亚洲柑橘木虱成虫和5龄若虫在感染黄龙病的柑橘上的取食行为及获菌效率比较

【目的】亚洲柑橘木虱Diaphorina citri是柑橘的毁灭性病害——黄龙病亚洲种‘Candidatus Liberibacter asiaticus’(‘C las’)的主要传播媒介。本研究的目的是明确木虱成虫和5龄若虫的取食行为、获菌效率是否有差异,以及寄主感染黄龙病是否对5龄若虫取食产生影响。【方法】利用直流型刺吸电位仪(DC-EPG Giga-4)记录柑橘木虱成虫和5龄若虫在携带黄龙病的酸橘Citrus reticulata cv.Sunki嫩梢上10 h的取食行为,用qPCR单头检测其获得黄龙病病原菌的效率,并比较5龄若虫在感病和健康植株嫩梢上的取食行为。【结果】柑橘木虱成虫与5龄若虫在感染黄龙病的酸橘上的取食行为有显著差异。5龄若虫比成虫更快地开始在韧皮部和木质部进行吸食,口针在韧皮部的总过程以及吸食时间显著长于成虫。此外,5龄若虫和成虫在EPG测定(同时饲菌)10 h后获菌率分别为37.5%和20.0%,若虫明显高于成虫。寄主植物感染黄龙病对5龄若虫的取食行为有一定的影响,表现在感病植株上的刺探次数、唾液分泌次数和韧皮部吸食次数都显著少于健康植株,而两者分泌唾液和韧皮部吸食时间没有显著差异。另外,在感病植株上首次韧皮部取食出现时间较在健康植株上要早。【结论】在同等时间下,柑橘木虱5龄若虫比成虫在感染黄龙病的酸橘上的取食能力更强、取食量更大、获菌率更高,原因可能是若虫需要更多的营养物质供其生长发育所致。寄主感染黄龙病对木虱5龄若虫的取食有利,使其更快地开始取食,而且更改取食位点次数变少,推测可能与黄龙病菌破坏了植物的防御有关。     

Enhanced proliferation and efficient transmission of Candidatus Liberibacter asiaticus by adult Diaphorina citri after acquisition feeding in the nymphal stage

We carried out a quantitative detection of Candidatus Liberibacter asiaticus, the bacterium associated with the disease of huanglongbing, in the vector psyllid Diaphorina citri by using a TaqMan real‐time PCR assay. The concentration of the bacterium was monitored at 5‐day intervals for a period of 20 days after psyllids were exposed as fifth instar nymphs or adults to a Ca. L. asiaticus‐infected plant for an acquisition access period of 24 h. When adults fed on Ca. L. asiaticus‐infected plant, the concentration of the bacterium did not increase significantly and the pathogen was not transmitted to any citrus seedlings. In contrast, when psyllids fed on infected plant as nymphs, the concentration of the pathogen significantly increased by 25‐, 360‐ and 130‐fold from the initial acquisition day to 10, 15 and 20 days, respectively. Additionally, the pathogen was successfully transmitted to 67% of citrus seedlings by emerging adults. Our data suggested that multiplication of the bacterium into the psyllids is essential for an efficient transmission and show that it is difficult for adults to transmit the pathogen unless they acquire it as nymphs.     

柑橘木虱两地理种群的内共生菌群落组成及传菌能力

柑橘黄龙病（Huanglongbing, HLB）是经柑橘木虱Diaphorina citri传播的最主要柑橘病害之一, 危害严重时能对柑橘产业造成毁灭性的破坏。为了鉴定福建和海南2个地理种群柑橘木虱的内共生菌群落组成, 本研究对16S rRNA部分保守序列进行PCR扩增, 并利用特异性引物对不同内共生菌进行了感染率检测; 另外, 还通过人工接虫的方法, 探索柑橘木虱成虫在带黄龙病菌蕉柑Citrus reticulata cv. Tankan上的获菌能力, 以及带菌柑橘木虱成虫对黄岩蜜橘C. reticulata cv. Subcompressa的传菌能力。研究发现, 这2个地理种群的柑橘木虱含有相同的内共生菌组成, 包括α-Proteobacteria, Wolbachia spp., γ-Proteobacteria, mycetocyte symbionts, β-Proteobacteria, Oxalobacter和β-Proteobacteria, Herbaspirillum, 而且这2个地理种群柑橘木虱的4种内共生菌的携带率均在95%以上。柑橘木虱成虫在带菌蕉柑上饲菌28 d后, 带菌率可达到82%, 而带菌柑橘木虱成虫在黄岩蜜橘上传菌75 d后, 可导致橘树整体带菌。本研究为柑橘木虱的进一步研究和防虫治病途径提供了一些理论依据。     

Sexual transmission of a plant pathogenic bacterium, Candidatus Liberibacter asiaticus, between conspecific insect vectors during mating

Candidatus Liberibacter asiaticus is a fastidious, phloem-inhabiting, gram-negative bacterium transmitted by Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The bacterium is the presumed causal agent of huanglongbing (HLB), one of the most destructive and economically important diseases of citrus. We investigated whether Las is transmitted between infected and uninfected D. citri adults during courtship. Our results indicate that Las was sexually transmitted from Las-infected male D. citri to uninfected females at a low rate (<4%) during mating. Sexual transmission was not observed following mating of infected females and uninfected males or among adult pairs of the same sex. Las was detected in genitalia of both sexes and also in eggs of infected females. A latent period of 7 days or more was required to detect the bacterium in recipient females. Rod shaped as well as spherical structures resembling Las were observed in ovaries of Las-infected females with transmission electron microscopy, but were absent in ovaries from uninfected D. citri females. The size of the rod shaped structures varied from 0.39 to 0.67 μm in length and 0.19 to 0.39 μm in width. The spherical structures measured from 0.61 to 0.80 μm in diameter. This investigation provides convincing evidence that a plant pathogenic bacterium is sexually transmitted from male to female insects during courtship and established evidence that bacteria persist in reproductive organs. Moreover, these findings provide an alternative sexually horizontal mechanism for the spread of Las within populations of D. citri, even in the absence of infected host trees.     

Infection of an Insect Vector with a Bacterial Plant Pathogen Increases Its Propensity for Dispersal

The spread of vector-transmitted pathogens relies on complex interactions between host, vector and pathogen. In sessile plant pathosystems, the spread of a pathogen highly depends on the movement and mobility of the vector. However, questions remain as to whether and how pathogen-induced vector manipulations may affect the spread of a plant pathogen. Here we report for the first time that infection with a bacterial plant pathogen increases the probability of vector dispersal, and that such movement of vectors is likely manipulated by a bacterial plant pathogen. We investigated how Candidatus Liberibacter asiaticus (CLas) affects dispersal behavior, flight capacity, and the sexual attraction of its vector, the Asian citrus psyllid (Diaphorina citri Kuwayama). CLas is the putative causal agent of huanglongbing (HLB), which is a disease that threatens the viability of commercial citrus production worldwide. When D. citri developed on CLas-infected plants, short distance dispersal of male D. citri was greater compared to counterparts reared on uninfected plants. Flight by CLas-infected D. citri was initiated earlier and long flight events were more common than by uninfected psyllids, as measured by a flight mill apparatus. Additionally, CLas titers were higher among psyllids that performed long flights than psyllid that performed short flights. Finally, attractiveness of female D. citri that developed on infected plants to male conspecifics increased proportionally with increasing CLas bacterial titers measured within female psyllids. Our study indicates that the phytopathogen, CLas, may manipulate movement and mate selection behavior of their vectors, which is a possible evolved mechanism to promote their own spread. These results have global implications for both current HLB models of disease spread and control strategies.     

Feeding behavior of Diaphorina citri and its transmission of ‘Candidatus Liberibacter asiaticus’ to citrus

Huanglongbing (HLB), also known as citrus greening, is currently the most destructive disease of citrus, responsible for huge economic losses in the world's major citrus production areas. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits ‘Candidatus Liberibacter asiaticus’ (Clas), the pathogen responsible to cause HLB. Understanding of vector, pathogen, and host plant interactions is important for the management of this vector‐disease complex. We used the direct‐current electrical penetration graph (DC‐EPG) system to evaluate feeding behavior of Clas‐infected D. citri adults, and their potential to transmit the pathogen to healthy citrus, Citrus reticulata Blanco cv. Sunki (Rutaceae), following a 24‐h inoculation access period. Plants were tested for the presence of Clas by qPCR 6 months after inoculation. Findings suggest that inoculation was associated with salivation into the phloem sieve elements (waveform E1). The minimum feeding time for successful transmission by a single adult was 88.8 min, with a minimum E1 duration of 5.1 min. Regression analysis indicated a significant relationship between E1 duration and transmission efficiency. The adults successful in transmitting Clas to healthy citrus were able to penetrate and feed in the phloem much earlier than those which did not transmit. The minimum duration of E1 for a female was shorter than that of a male, but transmission was higher. However, durations of other EPG parameters were not significantly different between male and female. Feeding by single Clas‐infected D. citri adults on 6‐month‐old plants (Sunki) resulted in 23% HLB‐positive plants 6 months after inoculation. Multiple nymphs or adults could transmit the pathogen more efficiently than individual adults in the field, and further enhance the severity of the disease. Effective tactics are warranted to control D. citri and disrupt transmission of Clas.     

Prophage-Mediated Dynamics of ‘Candidatus Liberibacter asiaticus’ Populations,the Destructive Bacterial Pathogens of Citrus Huanglongbing

Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of Candidatus Liberibacter asiaticus’ (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease development.     

Induced Release of a Plant-Defense Volatile ‘Deceptively’ Attracts Insect Vectors to Plants Infected with a Bacterial Pathogen

Transmission of plant pathogens by insect vectors is a complex biological process involving interactions between the plant, insect, and pathogen. Pathogen-induced plant responses can include changes in volatile and nonvolatile secondary metabolites as well as major plant nutrients. Experiments were conducted to understand how a plant pathogenic bacterium, Candidatus Liberibacter asiaticus (Las), affects host preference behavior of its psyllid (Diaphorina citri Kuwayama) vector. D. citri were attracted to volatiles from pathogen-infected plants more than to those from non-infected counterparts. Las-infected plants were more attractive to D. citri adults than non-infected plants initially; however after feeding, psyllids subsequently dispersed to non-infected rather than infected plants as their preferred settling point. Experiments with Las-infected and non-infected plants under complete darkness yielded similar results to those recorded under light. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen. Quantification of volatile release from non-infected and infected plants supported the hypothesis that odorants mediate psyllid preference. Significantly more methyl salicylate, yet less methyl anthranilate and D-limonene, was released by infected than non-infected plants. Methyl salicylate was attractive to psyllids, while methyl anthranilate did not affect their behavior. Feeding on citrus by D. citri adults also induced release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants. Infected plants were characterized by lower levels of nitrogen, phosphorus, sulfur, zinc, and iron, as well as, higher levels of potassium and boron than non-infected plants. Collectively, our results suggest that host selection behavior of D. citri may be modified by bacterial infection of plants, which alters release of specific headspace volatiles and plant nutritional contents. Furthermore, we show in a laboratory setting that this apparent pathogen-mediated manipulation of vector behavior may facilitate pathogen spread.     

柑橘木虱成虫趋光行为反应

柑橘木虱Diaphorina citri Kuwayama是柑橘黄龙病(huanglongbing,HLB)的重要传播媒介。为了利用灯光诱控技术防治柑橘木虱,本实验于室内条件下研究柑橘木虱对波长为360 nm、400 nm、440 nm、480 nm、520 nm、560 nm和600 nm的LED光源和不同光照强度趋光行为反应。结果表明:柑橘木虱对7种单色光都有正趋向性。其中雌雄混合存在时对400 nm的紫光趋向性最强,其次是560 nm的绿光;单独处理时,雌成虫对400 nm的紫光趋性最强,其次是520 nm的绿光,雄成虫则是对520 nm的绿光趋性最强,其次是400 nm的紫光。在200μw/cm 2到1000μw/cm 2的光照强度范围内,随着光照强度的增大,柑橘木虱雄成虫趋光行为逐渐增强,在光照强度为1000μw/cm 2时趋光行为最强,但雌成虫趋光行为变化不明显。该研究表明:柑橘木虱雌雄成虫具有明显的正趋光性,且对光谱和光强的反应存在差异。这一结果可为柑橘木虱田间的灯光诱控提供实验依据。     

Genetic Diversity of Candidatus Liberibacter asiaticus Based on Two Hypervariable Effector Genes in Thailand

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases of citrus worldwide. HLB is associated with three species of ‘Candidatus Liberibacter’ with ‘Ca. L. asiaticus’ (Las) being the most widely distributed around the world, and the only species detected in Thailand. To understand the genetic diversity of Las bacteria in Thailand, we evaluated two closely-related effector genes, lasA _I and lasA _II, found within the Las prophages from 239 infected citrus and 55 infected psyllid samples collected from different provinces in Thailand. The results indicated that most of the Las-infected samples collected from Thailand contained at least one prophage sequence with 48.29% containing prophage 1 (FP1), 63.26% containing prophage 2 (FP2), and 19.38% containing both prophages. Interestingly, FP2 was found to be the predominant population in Las-infected citrus samples while Las-infected psyllids contained primarily FP1. The multiple banding patterns that resulted from amplification of lasA _I imply extensive variation exists within the full and partial repeat sequence while the single band from lasA _II indicates a low amount of variation within the repeat sequence. Phylogenetic analysis of Las-infected samples from 22 provinces in Thailand suggested that the bacterial pathogen may have been introduced to Thailand from China and the Philippines. This is the first report evaluating the genetic variation of a large population of Ca. L. asiaticus infected samples in Thailand using the two effector genes from Las prophage regions.     

Effects of a particle film on biology and behavior of Diaphorina citri (Hemiptera: Psyllidae) and its infestations in citrus

Studies were conducted to investigate the effects of a kaolin-based hydrophilic particle film, Surround WP, on the biology and behavior of the psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae) and to assess population densities of D. citri in citrus subjected to monthly applications of Surround WP. Laboratory investigations indicated a 3% (wt:vol) suspension of Surround WP in water applied directly was not acutely toxic to eggs, older nymphs or adults. Presence of the dried particle film on leaves interfered with the ability of adults to grasp and walk on citrus leaves. During a 30-s period, adults spent an average of 5 s moving on leaves with particle film compared with 16 s on leaves without particle film. When leaves were inverted, a significantly higher percentage of adults fell or flew from treated leaves (53%) than untreated leaves (16%). In a 12-mo study investigating infestations of D. citri on citrus treated monthly with Surround WP, cumulative reductions of 78% in adult numbers on mature leaves and of 60% in adult numbers on flush shoots (immature leaves) were observed in treated trees compared with untreated trees. Numbers of eggs and nymphs per flush shoot were reduced by 85 and 78%, respectively, in trees treated with particle film. Reductions in infestation levels of D. citri in treated trees were attributed to the negative effects of the particle film on the ability of adults to grasp, move, and oviposit. The suppressive effects of a Surround treatment against adult psyllids were degraded by rain.     

Incidence of Candidatus Liberibacter asiaticus infection in abandoned citrus occurring in proximity to commercially managed groves

Huanglongbing is one of the most devastating diseases of citrus (Citrus spp.). One management tactic against huanglongbing is aggressive management of the vector, the Asian citrus psyllid (Diaphorina citri Kuwayama), with insecticide applications. However, D. citri in abandoned groves are not controlled and therefore pose a risk of reinfestation for nearby commercial citrus. These abandoned groves could serve as a reservoir for the vector, as well as a source of the presumed causal agent for huanglongbing in Florida, Candidatus Liberibacter asiaticus (Las). The current study was conducted to determine the degree to which Las is present in abandoned Florida citrus groves and to compare relative inoculum levels in nearby managed and abandoned groves during times of the year when D. citri are abundant (June, July, and August). In addition, the movement of Las by dispersing D. citri adults from inner and edge rows of abandoned grove plots to the corresponding rows of managed plots was quantified during the same 3 mo. The results of the current study confirmed the presence of Las in both D. citri and plant tissue in abandoned groves at statistically equivalent levels to those in nearby managed groves. The mean number of D. citri adults dispersing from abandoned to managed grove plots ranged from 7.25 +/- 1.70 to 70.25 +/- 21.25 per 4-d intervals. Of those, the mean number of dispersing D. citri adults that were carrying the Las pathogen ranged from 1.00 +/- 0.58 to 1.50 +/- 0.50. Our results indicate that abandoned citrus groves are a significant source of Ca. Las and that dispersing D. citri move this pathogen into nearby managed groves.     

Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing

Citrus huanglongbing (HLB, ex greening) is one of the most serious diseases of citrus. Different forms of the disease are caused by different Candidatus Liberobacter species, Candidatus Liberibacter asiaticus (Las), Ca. L. africanus (Laf) and Ca. L. americanus (Lam). The pathogen is transmitted by psyllid insects and by budding with contaminated plant materials. The vector psyllid Diaphorina citri can transmit both Las and Lam. Establishment of this vector into Florida, reports of Lam and Las in Brazil in 2004, and recent confirmation of HLB in Florida in September 2005 is of great concern to the citrus industry. Research on HLB has been hampered by the unculturable nature of the causal bacterium in artificial media. It has also been difficult to detect and identify the pathogens, possibly because of low concentration and uneven distribution in host plants and vector psyllids. In this study, we developed quantitative TaqMan PCR using 16S rDNA-based TaqMan primer-probe sets specific to the different Ca. Liberobacter spp. An additional primer-probe set based on plant cytochrome oxidase (COX) was used as a positive internal control to assess the quality of the DNA extracts. The assays do not cross-react with other pathogens or endophytes commonly resident in citrus plants, and are very sensitive. HLB pathogen DNA was successfully amplified from the equivalent of 20 ng of midrib tissue from symptomatic leaves. The consistent results of the assays with DNA extracted from plants infected by various Ca. Liberibacter species grown in greenhouses and in the field demonstrated a degree of reproducibility for these TaqMan assays. Inhibitors of the PCR that are frequently present in plant extracts did not affect the assay results. The population of the pathogens was estimated to be 5 x 10(7) and 2 x 10(6) cells/g of fresh midribs of symptomatic sweet orange leaves infected by Las and Lam, respectively. The ratio of pathogen DNA to host plant DNA was estimated by to be 1:13,000 (w/w) and 1:1000 (c/c: target copy/target copy) in DNA extracts obtained by a standard CTAB method. Our rapid, sensitive and specific TaqMan PCR assay for the detection, identification and quantification of Ca. Liberibacter species has been successfully used in the confirmation of HLB caused by Las in Florida, and will be very useful for a broad range of research programs as well as the regulatory response and management of HLB disease.     

Silencing of V-ATPase-E gene causes midgut apoptosis of Diaphorina citri and affects its acquisition of Huanglongbing pathogen

The Asian citrus psyllid, Diaphorina citri Kuwayama, is among the most important pests of citrus. It is the main vector of the Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas), which causes severe losses in citrus crops. Control of D. citri is therefore of paramount importance to reduce the spread of HLB. In this regard, using RNA interference (RNAi) to silence target genes is a useful strategy to control psyllids. In this study, using RNAi, we examined the biological functions of the V-ATPase subunit E (V-ATP-E) gene of D. citri, including its effect on acquisition of CLas. The amino acid sequence of V-ATP-E from D. citri had high homology with proteins from other insects. V-ATP-E was expressed at all D. citri life stages analyzed, and the expression level in mature adults was higher than that of teneral adults. Silencing of V-ATP-E resulted in a significant increase in mortality, reduced body weight, and induced cell apoptosis of the D. citri midgut. The reduced expression of V-ATP-E was indicated to inhibit CLas passing through the midgut and into the hemolymph, leading to a majority of CLas being confined to the midgut. In addition, double-stranded RNA of D. citri V-ATP-E was safe to non-target parasitic wasps. These results suggest that V-ATP-E is an effective RNAi target that can be used in D. citri control to block CLas infection.     

柑橘木虱蜜露排泄机制:进展与展望

半翅目昆虫柑橘木虱Diaphorina citri是柑橘类果树重要害虫,主要以高渗透压的植物韧皮部汁液为食,是柑橘毁灭性病害——柑橘黄龙病(HLB)的主要传播媒介。柑橘木虱取食韧皮部汁液时自身进化出一套完整的渗透调节机制调节其体内渗透压,将体内过量摄入的糖类转化成长链寡糖并以蜜露形式排出体外。本文从柑橘木虱蜜露排泄行为、蜜露组成成分以及影响蜜露排泄的多个因素进行了论述,同时综述了可能参与柑橘木虱蜜露排泄行为的渗透调节基因。研究表明,柑橘木虱雌雄成虫及若虫在蜜露排泄行为上存在显著差异,且排泄的蜜露在颜色、纹路及组成成分方面均有不同;寄主植物、杀虫剂、病原微生物及天敌化合物均会影响柑橘木虱蜜露排泄行为。分子机制探究发现,α-葡萄糖苷水解酶、水通道蛋白及糖基转移酶基因等关键渗透调节基因可能参与调控柑橘木虱蜜露排泄行为。本文可为未来有关柑橘木虱蜜露排泄行为方面的研究以及为研制柑橘木虱防治新型药剂开发新靶标提供参考。     

Morphological and molecular characterization of a Hirsutella species infecting the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), in Florida

The Asian citrus psyllid, Diaphorina citri Kuwayama, is an invasive pest that vectors citrus greening disease, which recently was detected in Florida. Mycosed adult D. citri were collected at four sites in central Florida between September 2005 and February 2006. Observation of the cadavers using scanning electron microscopy revealed that the pathogen had branched synnemata supporting monophiladic conidiogenous cells. A high-fidelity polymerase chain reaction (PCR) assay was used to amplify the 18S rRNA, 28S rRNA and beta-tubulin genes of the pathogen for phylogenetic analysis. The morphological and genetic data indicated that the pathogen was a novel isolate related to Hirsutella citriformis Speare. PCR assays using isolate-specific primers designed from the unique putative intron region of the beta-tubulin sequence distinguished the psyllid pathogen from five related Hirsutella species. The pathogen was maintained in vivo by exposing healthy D. citri to the synnemata borne on field-collected cadavers. Infected psyllids had an abundance of septate hyphal bodies in their hemolymph and exhibited behavioral symptoms of disease. In vitro cultures of the pathogen were slow-growing and produced synnemata similar to those found on mycosed D. citri. In laboratory bioassays, high levels of mortality were observed in D. citri that were exposed to the conidia-bearing synnemata produced in vivo and in vitro.     

Sex‐specific responses of Asian citrus psyllid to volatiles of conspecific and host‐plant origin

Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the primary vector of Candidatus Liberibacter spp. bacteria that cause citrus greening, a disease of worldwide importance. Olfactometry was employed to test responses of D. citri to odours from intact citrus plants (Mexican lime, Citrus aurantifolia, sour orange, Citrus aurantium, Marsh grapefruit, Citrus paradisi and Valencia orange, Citrus sinensis), citrus plants previously infested with D. citri, and odours of conspecifics including nymphs, adult insects of same and opposite sex, and their products (honeydew), both alone and in combination. In contrast to other studies, psyllids of both sexes were attracted to volatiles of undamaged Mexican lime leaves, whereas undamaged grapefruit attracted only females, and leaves of Valencia and sour orange did not attract either sex. All four plant species attracted female psyllids when previously infested, but only Mexican lime and sour orange‐attracted males. Thus, Citrus species appear to vary in the production of both constituitive and induced volatiles that attract adult psyllids. Volatiles emitted by nymphs did not attract either sex, but psyllid honeydew was attractive to males, likely due to female pheromone residues. Males oriented to the odour of females, whereas the reverse was not true, and neither males nor females oriented to same‐sex volatiles. The addition of conspecific cues (adults, nymphs or honeydew) did not increase female attraction to previously infested leaves, but male response was increased by the presence of adults and honeydew, regardless of plant species. Thus, female psyllids appear to orient more strongly to volatiles of plant origin, whereas males respond more strongly to cues emanating from females and conspecific excretions. These results suggest that female psyllids drive the initial colonization of host plants, whereas males orient to females and infested plants. Identification of the specific volatiles involved may permit their use in monitoring and management of this pest.     

Repellent effect of guava leaf volatiles on settlement of adults of citrus psylla, Diaphorina citri Kuwayama, on citrus

Abstract  The Asiatic citrus psyllid ( Diaphorina citri Kuwayama [Hemiptera: Sternorrhyncha: Psyllidae]) is a vector of huanglongbing (citrus greening), a devastating disease of citrus caused by phloem-limited bacteria. Growing guava ( Psidium guajava ) as an intercrop appears to be a successful means of reducing psyllid numbers within citrus orchards; however, the mechanism by which such a reduction is achieved is unknown. To determine the repellent effect of guava leaf and factors attributed to this activity, responses of adult psyllids to guava leaf and its odor were evaluated in cage tests and Y-tube olfactometer test. The results showed that guava leaf possessed a repellent effect against the adult citrus psyllids. Fewer psyllids were found on citrus leaves in the presence of guava foliage than in its absence. Young and old guava leaf showed equal repellent activity. By covering the guava shoots with net cloth, it was revealed that the repellent effect of guava leaf against adult psyllids on citrus was attributed to the volatile compounds, rather than physical factors. The olfactometer response of adult psyllids to guava leaf odor was dosage-dependent. Between guava odor and control, only 35.00%, 25.00% and 16.25% of the psyllids moved toward guava odor when presented with 5.0, 10.0 and 15.0 g of guava shoots, respectively. The olfactometer experiments also showed that both male and female psyllids responded similarly to the guava leaf odor.     

Herbivory by the insect diaphorina citri?induces greater change in citrus plant volatile profile than does infection by the bacterium,Candidatus Liberibacter asiaticus

The volatile organic compound (VOC) profile in plant leaves often changes after biotic and abiotic stresses. Monitoring changes in VOCs in plant leaves could provide valuable information about multitrophic interactions. In the current study, we investigated the effect of Asian citrus psyllid (ACP) infestation, citrus greening pathogen (Candidatus Liberibacter asiaticus [CLas]) infection, and simultaneous attack by ACP and CLas on the VOC content of citrus leaves. Leaf volatiles were extracted using hexane and analyzed with gas chromatography-mass spectrometry (GC-MS). Although ACP is a phloem-sucking insect that causes minimal damage to plant tissues, the relative amount of 21 out of the 27 VOCs increased 2- to 10-fold in ACP-infested plants. The relative amount of d-limonene, β-phelandrene, citronellal, and undecanal were increased 4- to 20- fold in CLas-infected plants. A principle component analysis (PCA) and cluster analysis (CA) showed that VOC patterns of ACP-infested and CLas-infected plants were different from each other and were also different from the controls, while the VOC pattern of double-attacked plants was more like that of the controls than that of ACP-infested or CLas-infected plants. VOC amounts from leaves were compromised when plants were attacked by ACP and CLas. The results of this study showed that a simple direct extraction of citrus leaf volatiles could be successfully used to discriminate between healthy and CLas-infected plants. Information about the effects of insect and pathogen attack on the VOC content profile of plants might contribute to a better understanding of biotic stress.     

Oral delivery of double-stranded RNAs and siRNAs induces RNAi effects in the potato/tomato psyllid, Bactericerca cockerelli

The potato/tomato psyllid, Bactericerca cockerelli (B. cockerelli), and the Asian citrus psyllid, Diaphorina citri (D. citri), are very important plant pests, but they are also vectors of phloem-limited bacteria that are associated with two devastating plant diseases. B. cockerelli is the vector of Candidatus Liberibacter psyllaurous (solanacearum), which is associated with zebra chip disease of potatoes, and D. citri is the vector of Ca. Liberibacter asiaticus, which is associated with the Huanglongbing (citrus greening) disease that currently threatens the entire Florida citrus industry. Here we used EST sequence information from D. citri to identify potential targets for RNA interference in B. cockerelli. We targeted ubiquitously expressed and gut-abundant mRNAs via injection and oral acquisition of double-stranded RNAs and siRNAs and were able to induce mortality in recipient psyllids. We also showed knockdown of target mRNAs, and that oral acquisition resulted primarily in mRNA knockdown in the psyllid gut. Concurrent with gene knockdown was the accumulation of target specific ～ 21 nucleotide siRNAs for an abundant mRNA for BC-Actin. These results showed that RNAi can be a powerful tool for gene function studies in psyllids, and give support for continued efforts for investigating RNAi approaches as possible tools for psyllid and plant disease control.