基于EPG技术分析茶树品种对茶小绿叶蝉的抗性及其相关的抗性物质

为深入探讨假眼小绿叶蝉在茶树上的刺探行为,进而筛选合适的刺探电位(electrical penetration graph,EPG)参数作为评判茶树抗感性指标,遂比较紫鹃、宁波黄金芽和黄观音等11个国家级和特色茶树良种对该叶蝉抗感性,并解析茶树抗感性与主要化学成分的相关性。使用刺探电位仪检测了该叶蝉在这11个茶树品种上的多种刺探行为及其参数、以聚类分析等方法解析各品种上EPG参数而判断每品种对于叶蝉的抗感性;再以HPLC检测茶叶主要成分,分析抗感性与化学成分的关联程度。结果如下:(1)进一步明确了代表该叶蝉在茶树上取食行为的7种EPG波形及其可能的生物学意义,即Np、A、S、C、E、F和R波,解析了各种波形的特征和出现规则,认为E波、F波以及未确定生物学意义的R波是抗性相关波。(2)在5h测试期间,叶蝉在黄观音和龙井43品种上的刺探次数最多,紫鹃、宁波黄金芽和金观音上的次之,铁观音和安吉白茶上的最少;E波平均持续时间在铁观音和黄牡丹上最长,乌牛早和安吉白茶上次之,紫鹃上最短;F波平均持续时间在黄观音上最长,紫鹃、龙井43和宁波黄金芽上次之,金观音上最短。(3)以刺探次数、韧皮部取食波E波历期、韧皮部取食困难波F波历期为指标,将11个品种聚为2类,即第Ⅰ类:安吉白茶、乌牛早、紫牡丹、中茶108、黄牡丹和铁观音;第Ⅱ类为黄观音、龙井43、紫鹃、宁波黄金芽和金观音;抗性强弱为:第Ⅱ类第Ⅰ类。(4)从感性至抗性品种,儿茶素类含量明显升高,且儿茶素总量与E波历期显著负相关(P为深入探讨假眼小绿叶蝉在茶树上的刺探行为,进而筛选合适的刺探电位（electrical penetration graph,EPG）参数作为评判茶树抗感性指标,遂比较紫鹃、宁波黄金芽和黄观音等11个国家级和特色茶树良种对该叶蝉抗感性,并解析茶树抗感性与主要化学成分的相关性。使用刺探电位仪检测了该叶蝉在这11个茶树品种上的多种刺探行为及其参数、以聚类分析等方法解析各品种上EPG参数而判断每品种对于叶蝉的抗感性;再以HPLC检测茶叶主要成分,分析抗感性与化学成分的关联程度。结果如下：（1）进一步明确了代表该叶蝉在茶树上取食行为的7种EPG波形及其可能的生物学意义,即Np、A、S、C、E、F和R波,解析了各种波形的特征和出现规则,认为E波、F波以及未确定生物学意义的R波是抗性相关波。（2）在5h测试期间,叶蝉在黄观音和龙井43品种上的刺探次数最多,紫鹃、宁波黄金芽和金观音上的次之,铁观音和安吉白茶上的最少;E波平均持续时间在铁观音和黄牡丹上最长,乌牛早和安吉白茶上次之,紫鹃上最短;F波平均持续时间在黄观音上最长,紫鹃、龙井43和宁波黄金芽上次之,金观音上最短。（3）以刺探次数、韧皮部取食波E波历期、韧皮部取食困难波F波历期为指标,将11个品种聚为2类,即第Ⅰ类：安吉白茶、乌牛早、紫牡丹、中茶108、黄牡丹和铁观音;第Ⅱ类为黄观音、龙井43、紫鹃、宁波黄金芽和金观音;抗性强弱为：第Ⅱ类 > 第Ⅰ类。（4）从感性至抗性品种,儿茶素类含量明显升高,且儿茶素总量与E波历期显著负相关（P < 0.05）;咖啡碱、氨基酸和可溶性糖含量皆未与E波、F波历期明显相关。认为：E波和F波历期以及刺探次数可作为茶树抗感性的主要参数;R波可能与韧皮部取食抗性相关;儿茶素类是抗性相关物质。研究可为深入探讨茶树抗虫机理提供参考。     

福建主要茶树品种理化特性与假眼小绿叶蝉种群数量的相关性分析

比较分析了福建4个主要茶树品种(福云6号、铁观音、毛蟹、黄棪)的理化特性与假眼小绿叶蝉种群数量之间的相关关系,结果显示,假眼小绿叶蝉的种群数量与茶树品种叶片的物理结构———叶片总厚度、上表皮、上表皮角质层、下表皮、海绵组织厚度之间的相关性不显著,而与叶片下表皮角质层和栅栏组织厚度之间存在显著负相关。在生化指标方面,假眼小绿叶蝉的种群数量与茶树叶片中茶多酚、水溶性糖、可溶性蛋白、儿茶素含量之间的相关性不显著;与咖啡碱的含量有一定负相关,相关关系接近显著水平;而与游离氨基酸含量呈显著正相关。     

迷迭香挥发物不同组合对假眼小绿叶蝉行为的调控

为探讨茶园群落中非茶植物气味引诱或驱避重要茶树害虫假眼小绿叶蝉的效应,遂将茶园常见杂草迷迭香挥发物中樟脑、α-松油醇和石竹烯分别配成10-2g/m L剂量,再等量组成混合物,在每个橡皮头上载100#L制成诱芯,附于素馨黄粘板上,于茶园中诱捕假眼小绿叶蝉,发现这种三组分诱芯明显地驱避假眼小绿叶蝉。将行为反应中分别对假眼小绿叶蝉呈现显著引诱效应的10-2g/m L樟脑、10-4g/m L石竹烯、10-6g/m Lα-水芹烯、10-4g/m Lα-松油醇和10-8g/m L桉树脑等量配成五组分混合物,同法制成诱芯,附于素馨黄粘板,诱捕假眼小绿叶蝉。发现五组分诱芯:(1)显著引诱假眼小绿叶蝉,诱效稍强于现价段茶园中使用的源于茶梢挥发物的诱芯;(2)每日诱捕的假眼小绿叶蝉数与天数呈抛物线关系;(3)加入液体石蜡作为缓释剂,可将诱效的半衰期延长2.5—4.0d;(4)18:00—6:00诱得的假眼小绿叶蝉数量占全天的70%—75%。分析认为:非茶植物迷迭香的挥发物中含有显著引诱假眼小绿叶蝉信息物质,这类信息物质制成的五组分诱芯的诱效较强,可以作为一种防治假眼小绿叶蝉的手段,早晨和黄昏是最佳诱捕时分。     

假眼小绿叶蝉对茶梢挥发物的行为反应

【目的】研究假眼小绿叶蝉Empoasca vitis G?the对健康茶梢和蝉害茶梢挥发物的行为反应,筛选出可引诱假眼小绿叶蝉的茶梢挥发物。【方法】以动态吸附法收集健康茶梢和蝉害茶梢挥发物,用气相色谱–质谱联用仪(GC-MS)进行定性定量分析,借助于Y形嗅觉仪检测多种挥发物引诱假眼小绿叶蝉成虫的活性。【结果】从健康茶梢和蝉害茶梢中共鉴定出30种挥发物组分,其中烯烃类含量较大。健康茶梢和蝉害茶梢挥发物中共有组分有13种,蝉害之后其含量皆上升,其中Z-b-罗勒烯和乙酸叶醇酯的含量分别是健康茶梢中的142.27倍、12.90倍。蝉害茶梢中新出现的组分有12种,其中紫苏烯含量较高。在10-2、10~(-4)、10-6 g/mL浓度下,乙酸叶醇酯表现出极显著的引诱水平(P<0.01);紫苏烯在10~(-2)和10~(-4) g/mL浓度下表现出极显著引诱水平(P<0.01);10~(-4) g/mL浓度下,Z-b-罗勒烯和D-柠檬烯极显著引诱假眼小绿叶蝉(P<0.01);10~(-4) g/mL芳樟醇、10~(-6) g/mL乙酸正丁酯和10~(-6) g/mL D-柠檬烯呈现出显著引诱水平(P<0.05);混合物组分Blend1和Blend2分别表现出极显著和显著引诱水平;而10~(-6) g/mLa-法尼烯显著排斥假眼小绿叶蝉(P<0.05)。【结论】假眼小绿叶蝉成虫对健康茶梢和蝉害茶梢挥发物多种组分具有不同的行为反应,引诱效果较强的单组分或混合组分的选定可为田间引诱效果试验提供参考。     

假眼小绿叶蝉自然天敌控制作用的评价

对假眼小绿叶蝉的卵期寄生蜂－缨小蜂进行了初步调查观察,并应用生命表方法对假眼小绿叶蝉的寄生性和捕食性天敌作用作了评价,提出了卵期寄生是一种重要的天敌控制因子,通过高峰期世代自然园与化防轩绿叶蝉生命表比较,结果表明,自然园种群趋势指数为１．０９,化防园为２．７１,模拟采摘试验,下一世代种群趋势指数均小于１,数量将下降。     

假眼小绿叶蝉触角化感器的扫描电镜观察

扫描电镜观察结果表明,假眼小绿蝉触角上只有一种毛状感器,而且,感器数量和分布在雌雄两性个体间相似。     

显著调控假眼小绿叶蝉行为的迷迭香挥发物鉴定

茶园生态系中生长着众多草本和木本植物,迷迭香就是其中常见的木本杂草。以其幼嫩部分为味源、茶园重要害虫假眼小绿叶蝉为试虫,用Y形管嗅觉仪做行为测定,以洁净空气为CK,发现随着味源剂量增加,其引诱的假眼小绿叶蝉数增至一个数值再减少,二者呈抛物线关系。从迷迭香挥发物中鉴定出46种化合物,包含醇类9种、酮类4种、酯类4种、酸类1种、烯烃类18种、烷烃类7种、醚类1种、醛类1种和酚类1种。醇类含量最大,占总量52.20%;单组分中桉树脑、左旋α-蒎烯和马鞭草烯酮的含量最大,分别占总量的42.54%、10.37%和6.82%。使用迷迭香挥发物中含量较大的25种组分为味源,每味源配制几种剂量,以正己烷为CK,用Y形管嗅觉仪进行行为测定,发现10-2g/mL樟脑、10-4g/mL石竹烯、10-6g/mL和10-8g/mLα-水芹烯、10-4g/mL和10-10g/mLα-松油醇、10-10g/mL和10-8g/mL桉树脑显著引诱假眼小绿叶蝉;而10-10g/mLβ-蒎烯显著排斥假眼小绿叶蝉。经分析认为:茶园中除了茶树之外,还有其它植物挥发物引诱假眼小绿叶蝉,可为筛选茶园中诱虫植物提供参考。     

假眼小绿叶蝉卵的寄生蜂种类及种群动态

通过田间采梢、室内镜下查卵和饲养观察,初步获得假眼小绿叶蝉Empoasca vitis Gthe卵寄生蜂2种,分别隶属于三棒缨小蜂属Stethynium sp.和裂骨缨小蜂属Schizophragma sp.,其中三棒缨小蜂属Stethyniumsp.是优势种。2种缨小蜂的寄生动态调查结果显示,除8月和12月寄生率较低外,其它月份的自然寄生率都在30%以上,特别是10~11月寄生率最高达65%,对小绿叶蝉的虫口数量具有重要控制作用,值得进一步研究和保护利用。     

假眼小绿叶蝉微卫星位点的生物信息学分析

【目的】为开发假眼小绿叶蝉Empoasca vitis分子标记,采用高通量测序技术对假眼小绿叶蝉DNA进行了测序与分析。【方法】本研究基于Illumina Hi Seq测序技术,构建了PE文库(~400bp),对获得的测序数据利用生物信息学分析手段完成全基因组扫描,并进一步使用MISA分析鉴定基因组序列中出现的微卫星序列(SSR)。针对微卫星序列共设计10对引物,并使用3步法进行引物多态性筛选。【结果】共计检测Scaffold数量为183 194条,其中包含SSR的Scaffold共计1 545条,共计筛选出1 569个SSR位点。在假眼小绿叶蝉的微卫星中,共包括87种重复基元类型,二核苷酸与三核苷酸重复序列为主要重复类型,分别占SSRs总数的70.26%和27.84%;二核苷酸重复基元CA/TG和三核苷酸重复基元AAT/ATT是优势重复基元,分别占SSRs总数的33.96%和5.86%。在设计的10对引物中,5对具有多态性,在8个假眼小绿叶蝉个体中共发现16个等位基因。【结论】结果说明假眼小绿叶蝉SSR位点在多态性方面具有极大的可开发性,具有多态性的SSR位点可对假眼小绿叶蝉种群间的分化,种群间的扩散机理和途径及影响因素等问题提供分子视角。     

茶假眼小绿叶蝉对不同茶香精和寄主植物的趋性反应

利用"T"形室观察了茶假眼小绿叶蝉Empoasca vitis Gthe对4种茶香精和3种寄主植物的趋性反应。结果表明,假眼小绿叶蝉对不同茶香精粉和寄主植物的趋性反应不同。叶蝉对绿茶和乌龙茶A香精有明显的趋向反应,而对乌龙茶B香精即铁观音茶香精则有明显的忌避反应,对红茶香精则无反应。假眼小绿叶蝉对茶梢和青菜的趋性反应无显著差异,而对茶梢和大豆的趋性反应有差异,表现出更喜好趋向茶梢。     

MacStylet, software to analyse electrical penetration graph data on the Macintosh

Conclusion Since the EPG method is increasingly utilized in the investigation of plant-Homoptera interactions, this software has been developed to enable fast processing of abundant data. The objective seems to have been achieved and, with a little practice, a 2-hour experiment may be analysed in about 10–15 minutes. Mac-Stylet is stand-alone shareware, freely distributed to all persons interested (request to G. Febvay, email: febvay@jouy.inra.fr).     

植食性同翅目昆虫寻找和接受植物韧皮部过程中的细胞与化学探试(英文)

植食性同翅目昆虫在最终接受其宿主植物的过程中,将感受从叶表层直到韧皮部筛管途中存在的多种化学物质,并导致不同的刺探和取食行为。本文就植物叶表层存在的糖类和生物碱类物质,叶肉层的多糖类、酸碱度和酚类物质以及韧皮部内的氨基酸、蛋白质、碳水化合物和植物次生物质等对昆虫寻找及取食韧皮部行为的影响作一综述,并且还探讨了植物叶肉层的细胞结构对昆虫刺探及取食行为的可能影响。此外,结合刺探电位(electrical penetration graphs,EPGs)的研究结果,还讨论了昆虫感受植物叶组织内部的各种化学和细胞结构信息的可能机制。     

CELLULAR AND CHEMICAL SAMPLING DURING PHLOEM FINDING AND HOST-PLANT ACCEPTANCE BY HOMOPTERAN INSECTS

Abstract The chemical compounds that exist in plant leaf tissue are of great importance for homopteran insects during the proccess of host-plant acceptance. From the leaf surface to the phloem tube, various groups of chemicals can be detected by insect's receptors which lead to different feeding responses. The effects of sugars and alkaloids in leaf surface; polysaccharides, pH gradients and phenolics in mesophyll; amino acids, proteins, carbohydrates and secondary substances in phloem tube on phloem finding and host-plant acceptance are reviewed respectively in this paper, which also includes the potential effects of mesophyll structures on insect's feeding preference. Furthermore the mechanism by which the insects perceive these chemical and mechanical cues, correlated with the EPG (electrical penetration graph) patterns (a series of visible signals showing the stylet activities in plant tissue) is also discussed.     

Probing and feeding characteristics of the greenhouse whitefly in association with host-plant acceptance and whitefly strains

Host-plant and whitefly strain effects and their interactions on the probing and sap feeding of the greenhouse whitefly, Trialeurodes vaporariorum (Westwood), have been investigated in this study using the DC-EPG (Electrical Penetration Graph) technique. Whiteflies generally displayed fewer but longer probes on highly acceptable cucumber than on less acceptable tomato. Both whitefly strains, the T(omato)-strain and the C(ucumber)-strain, showed a significantly lower number of phloem phases on cucumber than on tomato. However, the duration of total phloem phases achieved by either of the whitefly strains on these two host plants was not significantly different. These data indicate that a more continuous phloem feeding has occurred on cucumber plants. Indeed, the percentage of phloem feeding time after the first sustained phloem phase (longer than 15 min) was higher on cucumber for the C-strain whiteflies. When comparing these two whitefly strains, the T-strain whiteflies probed less frequently but longer than the C-strain whiteflies did on both host plants. Also, the T-strain whiteflies displayed a longer duration of total phloem phases on tomato. An interaction between the whitefly strain and plant effects was detected on a parameter, which showed that whiteflies probed significantly longer before reaching the first phloem phase on the host plants that had been previously experienced. In conclusion, both plant species and whitefly strains affect whitefly's probing and feeding behaviour, though plant effects are much stronger.     

Difference in feeding behaviors of two invasive whiteflies on host plants with different suitability: implication for competitive displacement

In China, Bemisia tabaci Q (commonly known as biotype Q) has rapidly displaced B (commonly known as biotype B) in the past 6 years. The mechanisms underlying such phenomenon have been studied extensively in recent years; however, we have not come to a definitive conclusion yet. In the present study, the differences in host suitability between B and Q whitefly adults to five host plants (cabbage, cotton, cucumber, poinsettia, and tomato) were evaluated based on their respective feeding behaviors using a direct-current electrical penetration graph (DC-EPG) system. Pair-wise comparisons of B. tabaci B and Q feeding on each of the five host plants clearly indicate that Q feeds better than B on tomato, cotton and poinsettia, while B feeds better than Q on cabbage and cucumber. The EPG parameters related to both phloem and non-phloem phases confirm that cabbage and cucumber are best suited to B, while tomato, cotton, and poinsettia are best suited to Q. Our present results support the contention that host suitability and adult feeding behavior contribute to the competitive displacement of biotype B by biotype Q. The discrepancy between field (previous studies) and laboratory results (this study), however, suggests that 1) whitefly displacement is apparently contributed by multiple factors; and 2) factor(s) other than the host plant suitability may play a vital role in dictating the whitefly biotypes in the field.     

Analysis of resistance in tomato and sweet pepper against the greenhouse whitefly using electrically monitored and visually observed probing and feeding behaviour

Different types of plant resistance against the greenhouse whitefly, Trialeurodes vaporariorum, were distinguished according to the tissue location of the resistance factors. The effects of resistance factors were compared by measuring the electrically monitored and visually observed probing and feeding behaviour of whiteflies on two resistant tomato genotypes (82207 and 82216), a susceptible tomato cultivar, and a non-host plant, sweet pepper. On sweet pepper, whiteflies displayed very short first probes, very long pathway probing and spent much time on non-feeding activities such as walking and standing still. Also, a high percentage of whiteflies rejected sweet pepper without ingesting substances from the phloem vessel. These data suggest a strong resistance that is based on the factors present in surface/epidermis and/or mesophyll layers of this plant. The behaviour of whiteflies on tomato 82207 was very different from that on sweet pepper. On tomato 82207 whiteflies apparently did not perceive resistance factors on the leaf surface and in the mesophyll. Resistance factors appeared to be present in the phloem tissue, because a higher number of phloem phases, longer phloem salivation periods and shorter phloem ingestion periods were observed when compared with the susceptible tomato cultivar. Tomato 82216 showed no clear resistance factor in the phloem. The importance of combined EPG and behavioural research for host-plant resistance studies are discussed.     

Effects of treatments for electrical penetration graph recordings on behaviour and biology of Aphis craccivora (Aphididae)

Abstract. Two laboratory studies were conducted to investigate effects of treatments for direct current electrical penetration graph (DC-EPG) recordings or 'tether effect', on behaviour and reproductive performance of cowpea aphid Aphis craccivora Koch. The experiments constituted a control study in application of DC-EPGs to analyse cowpea aphid feeding behaviour and host plant resistance mechanisms. Resistant (ICV-12) and susceptible (ICV-1) cultivars of cowpea Vigna unguiculata (L.) Walp were used. EPG treatments included two groups of aphids: tethered aphids that were exposed to DC electricity via an attachment of a thin, flexible gold wire on their dorsum using a droplet of adhesive silver paint, and 'free' (untethered) aphids with a dorsal spot of silver paint only. EPGs of the tethered aphids were recorded continuously for c. ! h, whereas from the 'free' aphids recordings were done only for brief periods of 2–5 min, by temporarily contacting a gold wire to the spot of silver paint. Waveform signals generated from resistance fluctuations and electromotive forces, and representing aphid stylet penetration behaviour were recorded. A separate experiment was conducted to investigate effects of EPG treatments on aphid survivorship and population growth. Overall, EPG treatments did not significantly affect aphid stylet penetration behaviour or life-table parameters. However, effects of crop cultivar on those characteristics were significant. Waveform E2, which denotes aphid ingestion in phloem sieve elements, and non-penetration behaviour were important indicators of aphid resistance in ICV-12. Also, apart from the number of aphid generations, other life-table parameters were useful indicators of ICV-12 resistance. Thus, DC-EPGs provided a reliable technique for studying aphid stylet behaviour, and investigation of aphid resistance in cowpeas.