7种松树挥发物分析及其主要萜烯类物质对红脂大小蠹行为选择的影响

入侵种红脂大小蠹在我国逐渐向北扩散至内蒙古和辽宁等地.同时在林间发现,相对于油松Pinus tabuliformis,其更偏好危害樟子松Pinus sylvestris var.mongolica.本文主要分析我国7种松树挥发性物质以及新发生地红脂大小蠹对其中主要物质的行为选择.本研究采用动态顶空采样法和气相色谱——质谱联用技术(GC-MS),对7种松树树干木段挥发性有机化合物(VOCs)进行采集和分析,包括油松、樟子松、华山松Pinus armandii、白皮松Pinus bungeana、马尾松 Pinus massoniana、红松 Pinus koraiensis和落叶松 Larix gmelinii.在此基础上参考以往研究中与红脂大小蠹行为相关的化合物,选择树种挥发物中7种主要烯烃类化合物:(1R)-(+)-α-蒎烯、(+)-3-蒈烯、(-)-β-蒎烯、月桂烯、(S)-(-)-柠檬烯,萜品油烯和莰烯,通过自制十通道行为仪器进行嗅觉反应试验,分析红脂大小蠹在不同浓度的化合物刺激下的行为选择反应.GC-MS结果表明,7种松树树干释放化合物中共包含烯烃类、醇类、酮类、醛类、酯类、酸类、烷烃和芳香烃8类化合物在内的54种化合物,其中烯烃类化合物相对含量最高.主成分分析及聚类分析结果表明,红脂大小蠹以往偏好寄主油松与新偏好寄主樟子松的挥发物相似度最高,(+)-3-蒈烯为其特有的挥发性物质,莰烯含量高于其他树种;4种受害寄主树种(油松、樟子松、白皮松和华山松)挥发性物质相似,均含有高比例的α-蒎烯,同时有β-蒎烯、柠檬烯、萜品油烯.选择上述6种化合物及月桂烯(华山松、白皮松中共有的萜烯类化合物)进行行为选择反应.结果显示,受害寄主树种中含有β-蒎烯以及油松、樟子松中的特征性挥发物质(+)-3-蒈烯对雌虫具有明显的吸引作用,低浓度(-)-柠檬烯(10-4mg/mL)对雄虫具有吸引作用;低浓度(10-4mg/mL)月桂烯对雌虫以及中浓度(10-2mg/mL)莰烯对雌雄成虫具有刺激性.综上所述,本研究证明了红脂大小蠹的寄主选择行为趋性与寄主树干挥发物的组成和含量有关.本试验结果将有助于理解植物挥发物对红脂大小蠹偏好寄主选择的影响,并进一步开发红脂大小蠹的引诱剂和趋避剂以应用于其防治.     

外来入侵害虫红脂大小蠹对寄主挥发物的反应

2002年,利用寄主挥发性物质(+)-α-蒎烯、(-)-β-蒎烯、3-蒈烯和(-)柠檬烯按不同配比制成诱芯,采用自行研制的挥发物释放装置,在山西省关帝山森林经营局西葫芦林场对危害油松Pinus tabulaeformis Carr.的重大外来入侵林业害虫红脂大小蠹Dendroctomus valens LeConte进行了引诱试验。结果表明,3蒈烯对红脂大小蠹的引诱作用最强,明显优于单独使用(+)-α-蒎烯和(-)-β-蒎烯的诱虫效果,也比北美应用的标准诱芯［(+)- α蒎烯(-)-β-蒎烯与3-蒈烯的比例为1∶1∶1］效果好;在标准诱芯中加入油松另一主要成分-(-)柠檬烯会明显降低诱虫效果。红脂大小蠹引诱剂最佳释放量为150 mg/d。该研究不仅对我国红脂大小蠹的监控具有实际应用意义,也从理论上探讨了红脂大小蠹与寄主油松协同进化的化学生态学过程。     

华山松大小蠹对几种寄主挥发物组分的EAG和行为反应

为筛选适宜配制华山松大小蠹(Dendroctonus armandi)引诱剂的有效化学成分,用触角电位仪和Y型嗅觉仪测定了华山松大小蠹雌、雄虫对华山松挥发物主要成分的触角电生理(EAG)和行为反应.结果表明:在0.01、0.1、1、10、100 μg 5种刺激剂量下,7种化合物在特定浓度下均有明确的EAG反应;雌、雄虫对月桂烯、(+)-3-蒈烯、β-石竹烯3种化合物及雄虫对(+)-α-蒎烯、柠檬烯的EAG相对电位的最大值均出现在10μg刺激条件下;在10 μg刺激剂量条件下,雌虫对(+)-α-蒎烯、β-蒎烯和(+)-3-蒈烯有明显的正趋向反应,正趋向率均高于72%且显著高于雄成虫对3种挥发物的正趋向率;在1 μg刺激下,只有雄虫对(+)-3-蒈烯有一定正趋向反应;而在100μg条件下,(-)-α-蒎烯对雌、雄虫均有一定驱避作用.该研究结果可为开发华山松大小蠹林间引诱剂提供借鉴.     

油松萜烯成分变化与红脂大小蠹的反应特性

采用顶空采样方法,比较健康油松、人工损伤油松以及抗性油松在单萜烯成分组成上的差异。GC-MS分析表明,在自然状况下,油松树干释放的萜烯类成分很少,以α-蒎烯占绝对优势(>97%);人工损伤后,油松萜烯类成分明显增多,除α-蒎烯为主要成分外,还包括β-蒎烯、月桂烯、柠檬烯、萜品油烯、β-水芹烯、长叶烯等;而抗性油松萜烯类成分更为复杂。对3类油松主要单萜类成分的相对含量方差分析表明,α-蒎烯的相对含量呈显著降低;3-蒈烯在损伤寄主中相对含量最高,在抗性寄主中相对含量与自然状态下没有差异。柠檬烯、莰烯、萜品油烯在抗性寄主中相对比率显著增加。而β-蒎烯、月桂烯、β-水芹烯相对含量在3个处理中变化不大。在此基础上,比较红脂大小蠹Dendroctonus valens LeConte对油松主要单萜类成分的触角电位及嗅觉行为反应。结果表明,室内触角电位、嗅觉试验与先前林间试验结果相一致,即红脂大小蠹对(+)-3-蒈烯表现出最强的电生理和行为反应。对R-(+)-α-蒎烯和S-(-)-α-蒎烯研究发现,红脂大小蠹对α-蒎烯2个对映体的触角电位、嗅觉行为无显著不同。结合油松单萜类含量变化特点与红脂大小蠹行为反应,认为3-蒈烯相对含量上升可能作为易感寄主特点;而柠檬烯、莰烯、萜品油烯相对比率增加则代表了抗性或者非适合寄主的特征。     

横坑切梢小蠹对植物挥发物的电生理和行为反应

为筛选出适宜配制横坑切梢小蠹(Tomicus minor Hartig)引诱剂的有效化合物,测定了横坑切梢小蠹雌、雄成虫对寄主云南松主要挥发性化合物的触角电位(EAG)和行为反应。结果表明:与对照相比,在10 g·L-1浓度刺激下所测试的9种植物挥发物都能引起横坑切梢小蠹雌、雄虫触角的EAG反应,且触角对不同挥发物的EAG反应差异显著(P0.05),其中雌、雄虫对(+)-α-蒎烯、(+)-柠檬烯、(+)-3-蒈烯、β-蒎烯和异松油烯的EAG反应较强;在剂量反应试验中,雌、雄虫的EAG反应都随着(+)-α-蒎烯、(+)-柠檬烯、(+)-3-蒈烯和β-蒎烯浓度的增加而增强,当浓度增加到10 g·L-1时,EAG反应最强;而雌、雄虫对异松油烯的EAG反应在1 g·L-1最强;除(+)-柠檬烯没有明显的定向作用外,(+)-α-蒎烯、β-蒎烯、(+)-3-蒈烯和异松油烯对横坑切梢小蠹均表现出了明显的引诱作用;行为反应多重比较表明,(+)-α-蒎烯在10 g·L-1浓度下对雌雄虫的引诱作用最大。该研究结果可为开发横坑切梢小蠹林间引诱剂提供参考。     

红脂大小蠹入侵机制与化学生态学研究

红脂大小蠹Dendroctonus valens LeConte是近来入侵我国的重大林业害虫,入侵山西后迅速在相邻省份扩展蔓延。文章围绕作者对红脂大小蠹入侵来源地、暴发机制以及红脂大小蠹化学生态学等领域开展的研究工作进展进行了总结。历史资料记载和分子证据均支持红脂大小蠹由北美(美国西海岸)地区传入。松树萜烯类化合物是红脂大小蠹寻找适合寄主的重要信号物质,近期在北美和中国几个地区同时开展的研究,确认了3-蒈烯对红脂大小蠹具有最强引诱作用,该研究结果简化和修正了先前北美一直使用的3组分植物源引诱剂配方;在筛选有效引诱成分的基础上,开展了引诱剂定量缓释载体的研究,该释放载体已经被授权专利,并得到广泛推广应用。红脂大小蠹信息素一直没有得到鉴定,近来通过对红脂大小蠹后肠提取物的分析,分离鉴定了5种电生理和行为上有生物活性的萜烯类氧化物,其中有3个组分对红脂大小蠹植物源引诱剂具有增效作用。在驱避剂方面,研究证实了马鞭草烯酮以及非寄主挥发物对红脂大小蠹具有驱避作用。同时,研究证明,马鞭草烯酮对红脂大小蠹的作用存在剂量效应。目前,作者正开展红脂大小蠹-共生菌-寄主松树三者相互关系以及红脂大小蠹与土著种之间化学通讯等方面的研究,以期阐明红脂大小蠹的入侵性及暴发机制,寻求更有效的控制途径。     

华山松木蠹象聚集信息素分离鉴定和引诱效果

为了研制华山松木蠹象Pissodes punctatus Langor et Zhang的引诱剂,对华山松木蠹象的虫粪和雄虫后肠挥发性物质进行了分析鉴定和室内外生物活性测试。经GC-MS测定,发现在华山松木蠹象的新鲜虫粪和雄虫后肠挥发性物质中,除了松树挥发性的单萜烯如α-蒎烯、β-蒎烯和3-蒈烯以外,还存在1-甲基-2-异丙烯基-环丁烷乙醇（grandisol）。室内Y-型嗅觉仪趋向实验表明,较低浓度的3-（+）-蒈烯、1-甲基-2-异丙烯基-环丁烷乙醇及其相应的醛1-甲基-2-异丙烯基-环丁烷2醛（grandisal）,引起华山松木蠹象的正趋向反应。林间引诱试验表明,1-甲基-2-异丙烯基-环丁烷乙醇和1-甲基2-异丙烯基-环丁烷2醛对华山松木蠹象具有一定的引诱作用。由此推断,1-甲基-2-异丙烯基-环丁烷乙醇可能是华山松木蠹象的集结信息素成分之一。     

青杨脊虎天牛对植物源挥发物的EAG和行为反应

测定了青杨脊虎天牛Xylotrechus rusticus （L.）雌、雄成虫对其寄主杨树中的水杨醛（0.95 μmol/μL）和非寄主植物中0.3 μmol/μL的叶绿醇、0.4 μmol/μL的水芹烯和0.6 μmol/μL的 R 型α-蒎烯、S 型α-蒎烯、S型β-蒎烯、3-蒈烯、罗勒烯、香草烯和松节油等10种植物挥发性气味物质的触角电位（EAG）反应。结果表明,与对照相比,这10种植物挥发物多能引起成虫明显的EAG反应( P<0.05,P<0.01 ),其中雌虫对松节油、水杨醛、R 型α-蒎烯和 S 型α-蒎烯的EAG反应较强; 雄虫对 R 型α-蒎烯的EAG反应最强,松节油次之。根据雌虫对这10种挥发物EAG反应的强弱,进一步测定了雌虫对0.00006、0.0006、0.006、0.06、0.6、0.12 μmol/μL的松节油、R 型α-蒎烯、S 型α-蒎烯以及0.000095、0.00095、0.0095、0.095、0.95、0.19 μmol/μL的水杨醛的EAG和行为反应。结果表明,雌虫对松节油、水杨醛和 R 型α-蒎烯的EAG反应随气味物质浓度的增加而增加,水杨醛浓度增加到0.95 μmol/μL、松节油和 R 型α-蒎烯浓度增加到0.6 μmol/μL以后,EAG反应值趋于平稳;对 S 型α-蒎烯的反应随浓度的增加而呈线性增加。水杨醛浓度低于0.095时,对雌虫没有明显的定向作用( P>0.05 ),高于此浓度时表现为驱避作用( P<0.05 ); 松节油在浓度低于或等于0.6 μmol/μL时对雌虫表现为驱避作用,浓度为0.6时驱避效果最佳（ P<0.01 ）。雌虫对 R 型α-蒎烯和 S 型α-蒎烯没有明显的定向行为反应。     

华山松不同部位挥发性单萜烯的含量及其对华山松木蠹象行为的影响

通过GC—MS分析,发现健康的与遭受华山松木蠹象Pissodes punctatus Langor et Zhang危害的华山松Pinusarmandii Franch树干以及华山松枝梢释放的挥发性单萜烯在含量上存在较大差别;利用Y-型双向选择嗅觉仪进行的室内趋向反应实验发现,处于补充营养期和交尾产卵期的华山松木蠹象对单萜烯表现出不同的趋向反应。在华山松枝梢挥发物中含量较高的（-）-β-蒎烯、月桂烯和柠檬烯引起补充营养期华山松木蠹象较大的趋向反应;在华山松树干韧皮部挥发物中含量较高的3-（＋）-蒈烯则引起交尾产卵期的华山松木蠹象较大的趋向反应。根据试验结果,对华山松和华山松木蠹象互作的机制进行了初步探讨。     

松墨天牛成虫对幼虫虫粪挥发性物质的触角电位反应及林间驱避试验

为了研制松墨天牛Monochamus alternatus Hope驱避剂,本文利用触角电位技术和田间试验的方法研究了松墨天牛雌雄成虫对幼虫虫粪挥发性物质的触角电位反应及幼虫虫粪己烷提取物、合成化合物混合物的林间驱避作用。结果表明：α-蒎烯、β-蒎烯、3-蒈烯、柠檬烯和长叶烯均能引起松墨天牛雌雄成虫一定的触角电生理反应,而4-甲基-2,6-二叔丁基苯酚不能引起松墨天牛雌雄成虫的触角电生理反应。林间驱避试验结果说明,幼虫虫粪的己烷提取物处理过的树干上的刻槽数明显少于对照树干上的刻槽数,二者具有极显著差异;有合成化合物的混合物存在的诱捕器诱捕到的雌虫数（平均值3.80±1.02）与对照诱捕到的雌虫数（平均值16.80±1.16）差异极显著。结果显示α-蒎烯、β-蒎烯、3-蒈烯、柠檬烯、长叶烯和4-甲基-2,6-二叔丁基苯酚的混合物对松墨天牛雌虫具有驱避作用,有望开发出一类新的驱避剂。     

Red turpentine beetle primary attraction to β-pinene or 3-carene (with and without ethanol) varies in western US pine forests

1. Lure attraction strength for red turpentine beetle, Dendroctonus valens (Coleoptera: Curculionidae: Scolytinae) observed previously in US Pacific Northwest ponderosa pine forests is (−)-β-pinene+ethanol > (+)-3-carene+ethanol, but untested elsewhere in its western US range. Thus, both were tested with (−)-β-pinene, (+)-3-carene, ethanol, and a blank in Oregon and California sites burned by wildfire, whereas in Arizona the first four lures were tested in a thinned-unburned site.
2. The D. valens responses in burned Oregon and California sites were similar, (−)-β-pinene+ethanol > (−)-β-pinene > 3-carene = 3-carene+ethanol > ethanol > blank, whereas in the cut-unburned Arizona site it was 3-carene+ethanol > 3-carene = (−)-β-pinene+ethanol > (−)-β-pinene. Whether this variation was influenced by beetle genetic differences, or chemical and physical parameters in the different environments and remaining stressed host resources 1-year post disturbance warrants additional study.
3. Responses to (−)-β-pinene varied, from a stronger attractant than (+)-3-carene in Oregon and California, to a weaker lure than (+)-3-carene in Arizona. This (−)-β-pinene variability was minimized when released in combination with ethanol, making (−)-β-pinene+ethanol the most consistent attractant of those tested across the three states, and a reliable lure for detection, monitoring, and management projects for D. valens in western US pine forests.

     

β-Pinene + ethanol attracts more red turpentine beetles than carene+ ethanol,with or without traces of frontalin,at prescribed burn or thinned sites

1. Red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae: Scolytinae), previously responded more strongly to (−)-β-pinene + ethanol than (+)-3-carene + ethanol lures at sites burned the prior year by wildfire in Oregon and northeastern California, whereas at a thinned-unburned Arizona site (+)-3-carene + ethanol was the stronger attractant. This discrepancy was further examined to tease apart whether D. valens attraction varies by region or previous forest disturbance types.
2. Here, (−)-β-pinene + ethanol and (+)-3-carene + ethanol lures were tested in pine stands at two Oregon sites disturbed the previous year by a prescribed burn or thinning only. Both lures were tested also with or without trace amounts of the pheromone frontalin, as its presence enhanced attractions in China but had not been tested in North America.
3. At both sites, regardless of prior forest disturbance, (−)-β-pinene + ethanol lures attracted the most beetles. Lures releasing trace frontalin attracted more beetles than their corresponding lures without it at both sites, except in one case.
4. Overall, previous year disturbances from disparate management treatments had minimal influence on lure attraction to D. valens. For detection, monitoring or management (−)-β-pinene + ethanol + frontalin in trace amounts attracts the most beetles of lures tested to date in Pacific Northwest pine forests.

     

Key structural features in cis-carane, (+)-3-carene,cis-pinane, (+)-α-pinene,and (−)-β-pinene influencing red turpentine beetle primary attraction when released with ethanol

1. In US Pacific Northwest ponderosa pine forests the primary attraction order shown previously for red turpentine beetle, Dendroctonus valens (Coleoptera: Curculionidae: Scolytinae), is (−)-β-pinene+ethanol > (+)-3-carene+ethanol > (+)-α-pinene+ethanol. The monoterpenes are bicyclic C₁₀H₁₆ isomers containing one 6-carbon ring with one double bond. Both pinenes have a 4-carbon second ring and differ only by their endocyclic or exocyclic double bond. The (+)-3-carene second ring has 3-carbons; its double bond is endocyclic like (+)-α-pinene.
2. Ring system and double bond influences on primary attraction were evaluated by hydrogenating (+)-3-carene and (+)-α-pinene to cis-carane and cis-pinane, respectively. Field test primary attraction strengths were (−)-β-pinene+ethanol > cis-carane+ethanol > cis-pinane+ethanol > ethanol.
3. In combination with ethanol (i) a double bond is not required in either ring system to attract D. valens, (ii) the cis-carane bicyclic 3, 6-carbon ring system provides stronger beetle attraction than the cis-pinane 4, 6-carbon bicyclic ring system, and likely structural basis for stronger (+)-3-carene attraction over (+)-α-pinene, (iii) adding an exocyclic double bond to the 4, 6-carbon ring system elevates attraction above the 3, 6-carbon ring system with no double bond, and (iv) the 4, 6-carbon ring system is a much stronger attractant with an exocyclic rather than endocyclic double bond.

     

The red turpentine beetle, <Emphasis Type="Italic">Dendroctonus valens</Emphasis> LeConte (Scolytidae): an exotic invasive pest of pine in China

An exotic invasive pest of pines, the red turpentine beetle, Dendroctonus valens LeConte (Scolytidae) (RTB), was first detected in Shanxi Province, northern China, in 1998 and started causing widespread tree mortality there in 1999. This outbreak continues and has spread to three adjacent provinces, causing unprecedented tree mortality. Although it is considered a minor pest of pines in North America, RTB has proven to be an aggressive and destructive pest of Pinus tabuliformis, China’s most widely planted pine species. The bionomics and occurrence, distribution, response to host volatiles, and host preference of this pine beetle in China are compared with what is known of the beetle in its native range in North America. Factors likely contributing to D. valens success in China and control of the beetle outbreak are discussed. (−)-β-pinene was shown to be the most attractive host volatile for D. valens from the Sierra Nevada of California, whereas 3-(+)-carene is the most attractive host volatile for beetles in China. Monocultures of Pinus tabuliformis, several consecutive years of drought conditions and warm winters have apparently factored D. valens invasion and establishment in China.     

Sexual variation of bacterial microbiota of Dendroctonus valens guts and frass in relation to verbenone production

Gut microbiota are widely involved in insect biology, and many factors can influence the microbiota in guts and frass. Dendroctonus valens is a very destructive forest pest in China, and the mass-attacking behavior is regulated by several semiochemicals, including verbenone, a multifunctional pheromone. The beetle harbors a variety of bacteria in its guts and frass and some of them are capable of verbenone production. D. valens is characterized by monogamy and female-initiated attacking behavior. Whether the bacterial communities fluctuate according to sex, and whether the variation influences the verbenone production, remains to be determined. In this study, the bacterial microbiota in D. valens guts and frass were analyzed, and verbenone production by their crude bacterial suspensions was compared in vitro. Bacterial diversity in female frass is more abundant compared to male frass, and the percentages and total amounts of main genera like Lactococcus and Pseudomonas in female frass are significantly higher than those in male frass. The verbenone produced by the female frass suspension is significantly higher than male frass. This study presents a comprehensive comparison of bacterial communities in guts and frass between both sexes of D. valens, highlighting the potential significance of female frass microbiota in verbenone production.     

Verbenone-releasing flakes protect individual Pinus contorta trees from attack by Dendroctonus ponderosae and Dendroctonus valens (Coleoptera: Curculionidae, Scolytinae)

1 In a study site in interior northern California, twenty individual lodgepole pines Pinus contorta were sprayed with a suspension of DISRUPT Micro‐Flake^® Verbenone (4,6,6‐trimethylbicyclo(3.1)hept‐3‐en‐2‐one) Bark Beetle Anti‐Aggregant flakes (Hercon Environmental, Emigsville, Pennsylvania) in water, with sticker and thickener, from ground level to a height of 7 m. Twenty trees sprayed with just water, sticker and thickener served as controls. All trees were baited immediately after spraying with mountain pine beetle Dendroctonus ponderosae aggregation pheromone lures, and lures were refreshed after 4 weeks. 2 Trees treated with verbenone had significantly lower attack density by D. ponderosae than controls at 2, 4, 6 and 8 weeks after application of flakes. 3 None of the treated trees was attacked by red turpentine beetle Dendroctonus valens, whereas control trees averaged nearly two D. valens attacks per tree, 8 weeks after treatment. 4 A dry frass index, used to predict ultimate tree mortality, was significantly higher in control trees than treated trees for all four sampling intervals. This index proved to be a significant predictor of ultimate tree mortality. 5 Ten months after application, treated trees showed significantly lower mortality than control trees.