华南地区柑橘木虱与柑橘粉虱内共生菌检测及其Wolbachia共生菌的系统发育关系分析

【目的】探明不同地理种群的柑橘木虱Diaphorina citri Kuwayama和柑橘粉虱Dialeurodes citri Ashmead体内昆虫内共生菌的种类及其感染率,并以Wolbachia共生菌为代表,对其系统发育关系进行分析,为今后自共生菌角度研发柑橘木虱和柑橘粉虱的新型防控技术奠定基础。【方法】以16S r DNA、23S r DNA以及wsp为目标基因,利用PCR技术检测采自于广州、湛江、南宁、桂林、厦门的柑橘木虱以及采自广州的柑橘粉虱体内共生菌的种类及其感染率;利用多位点序列分型(MLST)技术和MEGA 5.0软件对不同昆虫样本中的Wolbachia进行系统发育关系分析。【结果】本研究采集的柑橘木虱和柑橘粉虱均含有原生共生菌Portiera和次生共生菌Wolbachia、Cardinium、Rickettsia,但该3种次生共生菌在不同木虱与粉虱种群的感染率有所不同;Arsenophonus只在广州和湛江种群的柑橘木虱中检出。基于wsp基因及MLST基因序列的Wolbachia系统发育分析表明,华南地区柑橘木虱和柑橘粉虱体内的Wolbachia均属于Wolbachia的B大组Con亚组。【结论】不同地理种群的柑橘木虱与柑橘粉虱体内感染的共生菌种类及其感染率不同;Wolabchia共生菌与柑橘木虱寄主不存在协同进化关系,在同一采集点存在Wolbachia通过柑橘寄主在柑橘木虱之间、柑橘木虱与柑橘粉虱之间水平传播的可能性。     

Population Dynamics and Growth Rates of Endosymbionts During Diaphorina citri (Hemiptera,Liviidae) Ontogeny

The infection density of symbionts is among the major parameters to understand their biological effects in host–endosymbionts interactions. Diaphorina citri harbors two bacteriome-associated bacterial endosymbionts (Candidatus Carsonella ruddii and Candidatus Profftella armatura), besides the intracellular reproductive parasite Wolbachia. In this study, the density dynamics of the three endosymbionts associated with the psyllid D. citri was investigated by real-time quantitative PCR (qPCR) at different developmental stages. Bacterial density was estimated by assessing the copy number of the 16S rRNA gene for Carsonella and Profftella, and of the ftsZ gene for Wolbachia. Analysis revealed a continuous growth of the symbionts during host development. Symbiont growth and rate curves were estimated by the Gompertz equation, which indicated a negative correlation between the degree of symbiont–host specialization and the time to achieve the maximum growth rate (t*). Carsonella densities were significantly lower than those of Profftella at all host developmental stages analyzed, even though they both displayed a similar trend. The growth rates of Wolbachia were similar to those of Carsonella, but Wolbachia was not as abundant. Adult males displayed higher symbiont densities than females. However, females showed a much more pronounced increase in symbiont density as they aged if compared to males, regardless of the incorporation of symbionts into female oocytes and egg laying. The increased density of endosymbionts in aged adults differs from the usual decrease observed during host aging in other insect–symbiont systems.     

Molecular Characterization of Wolbachia Strains Associated with the Invasive Asian Citrus Psyllid Diaphorina citri in Brazil

Wolbachia is a symbiont intensively studied due to its ability to interfere with their host’s reproduction, and it has been recently proposed as an alternative tool to control insect pests or vectors of diseases. The Asian citrus psyllid Diaphorina citri is an important pest of citrus since it vectors the bacterium that causes the "Huanglongbing" disease in citrus. The frequency and diversity of Wolbachia associated with D. citri is unknown, limiting the utilization of Wolbachia as an alternative strategy for insect management. Thus, we aimed to determine the natural rate of infection, to characterize the Wolbachia strains associated with this psyllid by "multilocus sequencing typing” (MLST) and wsp analysis, and to verify the association of the symbiont to particular genotypes of the host. Analysis indicated Wolbachia infects 100 % of all specimens tested from all 15 sampled populations. MLST revealed the occurrence of five new sequence types (STs) of Wolbachia, while analysis based on the wsp sequences indicated only four different types of Wolbachia. ST-173 was predominant, while the remaining STs were population specific. Analysis of the host–symbiont relationship did not reveal any particular association of Wolbachia and haplotypes or a decrease in nucleotide diversity of D. citri in populations in which more than one ST was recorded. The consequences of the diversity of STs reported are still unknown, but the fact that Wolbachia infection is fixed and that there is one ST with a broad distribution highlights the use of this symbiont as an alternative strategy to control D. citri.     

Assembly of a Marine Viral Metagenome after Physical Fractionation

Metagenomic analyses of marine viruses generate an overview of viral genes present in a sample, but the percentage of the resulting sequence fragments that can be reassembled is low and the phenotype of the virus from which a given sequence derives is usually unknown. In this study, we employed physical fractionation to characterize the morphological and genomic traits of a subset of uncultivated viruses from a natural marine assemblage. Viruses from Kāne‘ohe Bay, Hawai‘i were fractionated by equilibrium buoyant density centrifugation in a cesium chloride (CsCl) gradient, and one fraction from the CsCl gradient was then further fractionated by strong anion-exchange chromatography. One of the fractions resulting from this two-dimensional separation appeared to be dominated by only a few virus types based on genome sizes and morphology. Sequences generated from a shotgun clone library of the viruses in this fraction were assembled into significantly more numerous contigs than have been generated with previous metagenomic investigations of whole DNA viral assemblages with comparable sequencing effort. Analysis of the longer contigs (up to 6.5 kb) assembled from our metagenome allowed us to assess gene arrangement in this subset of marine viruses. Our results demonstrate the potential for physical fractionation to facilitate sequence assembly from viral metagenomes and permit linking of morphological and genomic data for uncultivated viruses.     

Susceptibility of nymphs and adults of Diaphorina citri to the entomophathogenic fungus Hirsutella citriformis

The susceptibility of nymphs and adults of Diaphorina citri to infection by Hirsutella citriformis was evaluated. We also studied the ability of adult D. citri that had been contaminated with fungal conidia to transmit infection to nymphs. Diaphorina citri nymphs were more susceptible than adults. Adults were not able to transmit conidia to nymphs and cause infection.     

江西省柑橘木虱对五种常规药剂的抗药性

【目的】针对江西省柑橘木虱Diaphorina citri对常规杀虫剂抗药性水平的研究相对匮乏,研究不同地区柑橘木虱种群对常用杀虫剂的抗性水平,为生产实践合理化用药提供依据。【方法】采用浸叶法测定毒死蜱、联苯菊酯、噻虫嗪、烯啶虫胺和虫螨腈5种杀虫剂对上高县（SG）、宜黄（YH）、南丰（NF）、宁都（ND）、大余（DY）和寻乌县（XW）6个不同地区的田间柑橘木虱的毒力,结合毒力参考基线,得到抗性倍数。【结果】上高、宜黄和南丰县种群对毒死蜱的抗性倍数在中等水平（9.01-15.01）,而宁都、大余和寻乌县则是高水平抗性（128.36-308.82）。大余种群对联苯菊酯的抗性水平为133.71倍,其他5个地区均在中等水平（17.08-88.53）。不同地区柑橘木虱种群对噻虫嗪的抗性倍数在13.62-25.92,仅为中等偏低水平;而它们对烯啶虫胺的抗性倍数为77.37-257.71,已经达到极高水平。与联苯菊酯类似,除了大余种群对虫螨腈为高水平抗性外,其余5个地区种群均为中等水平。【结论】赣南脐橙产区的宁都县、大余县和寻乌县的柑橘木虱种群对5种常用杀虫剂均表现出高水平抗性,显著高于非赣南脐橙产区种群（上高县、宜黄县和南丰县）。推荐在高抗地区使用抗性水平较低的药剂,并注意轮换用药;在中等抗性地区应有限制地使用药剂,以延缓抗性增长。     

柑橘木虱抗药性研究进展

柑橘木虱Diaphorina citri Kuwayama主要危害柑橘等芸香科植物,传播柑橘黄龙病病菌。柑橘树感染黄龙病后,叶片黄化,果实早熟脱落,畸形,最终死亡,给柑橘产业带来了极大的损失。柑橘木虱对各种常见的杀虫剂已产生了不同程度的抗药性。本文从柑橘木虱的抗性测定方法、抗药性现状、交互抗性、抗药性机理和抗性治理等方面进行了综述,为今后柑橘木虱的防治提供一定的参考。     

The Influence of Learning on Host Plant Preference in a Significant Phytopathogen Vector,Diaphorina citri

Although specialist herbivorous insects are guided by innate responses to host plant cues, host plant preference may be influenced by experience and is not dictated by instinct alone. The effect of learning on host plant preference was examined in the Asian citrus psyllid, Diaphorina citri; vector of the causal agent of citrus greening disease or huanglongbing. We investigated: a) whether development on specific host plant species influenced host plant preference in mature D. citri; and b) the extent of associative learning in D. citri in the form of simple and compound conditioning. Learning was measured by cue selection in a 2-choice behavioral assay and compared to naïve controls. Our results showed that learned responses in D. citri are complex and diverse. The developmental host plant species influenced adult host plant preference, with female psyllids preferring the species on which they were reared. However, such preferences were subject to change with the introduction of an alternative host plant within 24–48 hrs, indicating a large degree of experience-dependent response plasticity. Additionally, learning occurred for multiple sensory modalities where novel olfactory and visual environmental cues were associated with the host plant. However, males and females displayed differing discriminatory abilities. In compound conditioning tasks, males exhibited recognition of a compound stimulus alone while females were capable of learning the individual components. These findings suggest D. citri are dynamic animals that demonstrate host plant preference based on developmental and adult experience and can learn to recognize olfactory and visual host plant stimuli in ways that may be sex specific. These experience-based associations are likely used by adults to locate and select suitable host plants for feeding and reproduction and may suggest the need for more tailored lures and traps, which reflect region-specific cultivars or predominate Rutaceae in the area being monitored.     

阿里食虱跳小蜂生物学特性及其应用研究进展

柑橘木虱Diaphorina citri Kuwayama是传播柑橘黄龙病病菌的重要媒介昆虫,对我国柑橘产业的健康发展造成了严重的威胁.阿里食虱跳小蜂Diaphorencyrtus aligarhensis(Shaffe,Alam and Agarwal)是柑橘木虱的一种优良内寄生蜂,在我国乃至世界多个国家均有分布,发挥着重要的生防作用.本文综述了阿里食虱跳小蜂分类地位、形态特征、地理分布、生长发育、生殖能力、寿命、寄生行为等生物学特性,以及人工繁育技术的研究与应用的最新研究进展,同时分析了目前其应用方面存在的问题,旨在为我国今后利用阿里食虱跳小蜂科学、高效和可持续防控柑橘木虱提供参考,不断推动我国柑橘产业健康、稳定和可持续发展.     

柑橘木虱及分泌物对胡瓜新小绥螨的吸引作用研究

在25℃温度条件下,在九里香(Murraya panicuata)嫩枝条上研究柑橘木虱Diaphorina citri Kuwayama混合虫态、卵、低龄若虫、高龄若虫、柑橘木虱分泌物对胡瓜新小绥螨Neoseiulus cucumeirs(Oudemans)雌螨的吸引作用,以探索与评价捕食螨对柑橘木虱各虫态及分泌物的趋向性。研究发现:1.柑橘木虱混合虫态对胡瓜新小绥螨的吸引作用在试验后第2小时经测验达到显著水平(P<0.05),第3小时达极显著水平(P<0.01),表明柑橘木虱混合虫态对胡瓜新小绥螨有很强的吸引作用;2.柑橘木虱卵对胡瓜新小绥螨雌成螨无吸引作用;3.试验开始后第1⁵小时在试验区胡瓜新小绥螨数量分别高于空白区25.00%、18.33%、13.33%、15.00%和23.33%,柑橘木虱低龄若虫对胡瓜新小绥螨有较强烈的吸引作用;4.柑橘木虱高龄若虫对胡瓜新小绥螨吸引作用在第1小时最高,第25小时在试验区胡瓜新小绥螨数量分别高于空白区25.00%、18.33%、13.33%、15.00%和23.33%,柑橘木虱低龄若虫对胡瓜新小绥螨有较强烈的吸引作用;4.柑橘木虱高龄若虫对胡瓜新小绥螨吸引作用在第1小时最高,第2³小时最低,第4小时开始回升,第5小时可达到51.67%。可见柑橘木虱高龄若虫对胡瓜新小绥螨有一定的吸引作用;5.柑橘木虱分泌物对胡瓜新小绥螨吸引作用在第1小时最高,试验区胡瓜新小绥螨数量高于空白区33.33%、胡瓜新小绥螨不能取食柑橘木虱分泌物,第2小时胡瓜新小绥螨向外逃跑的比率增加。但胡瓜新小绥螨在试验区数量仍高于试验区,柑橘木虱分泌物对胡瓜新小绥螨也有一定的吸引作用。本试验环境开放,模拟自然状态。在柑橘木虱混合虫态发生的情况下,胡瓜新小绥螨对其具备搜寻能力。     

Resistance of Citrus and Related Genera to Diaphorina citri Kuwayama (Hemiptera: Liviidae)

The present study was developed to evaluate the resistance of the following genotypes of Citrus and related genera to this pest: ‘Pera,’ ‘Natal’, and ‘Washington Navel’ oranges (Citrus sinensis), ‘Marsh Seedless’ grapefruit (Citrus paradisi), hardy orange ‘Rubidoux’ (Poncirus trifoliata), kumquat (Fortunella margarita Swingle), citrumelo ‘Swingle’ (C. paradisi x P. trifoliata), and citrange ‘Troyer’ (P. trifoliata x C. sinensis). The experiments were performed in greenhouses with plants grafted onto ‘Rangpur’ lime (Citrus limonia) and placed individually in voile cages. The preference for oviposition in a no-choice test, and the effect of genotype were evaluated. The egg-adult cycle was monitored to determine the effect of genotype on the biology of the insect. Poncirus ‘Rubidoux’ was the least preferred genotype for oviposition; reduced number of eggs was also found to occur on citrange ‘Troyer’, and ‘Marsh Seedless’ was the genotype with the most eggs. No significant variation in the duration of the embryonic period was observed; however, a difference in the viability of eggs was found, with the lowest egg viabilities on ‘Swingle.’ Kumquat and ‘Marsh Seedless’ genotypes were correlated with increased durations of the nymphal phase, however, there was no difference in the survival of this phase. Fecundity of females on ‘Troyer’, ‘Swingle’, and kumquat was reduced. Considering all of the evaluated parameters, it was concluded that cultivars of sweet orange are the most susceptible genotypes to Diaphorina citri. Regarding oviposition, P. trifoliata ‘Rubidoux’ showed resistance of the antixenosis type.