The recent emergence of several plant diseases caused by psyllid-borne bacterial pathogens worldwide (Candidatus Liberibacter spp.) has created renewed interest on the interaction between psyllids and bacteria.In spite of these efforts to understand psyllid association with bacteria,many aspects of their interactions remain poorly understood.As more organisms are studied,subtleties on the molecular interactions as well as on the effects of the bacteria on the psyllid host are being uncovered.Additionally,psyllid-borne bacterial phytopathogens can also affect the host plant,which in turn can impact psyllid physiology and behavior.Here,we review the current literature on different aspects of the influence of bacteria on multitrophic interactions among plants,psyllids,and pathogens.We then highlight gaps that need to be addressed to advance this field,which can have significant implications for controlling these newly emergent and other plant diseases.

为探明柑橘木虱体内黄龙病菌psy62株系中原噬菌体遗传多样性及与柑橘株系的遗传差异,本研究将利用3对引物在psy62基因组中2个噬菌体FP1和FP2的同源序列区域内对赣南地区阳性样品进行PCR扩增和测序.结果显示,赣南地区阳性样品在此基因区域内有28头柑橘木虱检测到原噬菌体,共有5种类型,分别为A, A2,B, C,C1.其中A, B的检测率相对较高;C1检测率最低,为7.1%.由此可,见在此基因位点不同地理位置的柑橘木虱原噬菌体种群有较大的遗传差异,且与美国佛罗里达的"Ca.L.asiaticus"株系,中国柑橘叶片"Ca.L.asiaticus"株系均有显著的遗传差异,这有助于进一步的研究分析原噬菌体的种类差异对柑橘木虱的传毒机制的影响.

Wolbachia can profoundly influence the survival,reproduction,and defenses of insect hosts.These interactions could potentially be harnessed for managing pests or insecttransmitted diseases.Diaphorina citri Kuwayama is a phloem-feeding pest capable of transmitting the putative causal agent of citrus greening,Candidatus Liberibacter asiaticus (CLas).Like many insects,D.citri is also infected with Wolbachia (wDi).Recent studies indicate that the relative abundance of wDi could be associated with the abundance of CLas,and that wDi may contribute to regulating expression of phage lytic cycle genes in CLas,suggesting the need for better understanding of wDi biology in general.This study investigated the genetic diversity of wDi among D.citri in populations spanning eleven countries and two U.S.territories.Six Wolbachia genes,wsp,coxA,fbpA,ftsZ,gatB,and hcpA,were sequenced and compared across samples.Two prevalent wDi strains were identified across the samples,and screening of clone libraries revealed possible coinfection of wDi strains in specific populations.D.cirri mitochondrial cytochrome oxidase subunit Ⅰ gene (mtCOI) were more divergent between D.citri populations that were infected with different wDi strains or had different infection statuses (single infection vs.coinfection).While we could not eliminate the possibility that maternal transmission may contribute to such patterns,it is also possible that wDi may induce cytoplasmic incompatibility in their host.These findings should contribute to the understanding of wDi population ecology,which may facilitate manipulation of this endosymbiont for management of citrus greening disease worldwide.

Citrus is one of the top three fruit crops worldwide.Citrus production is facing an unprecedented crisis due to Huanglongbing (HLB,also known as citrus greening),which is the most devastating disease of citrus.HLB was first reported in Asia a century ago.HLB is associated with Candidatus Liberibacter asiaticus (Las),Ca.L.americanus (Lam),and Ca.L.africanus (Bové,2006),which belong to the gram-negative α-proteobacteria.Among them,Las is the most widely spread.Las and Lam are vectored by Diaphorina citri Kuwayama (ACP),whereas Laf is transmitted by Trioza erytreae.HLB and ACP are rapidly spreading to and having a devastating impact on citrus production in other citrusproducing areas such as Brazil,Caribbean counties,and the US.To date,HLB has been confirmed in 51 of the 140 citrusproducing countries.The major citrus-producing regions without HLB,such as the Mediterranean region and Australia,are at great risk for ACP establishment and HLB spread (Gutierrez and Ponti,2013).In addition,Trioza erytreae has been found in Spain and poses immediate threat of spread of HLB in the Mediterranean region.Thus,HLB has become a global problem.

[背景]柑橘黄龙病是世界柑橘生产上最具毁灭性的病害之一,主要由候选韧皮部杆菌属亚洲种(“Candidatus Liberibacter asiaticus”,CLas)引起.CLas全基因组测序已经完成,因而该病原菌的基因表达研究和功能验证得以进行.[目的]筛选CLas内参基因并评估其不同侵染时期和在不同品种植物寄主中的表达稳定性.[方法]基于基因功能类别,利用实时荧光定量PCR技术分析23个CLas的候选内参基因相对表达情况(16S rRNA基因作为参照基因).结合Ct值标准差和geNorm、NormFinder、RefFinder软件,评价内参基因的表达稳定性.[结果]在感染黄龙病不同时期和不同品种的植物寄主样本中,14对引物表现出较强的特异性和稳定性.内参基因稳定性排名:ftsZ＞gyrA＞rpoB1＞ftsA＞secA＞gap＞zapE＞gmk2＞rpoD＞secY＞rpoO＞ftsW＞gmk1＞recA,根据geNorm配对变异值Vn/n+1选择稳定性最好的ftsZ和gyrA作为内参基因作进一步评估.以ftsZ+gyrA以及16S rRNA基因分别作为内参基因检测柑橘黄龙病菌致病基因Las△5313的表达水平,所得的表达模式相同.[结论]柑橘黄龙病菌中涉及DNA复制和细胞分裂功能的管家基因表达较稳定,在CLas的基因表达研究中可选择ftsZ+gyrA的基因组合作为内参.本研究为后续利用实时荧光定量PCR分析CLas基因表达及研究CLas致病机理奠定基础.

为获得长春花(Catharanthus roseus)过氧化物酶(peroxidase,POD)基因的全长cDNA序列以及黄龙病菌(Candidatus Liberibacter asiaticus)侵染后长春花POD基因(PwPOD)的表达情况,本研究根据不同作物POD基因的同源性,筛选出长春花过氧化物酶基因的引物,采用SMARTer-RACE方法扩增其3’和5’末端,运用qRT-PCR的分析病原感染后不同时间长春花叶、主茎和根中PwPOD基因的mRNA转录水平的变化.经拼接后获得PwPOD的全长cDNA序列,该基因全长1 611 bp (GenBank登录号KT032115).生物信息学分析表明,该基因与其他已知物种的POD基因同源性达89％,编码一个无跨膜结构域,N末端含有信号肽的稳定蛋白.分析染病后长春花各组织中Pw POD的表达发现其表达量随着感染时间的增加呈先升后降的趋势,分别在嫁接后第30天(叶片)、第35天(主茎)和第35天(根)达到峰值,表达量分别上调12.8倍、3.96倍和5.53倍.证实PwPOD的表达确实受到黄龙病菌侵染的诱导,其表达量可能与寄主的抗病能力相关.

黄龙病是全球广泛发生的毁灭性病害,柑橘木虱为其主要媒介昆虫.越南最早于上个世纪六十年代有相关报道.通过一些国际合作项目,越南对柑橘木虱和黄龙病进行了一系列研究并积极采取措施有效防控:包括消除病株、筛选抗病虫品种、改变种植密度和时间、化学防治与生物防治、作物间种、喷施矿物油乳剂、施用有机肥、使用无病苗和无病接穗并在运输过程中封闭保护等.其中控制黑臭蚁Dolichoderus thoracicus增殖黄猄蚁Oecophylla smaragdina、交错式间种番石榴以控制柑橘木虱和黄龙病值得中国借鉴和参考.

"Candidatus Liberibacter solanacearum" (Lso) are phloem-restricted and unculturable Gram-negative bacteria.Presently five haplotypes have been identified worldwide;but only haplotypes A and B are associated with the vector Bactericera cockerelli ((S)ulc.) in the Americas.Previous studies showed that Lso-infection reduces B.cockerelli reproductive output and that Lso haplotype B is more pathogenic than Lso haplotype A.To understand the interaction ofLso haplotype B and B.cockerelli,the fitness of Lso-free and Lso B-infected insects,and the expression of vitellogenin (Bc Vgl-like),a gene involved directly in the insect reproduction were analyzed.Statistical differences in the number of eggs oviposited,and the total number of progeny nymphs and adults were found among crosses of insects with or without Lso.Significant differences in sex proportions were found between Lso B-infected and Lso-free crosses:a higher proportion of F1 adult females were obtained from Lso B-infected mothers.A significant reduction of BcVgl-like was observed in crosses performed with Lso B-infected females compared to the Lso-free insects.In female cohorts of different age,a significant reduction of BcVgl-like expression was measured in 7-d-old Lso B-infected females (virgin and mated) compared with 7-d-old Lso-free females (virgin and mated),respectively.The reduction of Bc Vgl-like transcript was associated with a lower number of developing oocytes observed in female's reproductive systems.Overall,this study represents the first step to understand the interaction of Lso B with B.cockerelli,highlighting the effect of Lso B infection on egg production,Bc Vgl-like expression,and oocyte development.

Bacillus thuringiensis (Bt) toxins are effective in controlling insect pests either through the spraying of products or when expressed in transgenic crops.The discovery of endophytic Bt strains opened new perspectives for studies aimed at the control of sapsucking insects,such as the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera:Liviidae),a vector of"Candidatus Liberibacter spp.," associated with citrus huanglongbing (HLB).In this study,transloeation of endophytic Bt strains in citrus seedlings inoculated with Bt suspension delivered by soil-drench,and their systemic pathogenicity to D.citri nymphs were investigated.The pathogenicity of three wild-type Bt strains against D.citri third-instar nymphs was demonstrated.Among the 10 recombinant strains tested (each of them harboring a single cry or cyt gene),3 can be highlighted,causing 42％-77％ and 66％-90％ nymphal mortality at 2 and 5 d after inoculation,respectively.The isolation of Bt cells from young citrus shoots and dead nymphs,and PCR performed with specific primers,confirmed the involvement of the Bt strains in the psyllid mortality.This is the first report showing the translocation of Bt strains from citrus seedling roots to shoots and their potential to control D.citri nymphs that fed on these soil-drench inoculated seedlings.The Bt strains that caused the highest mortality rates have the potential to be used as bioinsecticides to control D.citri and the identified genes can be used for the production of transgenic Bt citrus.

Immunofluorescence has been widely used to localize microbes or specific molecules in insect tissues or cells.However,significant autofluorescence is frequently observed in tissues which can interfere with the fluorescent identification of target antigens,leading to inaccurate or even false positive fluorescent labeling.The alimentary canal of the potato psyllid,Bactericera cockerelli (S)ulc,exhibits intense autofluorescence,hindering the application of immunolocalization for the detection and localization of the economically important pathogen transmitted by this insect,"Candidatus Liberibacter solanacearum" (Lso).In the present study,we tested the use of irradiation,hydrogen peroxide (H2O2) and Sudan black B (SBB) treatments to reduce the autofluorescence in the B.cockerelli alimentary canal tissues.Furthermore,we assessed the compatibility of the above-mentioned treatments with Lso immunolocalization and actin staining using phalloidin.Our results showed that the autofluorescence in the alimentary canal was reduced by irradiation,H2O2,or SBB treatments.The compatibility assays indicated that irradiation and H2O2 treatment both greatly reduced the fluorescent signal associated with Lso and actin.However,the SBB incubation preserved those target signals,while efficiently eliminating autofluorescence in the psyllid alimentary canal.Therefore,herein we propose a robust method for reducing the autofluorescence in the B.cockerelli alimentary canal with SBB treatment,which may improve the use of immunofluorescence labeling in this organism.This method may also have a wide range of uses by reducing the autofluorescence in other arthropod species.