亚洲柑橘木虱与柑橘黄龙病菌互作的研究进展

亚洲柑橘木虱Diaphorina citri Kuwayama作为柑橘产业重要病害柑橘黄龙病的主要传播媒介,已经成为重点防治对象。该害虫与黄龙病之间的互作一直是相关研究的热点,本文就近年来该领域的研究进展做了一个总结,从亚洲柑橘木虱的获菌与传病机制、病原菌与柑橘木虱之间的互作以及病原菌感染寄主植物后对木虱的影响等方面进行了综述。期望为深入开展黄龙病相关研究、寻找防控新途径提供依据。     

Proteomic maps of subcellular protein fractions of the Asian citrus psyllid Diaphorina citri,the vector of citrus huanglongbing

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an insect vector that transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas) associated with the destructive citrus disease, citrus huanglongbing (HLB). Currently, D. citri is the major target in HLB management, although insecticidal control and disruption of the D. citri–CLas interactions both face numerous challenges. The present study reports the subcellular proteomic profiles of D. citri, encompassing the three main subcellular protein fractions: cytosol, mitochondria and microsomes. After optimization, subcellular proteins of both high and low abundance are obtained by two‐dimensional gel electrophoresis (2‐DE). A total of 1170 spots are detected in the 2‐DE gels of the three subcellular fractions. One hundred and sixty‐four differentially expressed proteins are successfully identified using liquid chromatography‐dual mass spectroscopy. An efficient protocol for subcellular protein fractionation from D. citri is established and a clear protein separation is achieved with the chosen protein fractionation protocol. The identified cytosolic proteins are mainly metabolic enzymes, whereas a large portion of the identified proteins in the mitochondrial and microsomal fractions are involved in ATP biosynthesis and protein metabolism, respectively. Protein–protein interaction networks are predicted for some identified proteins known to be implicated in pathogen–vector interactions, such as actin, tubulin and ATP synthase, as well as insecticide resistance, such as the cytochrome P450 superfamily. The findings should provide useful information to help identify the mechanism responsible for the CLas–D. citri interactions and eventually contribute to D. citri control.     

Diaphorina citri populations carrying the bacterial plant pathogen Candidatus Liberibacter asiaticus are more susceptible to infection by entomopathogenic fungi than bacteria‐free populations

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Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), and huanglongbing disease do not exist in the Stapleton Station area of the Northern Territory of Australia

Abstract  A series of specimens of the Asian citrus psyllid, Diaphorina citri , collected from the Northern Territory (NT) in 1915 was recently rediscovered in the Natural History Museum, London. Surveys were conducted in 2002 on suitable hosts in the locality of the 1915 collections to see if the infestation had persisted. These failed to detect either D. citri or the bacterium that it transmits and that causes huanglongbing disease in citrus. It is presumed that D. citri was eradicated fortuitously by the removal of all citrus plants above latitude 19°S during an eradication program for citrus canker in the NT from 1916 until 1922.     

Twelve polymorphic microsatellite loci from the Asian citrus psyllid,Diaphorina citri Kuwayama,the vector for citrus greening disease,huanglongbing

Twelve polymorphic microsatellite markers were developed from microsatellite‐enriched DNA libraries and mined from an expressed sequence tags library of Diaphorina citri, the vector of the citrus greening disease (huanglongbing). Analysis of 288 individuals from Florida, Texas, and Brazil showed that allelic diversity ranged from three to eight alleles per locus and observed and expected heterozygosities ranged from 0.014 to 0.569 and from 0.052 to 0.653, respectively. These variable microsatellite loci can provide means for assessing overall genetic variation and migration patterns for this agriculturally important pest species. This information can be used to aid in developing successful management strategies.     

In vitro egg hatching of Diaphorina citri,the vector of Huanglongbing

Citrus greening (Huanglongbing, HLB) is a destructive disease associated with the uncultivable, phloem-limited, gram-negative bacteria Candidatus Liberibacter spp., which affects citrus crops and other Rutaceae species. HLB is rapidly transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). We developed an artificial medium on which D. citri eggs can hatch and first-instar nymphs can feed. The medium could be used to study insect physiology and screen molecules that may interfere with egg hatching and nymph development.     

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.     

柑橘木虱对12种寄主植物嫩梢及其挥发物成分的趋性反应

为了发现对柑橘木虱Diaphorina citri Kuwayama具有引诱活性的寄主植物嫩梢挥发物,采用Y型嗅觉仪在室内测定柑橘木虱对12种寄主植物嫩梢及其挥发物成分的趋性反应,采用固相微萃取技术(SPME)和气-质联用分析技术(GC-MS)分析鉴定嫩梢挥发物的共同成分.结果表明,柑橘木虱对九里香Murraya ex...     

Development of an artificial diet and feeding system for juvenile stages of the Asian citrus psyllid,Diaphorina citri

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the main vector for the plant pathogen Candidatus Liberibacter asiaticus Jagoueix et al. (CLas). To date, attempts to develop an artificial diet for ACP have focused solely on the adult life stage, ignoring the juvenile stages. We developed a feeding system and artificial diet that is compatible with the juvenile stages of ACP and tested growth rates when exposed to varying concentrations of sucrose and amino acids. We found a surprisingly high tolerance for high sucrose concentrations in ACP when compared to known tolerances of sucrose concentrations in other phloem‐feeding insects. This is indicative that ACP may be physiologically adapted to a broad range of sucrose concentrations and osmotic stresses. The growth rate of juvenile ACP was optimized when amino acids were in a global concentration of 150 mM, with higher concentrations not appreciably impacting growth. This finding corresponds with the optimal amino acid concentrations required in other phloem feeders, notably the pea aphid. The development of this feeding system, with the diet optimized for growth, will allow for future experiments to be undertaken into the uptake of CLas by the psyllid and into the nutritional requirements of ACP. This feeding system will also allow for physiological comparisons among the phloem‐feeding insects.     

Citrus Vascular Proteomics Highlights the Role of Peroxidases and Serine Proteases during Huanglongbing Disease Progression

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Highlights

• •HLB is the most devastating citrus disease associated with the pathogen CLas.
• •Proteases and peroxidases accumulated in plant vascular tissue after CLas infection.
• •Dynamic changes in serine protease activity occur across genotypes and environments.

     

Comparative microbiome analysis reveals bacterial communities associated with Candidatus Liberibacter asiaticus infection in the Huanglongbing insect vector Diaphorina citri

The Asian citrus psyllid (Diaphorina citri) is a major pest of the citrus industry and is also the vector for Candidatus Liberibacter asiaticus (CLas), a destructive Huanglongbing (HLB) disease of citrus trees. Insect endosymbionts and gut bacteria play important roles in vector-pathogen interactions and host immunity. Thus, our aim was to evaluate the correlation between CLas infection and the microbiome in D. citri by conducting 16S rRNA amplification sequencing on insects successfully and unsuccessfully infected with CLas (CLas-infected and non-infected). Genera Candidatus Profftella, Wolbachia, and Candidatus Carsonella were highly abundant genera in all tested samples. Compared with the non-infected and control groups, CLas-infected samples harboured more observed OTUs and showed higher alpha diversity metrics. Principal coordinate analysis based on beta-diversity metrics indicated two distinct clusters between the CLas-infected samples and non-infected/control samples. Subsequent LEfSe analysis revealed that Candidatus Profftella was more abundant in the non-infected group than in the control and CLas-infected groups. The interaction network also indicated a co-exclusion relationship between Candidatus Profftella and CLas, while CLas co-existed with Wolbachia, several Enterobacteriaceae spp., and multiple other bacteria. Our study provides insight into the interaction between the microbiome community in D. citri and CLas, which can facilitate the management of this pest and its associated pathogen.     

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.     

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.     

Comparative proteomic analysis between fifth‐instar nymphs and adults of Asian citrus psyllid Diaphorina citri

Asian citrus psyllid Diaphorina citri Kuwayama is extremely problematic worldwide, particularly where Huanglongbing (HLB) disease, the most serious and devastating of citrus diseases, is found. The threat is a result of its ability to transmit the causal agent of HLB, Candidatus Liberibacter asiaticus (CLas) bacterium. Improvements in proteomics, mass spectrometry, bioinformatics tools and gene ontology annotation facilitate the mapping and large‐scale identification and quantification of proteins. To date, only a few comparative proteomic studies report the developmental proteomic changes of hemimetabolous and plant–disease vector insects. Two‐dimensional gel electrophoresis analysis of D. citri total protein is able to detect qualitative and quantitative developmental differences. Liquid chromatography‐tandem mass spectrometry identifies 89 protein spots. Most proteins are metabolism and bioenergetics‐related. Nineteen protein spots are found to be implicated in stress/defence/immunity; 7 in development regulation; 9 in nervous system functions; 4 in the reproductive system; 23 in cytoskeleton and muscle organization; and 4 in movement, flight and other processes. Significant increases in the level of proteins related to structural constitution of the skeleton, stress/defence/immunity, reproduction system, muscles, locomotion and flight are found in adults, consistent with the fact that D. citri is a hemimetabolous insect, whereas proteins involved in developmental regulation are higher in the nymphal stage. The identification of these variably expressed proteins between the nymph and adult stages, linked with the basis of their physiological roles, will lead to a better understanding of the factors influencing development in D. citri and the regulation of some crucial metabolic pathways. It may also help to identify targets for genetic manipulation using RNA interference or other techniques to disrupt Asian citrus psyllid development, lifespan or its ability to transmit CLas.     

Metabolomic analyses of the haemolymph of the Asian citrus psyllid Diaphorina citri,the vector of huanglongbing

The Asian citrus psyllid Diaphorina citri Kuwayama is currently threatening the citrus industry by transmitting the causative agent Candidatus Liberibacter asiaticus (CLas) of huanglongbing. Multiplication of CLas in haemolymph of D. citri indicates that it contains the necessary nutrients for CLas. Although many studies examine D. citri, the haemolymph composition of this dangerous pest remains to be investigated. In the present study, the haemolymph of D. citri is collected using a nanolitre syringe after the removal of one of its forelegs. The haemolymph is either derivatized with methyl chloroformate (MCF) or trimethylsilyl (TMS) derivatizing reagent and analyzed with gas chromatography–mass spectrometry. Nineteen amino acids, two organic acids and seven fatty acids are detected in the haemolymph after MCF derivatization. More metabolites are detected after TMS derivatization. Sugars are the most abundant metabolites in the haemolymph. Glucose and fructose are the main monosaccharides. Trehalose and sucrose are the major disaccharides. Furthermore, three inositol isomers (myo‐inositol, scyllo‐inositol and chiro‐inositol) are detected in the haemolymph. Organic acids are found in low amounts, whereas phosphoric acid is found at a higher concentration. Twenty‐four nucleotides and sugar nucleotides, including ATP, ADP and AMP, are detected using high‐performance anion‐exchange chromatography. Adenine nucleotides are the most abundant nucleotides followed by uridine and guanosine. The adenylate energy charge for the haemolymph is 0.77. Our results show that many metabolites found in the citrus phloem sap are also found in the haemolymph of D. citri.     

不同离体寄主及其成熟度对亚洲柑桔木虱存活的影响

【目的】亚洲柑桔木虱Diaphorina citri Kuwayama的虫口密度及活动与柑桔黄龙病的田间传播、流行有十分密切的关系,寻找合适的室内饲养条件能够便于观察和研究其生物学特性和传病机制。【方法】本文以九里香Murraya exotica(L.)Mant.、酸桔Citrus sunki Hort.ex Tanaka和马水桔Citrus reticulata Blanco.cv.Mashuiju 3种寄主植物不同成熟度离体梢为研究材料,(1)比较亚洲柑桔木虱卵分别在3种寄主植株和离体嫩梢上的孵化率;(2)比较3种寄主不同成熟度离体梢对各龄若虫存活率和蜕皮的影响;(3)比较3种寄主不同成熟度离体梢对成虫存活率的影响。【结果】(1)亚洲柑桔木虱在植株嫩梢上卵的孵化率高于离体嫩梢,九里香表现最明显;(2)低龄若虫在叶片未完全展开的离体嫩梢上存活率最高,而高龄若虫在完全展开的嫩梢上最高;(3)用离体成熟梢饲养柑桔木虱成虫存活率高于离体嫩梢,酸桔和马水桔离体梢饲养的存活率较九里香高。【结论】用寄主植物离体梢饲养的亚洲柑桔木虱卵孵化率和若虫、成虫存活率都较高而且较稳定,该方法可用于这种木虱的室内繁殖中。