathogenicity. Method Through the artificial inoculation, 12 species of ophiostomatoid fungi isolated from the body and tunnel of I. subelongatus were inoculated on Larix olgensis. After 60 days, the lesion sizes were observed and measured, and the host resistance responses were tested by inoculation of Endoconidiophora fujiensis and Endoconidiophora laricicola on larch. Result The results showed that the area of lesions caused by E. laricicola, E. fujiensis, Ophiostoma hongxingense, Ophiostoma peniculi and Ophiostoma xinganense were larger than those of the control. The lesion areas produced by the other seven species of Ophiostoma showed no significant difference comparing with the control. The average lesion area produced by E. laricicola were the largest with 3.51 ± 0.18 cm2, and the content of terpenoids in the host showed a trend of first increasing and then decreasing, showing a strong pathogenicity. However, the average lesion area of E. fujiensis was 1.70 ± 0.08 cm2, and the content of host terpenoids showed a decreasing trend, exhibiting weak pathogenicity. Conclusion E. laricicola is highly pathogenic to larch, E. fujiensis, O. hongxingense, O. peniculi and O. xinganense are weakly pathogenic, while the other seven Ophiostoma fungi show no obvious pathogenicity. -
Key words:
- Ips subelongatus
- / Endoconidiophora
- / Ophiostoma
- / monoterpene
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表 1 供试菌株信息
Table 1. The information of tested strains
种名
Species菌株号
Strain No.寄主
Host采集地
LocationEndoconidiophora fujiensis MJG55 日本落叶松 Larix kaempferi 辽宁抚顺 Fushun, Liaoning MJG29 日本落叶松 L. kaempferi 辽宁抚顺 Fushun, Liaoning E. laricicola TRL63 西伯利亚落叶松 L. sibirica 新疆阿勒泰 Altay, Xinjiang KBLK9 西伯利亚落叶松 L. sibirica 新疆阿勒泰 Altay, Xinjiang Ophiostoma hongxingense HXS70 兴安落叶松 L. gmelinii 黑龙江哈尔滨 Harbin, Heilongjiang HXS72 兴安落叶松 L. gmelinii 黑龙江哈尔滨 Harbin, Heilongjiang O. peniculi CFCC52688 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia GH77 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia O. pseudobicolor CFCC52683 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia CFCC52684 兴安落叶松 L. gmelinii 内蒙古赤峰 Chifeng, Inner Mongolia O. subelongati CFCC52693 兴安落叶松 L. gmelinii 黑龙江哈尔滨 Harbin, Heilongjiang ZHS37 兴安落叶松 L. gmelinii 黑龙江哈尔滨 Harbin, Heilongjiang O. genhense CFCC52675 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia CFCC52676 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia O. lotiforme CFCC52691 樟子松 Pinus sylvestris var. mongholica 内蒙古海拉尔 Hailar, Inner Mongolia CFCC52692 樟子松 P. sylvestris var. mongholica 内蒙古海拉尔 Hailar, Inner Mongolia O. rufum CFCC52681 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia CFCC52682 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia O. minus CFCC52697 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia CFCC52698 樟子松 P. sylvestris var. mongholica 内蒙古海拉尔 Hailar, Inner Mongolia O. multisynnematum CFCC52677 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia CFCC52678 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia O. xinganense CFCC52679 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia CFCC52680 兴安落叶松 L. gmelinii 内蒙古呼伦贝尔 Hulunbuir, Inner Mongolia -
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