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Huanglongbing (HLB) is a fatal citrus disease that is currently threatening citrus varieties worldwide. One putative causative agent, Candidatus Liberibacter asiaticus (CLas), is vectored by Diaphorina citri, known as the Asian citrus psyllid (ACP). Understanding the details of CLas infection in HLB disease has been hindered by its Candidatus nature and the inability to confidently detect it in diseased trees during the asymptomatic stage. To identify early changes in citrus metabolism in response to inoculation of CLas using its natural psyllid vector, leaves from Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) trees were exposed to CLas-positive ACP or CLas-negative ACP and longitudinally analyzed using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry; data available in Dryad: 10.25338/B83H1Z), and metabolomics (proton nuclear magnetic resonance). At 4 weeks postexposure (wpe) to psyllids, the initial HLB plant response was primarily to the ACP and, to a lesser extent, the presence or absence of CLas. Additionally, analysis of 4, 8, 12, and 16 wpe identified 17 genes and one protein as consistently differentially expressed between leaves exposed to CLas-positive ACP versus CLas-negative ACP. This study informs identification of early detection molecular targets and contributes to a broader understanding of vector-transmitted plant pathogen interactions.

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama is the leading vector of Candidatus Liberibacter asiaticus (CLas), the causative agent of citrus Huanglongbing (HLB) disease. The distribution and dynamics of CLas within ACP are critical to understanding how the transmission, spread and infection of CLas occurs within its host vector in nature. In this study, the distribution and titer changes of CLas in various tissues of ACP 5th instar nymphs and adults were examined by fluorescence in situ hybridization (FISH) and real-time quantitative PCR (qPCR) techniques. Results demonstrated that 100% of ACP 5th instar nymphs and adults were infected with CLas following feeding on infected plants, and that CLas had widespread distribution in most of the tissues of ACP. The titers of CLas within the midgut, salivary glands and hemolymph tissues were the highest in both 5th instar nymphs and adults. When compared with adults, the titers of CLas in these three tissues of 5th instar nymphs were significantly higher, while in the mycetome, ovary and testes they were significantly lower than those of adults. FISH visualization further confirmed these findings. Dynamic analysis of CLas demonstrated that it was present across all the developmental ages of ACP adults. There was a discernible upward trend in the presence of CLas with advancing age in most tissues of ACP adults, including the midgut, hemolymph, salivary glands, foot, head, cuticula and muscle. Our findings have significant implications for the comprehensive understanding of the transmission, dissemination and infestation of CLas, which is of much importance for developing novel strategies to halt the spread of CLas, and therefore contribute to the efficient prevention and control of HLB. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid, Diaphorina citri, is the primary vector of the HLB pathogen, Candidatus Liberibacter asiaticus (CLas). The acquisition of CLas shortens the developmental period of nymphs, accelerating the emergence into adulthood and thereby facilitating the spread of CLas. Cuticular proteins (CPs) are involved in insect emergence. In this study, we investigated the molecular mechanisms underlying CLas‐promoted emergence in D. citri via CP mediation. Here, a total of 159 CP genes were first identified in the D. citri genome. Chromosomal location analysis revealed an uneven distribution of these CP genes across the 13 D. citri chromosomes. Proteomic analysis identified 54 differentially expressed CPs during D. citri emergence, with 14 CPs exhibiting significant differential expression after CLas acquisition. Five key genes, Dc18aa‐1, Dc18aa‐2, DcCPR‐24, DcCPR‐38 and DcCPR‐58, were screened from the proteome and CLas acquisition. The silencing of these 5 genes through a modified feeding method significantly reduced the emergence rate and caused various abnormal phenotypes, indicating the crucial role that these genes play in D. citri emergence. This study provides a comprehensive overview of the role of CPs in D. citri and reveals that CLas can influence the emergence process of D. citri by regulating the expression of CPs. These key CPs may serve as potential targets for future research on controlling huanglongbing (HLB) transmission. [ABSTRACT FROM AUTHOR]

Extensive microbial interactions occur within insect hosts. However, the interactions between the Huanglongbing (HLB) pathogen and endosymbiotic bacteria within the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) in wild populations remain elusive. Thus, this study aimed to detect the infection rates of HLB in the ACP across five localities in China, with a widespread prevalence in Ruijin (RJ, 58%), Huidong (HD, 28%), and Lingui (LG, 15%) populations. Next, microbial communities of RJ and LG populations collected from citrus were analyzed via 16S rRNA amplicon sequencing. The results revealed a markedly higher microbial diversity in the RJ population compared to the LG population. Moreover, the PCoA analysis identified significant differences in microbial communities between the two populations. Considering that the inter-population differences of Bray–Curtis dissimilarity in the RJ population exceeded those between populations, separate analyses were performed. Our findings indicated an increased abundance of Enterobacteriaceae in individuals infected with HLB in both populations. Random forest analysis also identified Enterobacteriaceae as a crucial indicator of HLB infection. Furthermore, the phylogenetic analysis suggested a potential regulatory role of ASV4017 in Enterobacteriaceae for ACP, suggesting its possible attractant activity. This research contributes to expanding the understanding of microbial communities associated with HLB infection, holding significant implications for HLB prevention and treatment. [ABSTRACT FROM AUTHOR]

The evolution of insect vector‐pathogen relationships has long been of interest in the field of molecular ecology. One system of special relevance, due to its economic impacts, is that between Diaphorina citri and 'Candidatus Liberibacter asiaticus' (CLas), the cause of the severe Asian form of huanglongbing. CLas‐positive D. citri are more fecund than their CLas‐negative counterparts, boosting opportunities for pathogens to acquire new vector hosts. The molecular mechanism behind this life‐history shift remains unclear. Here, we found that CLas promoted ovarian development and increased the expression of the vitellogenin receptor (DcVgR) in ovaries. DcVgR RNAi significantly decreased fecundity and CLas titer in ovaries, extended the preoviposition period, shortened the oviposition period and blocked ovarian development. Given their importance in gene regulation, we explored the role of miRNAs in shaping these phenotypes and their molecular triggers. Our results showed that one miRNA, miR‐275, suppressed DcVgR expression by binding to its 3' UTR. Overexpression of miR‐275 knocked down DcVgR expression and CLas titer in ovaries, causing reproductive defects that mimicked DcVgR knockdown phenotypes. We focused, further, on roles of the Juvenile Hormone (JH) pathway in shaping the observed fecundity phenotype, given its known impacts on ovarian development. After CLas infection, this pathway was upregulated, thereby increasing DcVgR expression. From these combined results, we conclude that CLas hijacks the JH signalling pathway and miR‐275, thereby targeting DcVgR to increase D. citri fecundity. These changes simultaneously increase CLas replication, suggesting a pathogen‐vector host mutualism, or a seemingly helpful, but cryptically costly life‐history manipulation. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide. It is associated with the non-culture bacteria Candidatus Liberibacter spp., which can be transmitted by grafting and/or the psyllid vectors Diaphorina citri (ACP) and Trioza erytreae (AfCP). Although HLB has not been reported in the Mediterranean Basin to date, both vectors are present, and thus represent a serious threat to the citrus industry in this region. Resistant citrus cultivars or effective therapeutic treatments are not currently available for HLB. Nevertheless, area-wide pest management via coordinated management efforts over large areas has been implemented in Brazil, China and the USA for HLB control. This study proposes an open access flexible methodology to address area-wide management of both HLB vectors in the Mediterranean Basin. Based on a risk-based approach which considers climatic information and other variables that may influence vector introduction and spread, such as conventional, organic, abandoned and residential citrus areas as well as transportation corridors, an area-wide management division in pest management areas (PMAs) is proposed. The size and location of these PMAs were estimated by means of a hierarchical clustering algorithm with spatial constraints whose performance was assessed under different configuration scenarios. This proposal may assist policymakers and the citrus industry of the citrus-growing areas of the Mediterranean Basin in risk management planning in the case of the spread of HLB vectors or a possible introduction of the disease. Additionally, it may be a valuable resource to inform opinion dynamic models, enabling the identification of pivotal factors for the success of control measures. [ABSTRACT FROM AUTHOR]

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the bacteria Candidatus Liberibacter americanus (CLam) and Candidatus Liberibacter asiaticus (CLas), which are phloem-restricted and associated with the most important and destructive worldwide citrus disease, Huanglongbing (HLB). Currently, no cure for HLB has been described. Therefore, measures have focused on reducing D. citri populations. In these insects, cathepsin B (DCcathB) and L (DCcathL) enzymes play an important role in digestion, and are involved in embryogenesis, immune defense, and ecdysis. In this study, we used a CTV-based vector to deliver dsRNA (CTV-dsRNA) into Citrus macrophylla plants targeting DCcathB and DCcathL genes in D. citri that fed on the phloem of these CTV-RNAi infected plants. Subsequently, we evaluated expression of DCcathB and DCcathL genes as well as the Vitellogenin (Vg) gene by RT-qPCR in D. citri fed on CTVdsRNA occurring in plant phloem. It was found that a defective phenotype in D. citri females as a result of knockdown of DCcathB and DCcathL genesmediated by CTV dsRNA. These results showed that Psyllids fed on plants treated with the CTV-dsRNA exhibited downregulation of the Vg gene, one of the most important genes associated with embryogenic and female development, which was associated with dsRNA-mediated silencing of the two cathepsin genes. Based on our findings, a CTV-based strategy for delivering RNAi via plants that targets DCcathB and DCcathL genesmay represent a suitable avenue for development of dsRNA-based tools to manage D. citri that limits the spread of HLB. [ABSTRACT FROM AUTHOR]

Extensive microbial interactions occur within insect hosts. However, the interactions between the Huanglongbing (HLB) pathogen and endosymbiotic bacteria within the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) in wild populations remain elusive. Thus, this study aimed to detect the infection rates of HLB in the ACP across five localities in China, with a widespread prevalence in Ruijin (RJ, 58%), Huidong (HD, 28%), and Lingui (LG, 15%) populations. Next, microbial communities of RJ and LG populations collected from citrus were analyzed via 16S rRNA amplicon sequencing. The results revealed a markedly higher microbial diversity in the RJ population compared to the LG population. Moreover, the PCoA analysis identified significant differences in microbial communities between the two populations. Considering that the inter-population differences of Bray–Curtis dissimilarity in the RJ population exceeded those between populations, separate analyses were performed. Our findings indicated an increased abundance of Enterobacteriaceae in individuals infected with HLB in both populations. Random forest analysis also identified Enterobacteriaceae as a crucial indicator of HLB infection. Furthermore, the phylogenetic analysis suggested a potential regulatory role of ASV4017 in Enterobacteriaceae for ACP, suggesting its possible attractant activity. This research contributes to expanding the understanding of microbial communities associated with HLB infection, holding significant implications for HLB prevention and treatment.

Huanglongbing (HLB) is a devastating disease that has led to an acute crisis for growers of citrus, one of the world's most important fruit crops. The phloem-feeding Asian citrus psyllid (ACP), Diaphorina citri, is the main pest at the new shoot stage and is the only natural vector of HLB pathogenic bacteria. Little is known about how plants perceive and defend themselves from this destructive pest. Here, we characterized changes in the expression of various genes in citrus plants that were continuously infested by D. citri for different durations (12, 24, and 48 h). A total of 5 219 differentially expressed genes (DEGs) and 643 common DEGs were identified across all time points. Several pathways related to defense were activated, such as peroxisome, alpha-linolenic acid metabolism, and phenylpropanoid and terpenoid biosynthesis, and some pathways related to growth and signal transduction were suppressed in response to D. citri infestation. The expression of genes including kinases (CML44, CIPK6, and XTH6), phytohormones (SAMT, LOX6, and NPR3), transcription factors (bHLH162, WRKY70, and WRKY40), and secondary metabolite synthesis-related genes (PAL, 4CL2, UGT74B1 and CYP82G1) was significantly altered in response to D. citri infestation. The findings of this study greatly enhance our understanding of the mechanisms underlying the defense response of citrus plants to D. citri infestation at the molecular level. Functional characterization of the candidate defense-related genes identified in this study will aid the molecular breeding of insect-resistant citrus varieties.

Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide. It is associated with the non-culture bacteria Candidatus Liberibacter spp., which can be transmitted by grafting and/or the psyllid vectors Diaphorina citri (ACP) and Trioza erytreae (AfCP). Although HLB has not been reported in the Mediterranean Basin to date, both vectors are present, and thus represent a serious threat to the citrus industry in this region. Resistant citrus cultivars or effective therapeutic treatments are not currently available for HLB. Nevertheless, area-wide pest management via coordinated management efforts over large areas has been implemented in Brazil, China and the USA for HLB control. This study proposes an open access flexible methodology to address area-wide management of both HLB vectors in the Mediterranean Basin. Based on a risk-based approach which considers climatic information and other variables that may influence vector introduction and spread, such as conventional, organic, abandoned and residential citrus areas as well as transportation corridors, an area-wide management division in pest management areas (PMAs) is proposed. The size and location of these PMAs were estimated by means of a hierarchical clustering algorithm with spatial constraints whose performance was assessed under different configuration scenarios. This proposal may assist policymakers and the citrus industry of the citrus-growing areas of the Mediterranean Basin in risk management planning in the case of the spread of HLB vectors or a possible introduction of the disease. Additionally, it may be a valuable resource to inform opinion dynamic models, enabling the identification of pivotal factors for the success of control measures.

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the bacteria Candidatus Liberibacter americanus (CLam) and Candidatus Liberibacter asiaticus (CLas), which are phloem-restricted and associated with the most important and destructive worldwide citrus disease, Huanglongbing (HLB). Currently, no cure for HLB has been described. Therefore, measures have focused on reducing D. citri populations. In these insects, cathepsin B (DCcathB) and L (DCcathL) enzymes play an important role in digestion, and are involved in embryogenesis, immune defense, and ecdysis. In this study, we used a CTV-based vector to deliver dsRNA (CTV-dsRNA) into Citrus macrophylla plants targeting DCcathB and DCcathL genes in D. citri that fed on the phloem of these CTV-RNAi infected plants. Subsequently, we evaluated expression of DCcathB and DCcathL genes as well as the Vitellogenin (Vg) gene by RT-qPCR in D. citri fed on CTV-dsRNA occurring in plant phloem. It was found that a defective phenotype in D. citri females as a result of knockdown of DCcathB and DCcathL genes mediated by CTV dsRNA. These results showed that Psyllids fed on plants treated with the CTV-dsRNA exhibited downregulation of the Vg gene, one of the most important genes associated with embryogenic and female development, which was associated with dsRNA-mediated silencing of the two cathepsin genes. Based on our findings, a CTV-based strategy for delivering RNAi via plants that targets DCcathB and DCcathL genes may represent a suitable avenue for development of dsRNA-based tools to manage D. citri that limits the spread of HLB.

Huanglongbing (HLB) is a global devastating citrus disease that is mainly caused by "Candidatus Liberibacter asiaticus" (CLas). It is mostly transmitted by the insect Asian citrus psyllid (ACP, Diaphorina citri) in a persistent and proliferative manner. CLas traverses multiple barriers to complete an infection cycle and is likely involved in multiple interactions with D. citri. However, the protein-protein interactions between CLas and D. citri are largely unknown. Here, we report on a vitellogenin-like protein (Vg_VWD) in D. citri that interacts with a CLas flagellum (flaA) protein. We found that Vg_VWD was upregulated in CLas-infected D. citri. Silencing of Vg_VWD in D. citri via RNAi silencing significantly increased the CLas titer, suggesting that Vg_VWD plays an important role in the CLas-D. citri interaction. Agrobacterium-mediated transient expression assays indicated that Vg_VWD inhibits BAX- and INF1-triggered necrosis and suppresses the callose deposition induced by flaA in Nicotiana benthamiana. These findings provide new insights into the molecular interaction between CLas and D. citri. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB), or citrus greening, is the most destructive disease of cultivated citrus worldwide. Candidatus Liberibacter asiaticus (C Las), the putative causal agent of HLB, is transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). In Florida, D. citri was first reported in 1998, and C Las was confirmed in 2005. Management of HLB relies on the use of insecticides to reduce vector populations. In 2016, antibiotics were approved to manage C Las infection in citrus. Diaphorina citri is host to several bacterial endosymbionts and reducing endosymbiont abundance is known to cause a corresponding reduction in host fitness. We hypothesized that applications of oxytetracycline and streptomycin would reduce: C Las populations in young and mature citrus trees, C Las acquisition by D. citri , and D. citri abundance. Our results indicate that treatment of citrus with oxytetracycline and streptomycin reduced acquisition of C Las by D. citri adults and emerging F1 nymphs as compared with that observed in trees treated only with insecticides, but not with antibiotics. However, under field conditions, neither antibiotic treatment frequency tested affected C Las infection of young or mature trees as compared with insecticide treatment alone (negative control); whereas trees enveloped with mesh screening that excluded vectors did prevent bacterial infection (positive control). Populations of D. citri were not consistently affected by antibiotic treatment under field conditions, as compared with an insecticide only comparison. Collectively, our results suggest that while foliar application of oxytetracycline and streptomycin to citrus reduces acquisition of C Las bacteria by the vector, even high frequency applications of these formulations under field conditions do not prevent or reduce tree infection. [ABSTRACT FROM AUTHOR]

The citrus industry of Florida faces insurmountable challenges against the destructive diseases citrus tristeza and Huanglongbing (HLB, or citrus greening). Though the tristeza causal agent, citrus tristeza virus (CTV), has been in Florida decades longer than HLB, growers have concentrated most of their efforts on combating the more detrimental HLB. The Asian citrus psyllid (Diaphorina citri; ACP) is the insect vector of the bacterial pathogen Candidatus Liberibacter asiaticus and transmits the incurable HLB to all commercial citrus. During our searches for biological and viral controls against the ACP, we consistently detected sequences of CTV in Florida field populations of ACP. This unexpected finding led us to investigate whether ACPs collected from young shoots could be used as a tool to survey CTV in Florida citrus groves. We first surveyed for the most common CTV strains in Florida (T30, T36, and VT/T68) in citrus trees on mostly sour orange (Citrus aurantium) rootstock, the rootstock susceptible to CTV decline. Out of 968 trees sampled across five years (2018-2022), approximately 8.2% were positive for CTV, with more than half of the CTV-positive trees infected with strain T30. Simultaneously, we looked at CTV strains in ACPs during this time and found that approximately 88% of pooled adult and nymph ACPs also had CTV, with over half the positive samples having the T36 strain. As a result of the much higher CTV incidences in the ACPs, we conducted a second investigation into whether we could more easily detect the same CTV strains in ACP nymphs as in CTV-infected citrus tissue. After individually sampling 43 trees and pooling the nymphs from each tree, we detected CTV at about the same incidence in the citrus tissue and the nymphs, but with much less ACP tissue, time, and resources required for detection compared to citrus tissue. Results from this study illustrate the sustained threat of CTV to Florida citrus and demonstrate the ACP as a potential bioindicator for CTV. K [ABSTRACT FROM AUTHOR]

Simple Summary: The severe Asian form of huanglongbing is the most serious disease of citrus in Malaysia. It is caused by a phloem-limited bacterium 'Candidatus Liberibacter asiaticus' and transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama. Minimising the devastating impacts of the disease requires effective integrated pest management (IPM) programs to limit detrimental environmental and health consequences of over-reliance on synthetic pesticides. Key components of such programs include grower awareness, planting of pathogen-free trees, monitoring psyllid and disease incidence, removal of diseased trees, and judicious use of insecticides, mineral oils, and natural enemies to suppress incidence of the psyllid. Significant progress has been made towards implementing the programs. The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, transmits 'Candidatus Liberibacter asiaticus' (CLas), a phloem-limited bacterium associated with the severe Asian form of huanglongbing (HLB), and the most destructive disease of citrus. The pathogen and the psyllid, both of South Asian origin, are now widespread in citrus regions of Asia and the Americas. There is no cure for the disease. Application of synthetic pesticides, in some instances more frequently than fortnightly, to minimise incidence of ACP in citrus orchards, has not prevented inevitable impacts of the disease in regions of Asia where CLas is present. Despite the inevitable spread of the disease, significant progress has been made in Sarawak since the mid-1990s towards effectively implementing integrated pest management (IPM) programs for stemming the impact of the disease and detrimental consequences of over-reliance on synthetic pesticides. Growers are encouraged to plant pathogen-free trees, remove diseased trees, monitor incidence of the psyllid, and to use pesticides judiciously to reduce their detrimental impacts on natural enemies. Knowledge has been enhanced through research on seasonal incidence of the psyllid, use of mineral oils, development of protocols and iodine–starch test kits for detecting infected trees, PCR for confirming the presence of CLas in symptomatic leaves, methods for monitoring incidence the psyllid, and training extension staff and growers. However, major impediments to increasing the average longevity of trees beyond <5 years in poorly managed orchards, based on marcotting (air layering), and >12 years in well-managed orchards, based on pathogen-free trees, still need to be addressed. These include grower knowledge, marcotting, aggressive marketing of synthetic pesticides, high prices of mineral oils, spray application procedures, and better reliance on natural enemies of the psyllid. [ABSTRACT FROM AUTHOR]

The citrus industry of Florida faces insurmountable challenges against the destructive diseases citrus tristeza and Huanglongbing (HLB, or citrus greening). Though the tristeza causal agent, citrus tristeza virus (CTV), has been in Florida decades longer than HLB, growers have concentrated most of their efforts on combating the more detrimental HLB. The Asian citrus psyllid (Diaphorina citri; ACP) is the insect vector of the bacterial pathogen Candidatus Liberibacter asiaticus and transmits the incurable HLB to all commercial citrus. During our searches for biological and viral controls against the ACP, we consistently detected sequences of CTV in Florida field populations of ACP. This unexpected finding led us to investigate whether ACPs collected from young shoots could be used as a tool to survey CTV in Florida citrus groves. We first surveyed for the most common CTV strains in Florida (T30, T36, and VT/T68) in citrus trees on mostly sour orange (Citrus aurantium) rootstock, the rootstock susceptible to CTV decline. Out of 968 trees sampled across five years (2018-2022), approximately 8.2% were positive for CTV, with more than half of the CTV-positive trees infected with strain T30. Simultaneously, we looked at CTV strains in ACPs during this time and found that approximately 88% of pooled adult and nymph ACPs also had CTV, with over half the positive samples having the T36 strain. As a result of the much higher CTV incidences in the ACPs, we conducted a second investigation into whether we could more easily detect the same CTV strains in ACP nymphs as in CTV-infected citrus tissue. After individually sampling 43 trees and pooling the nymphs from each tree, we detected CTV at about the same incidence in the citrus tissue and the nymphs, but with much less ACP tissue, time, and resources required for detection compared to citrus tissue. Results from this study illustrate the sustained threat of CTV to Florida citrus and demonstrate the ACP as a potential bioindicator for CTV.

This system was not well characterized in I D. citri. i Our comprehensive in silico and bioinformatic analyzes using protein-protein BLAST of I Drosophila melanogaster i as a model against I D. citri i in the NCBI and I Diaphorina citri i OGS v2.0 protein databases revealed seven Toll pathway-related genes in I D. citri. In addition, the presence of a death domain in the Tube protein of I D. citri i and I D. melanogaster i does not require their association with a protein kinase family protein, as death domains are observed in many proteins lacking catalytic function or a protein kinase domain, and this result agrees with findings by Towb et al. [[78]]. The BLASTp search of I D. melanogaster i produced significant similarities to the embryonic polarity protein Dorsal-like isoform X1 (XP 017305228.1), embryonic polarity protein Dorsal-like isoform X2 (XP 026675878.1) and embryonic polarity protein Dorsal-like isoform X3 (XP 017305229.1). In our protein-protein BLAST search of the Dif/dI protein sequence from I D. melanogaster i against the I D. citri i , both genes hit the same protein of embryonic polarity protein Dorsal-like. The protein sequences from Pelle gene were identified using protein-protein BLAST (BLASTp) using Pelle gene (Pll, AAA28750.1) from D. melanogaster, as a query sequence against D. citri and other insects available in GeneBank, the National Center for Biotechnology Information website (NCBI,, 15 April 2021). [Extracted from the article]

Abstract Background The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an insect pest species responsible for damages of citrus fruit quality and tree health. This insect is a vector of bacteria ‘Candidatus Liberibacter’ spp. a putative causal agent of citrus greening disease known as Huanglongbing (HLB), considered one of the most destructive diseases of citrus orchards worldwide. Disease management is mainly based on vector control using pesticides which can affect natural enemies that play an important role in pest control. The entomopathogenic fungi (EPF) Beauveria bassiana (2067 and 2121) and Metarhizium anisopliae (2411) were achieved by applying a suspension of 1 × 107 spores against D. citri nymphs and artificially infected the citrus seedlings under controlled and semi-field conditions. Also, the activity of these EPFs on the coccinellid and lacewing predators was evaluated. The effects of the main chemicals used in citrus plantations on the selected fungi for possible combined use was also investigated. Results The obtained results showed that under controlled conditions, the percentage of insect mortality produced by EPF varied between 82.8 and 85.9%. Under semi-field conditions, the infection and mortality of D. citri caused by the M. anisopliae 2411 strain was significantly higher than that of B. bassiana 2067, 78.9 and 51%, respectively. Non-significant effect of EPF on the natural enemies of D. citri was observed, suggesting that EPF and predators had compatible effects. Conclusion This study clearly demonstrated that EPF were able to kill D. citri and can be explored as a promissory biocontrol candidate. Simultaneous use in an integrated pest management program could be possible by applying EPF combined with pesticides.

[Display omitted] • The toxicity of 17 pesticidal proteins was tested against D. citri adults. • Of these Tpp78Aa1 was toxic to D. citri adults with an LC 50 of 204 µg/mL. • CLas infection did not impact Tpp78Aa1 toxicity to D. citri. The Asian citrus psyllid (ACP) Diaphorina citri transmits the causative agent of huanglongbing, or citrus greening disease, that has decimated global citrus production. Pesticidal proteins derived from bacteria such as Bacillus thuringiensis (Bt) can provide effective and environmentally friendly alternatives for management of D. citri , but few with sufficient toxicity to D. citri have been identified. Here, we report on the toxicity of 14 Bt-derived pesticidal proteins from five different structural groups against D. citri. These proteins were selected based on previously reported toxicity to other hemipteran species and on pesticidal protein availability. Most of the proteins were expressed in Escherichia coli and purified from inclusion bodies or His-tag affinity purification, while App6Aa2 was expressed in Bt and purified from spore/crystal mixtures. Pesticidal proteins were initially screened by feeding psyllids on a single dose, and lethal concentration (LC 50) then determined for proteins with significantly greater mortality than the buffer control. The impact of CLas infection of D. citri on toxicity was assessed for selected proteins via topical feeding. The Bt protein Tpp78Aa1 was toxic to D. citri adults with an LC 50 of approximately 204 µg/mL. Nymphs were more susceptible to Tpp78Aa1 than adults but no significant difference in susceptibility was observed between healthy and CLas-infected nymphs or adults. Tpp78Aa1 and other reported D. citri -active proteins may provide valuable tools for suppression of D. citri populations. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis (Bt) can be effective in controlling insect pests either in spray products or by their toxins expressed in transgenic crops. The discovery of Bt as endophytic in citrus opened new perspectives for the control of Diaphorina citri Kuwayama, the vector of the bacteria Candidatus Liberibacter spp. associated with Huanglongbing. In this study, the endophytic translocation of Bt in citrus seedlings (small and large plants) and in different scion-rootstock combinations were investigated. We also assessed the lethal concentration of D. citri nymphs caused by systemic Bt in citrus and, moreover, the binding of Cry toxins with the Asian citrus psyllid (ACP) gut receptors. Endophytic translocation of Bt in citrus seedlings and nursery trees was confirmed by colony isolation, PCR detection, and Btk-GFP microscopy analyses, which demonstrated that, after Bt inoculation into the substrate, the bacteria enter through lateral root emergence sites and are localized in stem xylem vessel elements. Mean lethal concentrations for D. citri third instar nymphs were 4.92 × 104 and 2.19 × 104 spores mL−1 for S1450 and S1302 strains, respectively. Nymphal mortality induced by the bacteria was higher in citrus seedlings than in citrus nursery trees. All Cry-toxins tested were able to bind to D. citri brush border membrane vesicles, suggesting the potential pathogenicity of Bt strains against psyllid. This study clearly demonstrates the penetration and Bt translocation in the xylem of small and large citrus plants and the potential of the bacteria to cause ACP nymphs mortality that fed on shoots from Bt-treated citrus. Bacillus thuringiensis (Bt) as endophytic in citrus plants has a potential to control the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. In this study, the endophytic translocation and pathogenicity of Bt against ACP nymphs feeding on citrus under different citrus scion-rootstock combinations were assessed, and Bt-toxins binding with D. citri gut receptors, were investigated. Our results clearly demonstrated the penetration and endophytic Bt translocation in the citrus vessels and the potential of the Bt to control psyllid nymphs that fed on shoots from citrus Bt-treated. [Display omitted] • Bacillus thuringiensis strains endophytically translocate in the xylem vessel elements of citrus seedlings and citrus nursery trees grafted on different scion-rootstocks combinations. • Cry toxins bind to brush border membrane vesicles obtained from D. citri nymphs. • Bt systemic product could be an alternative tool for the D. citri management programs. [ABSTRACT FROM AUTHOR]

Background: Plant immunity against pathogens and pests is comprised of complex mechanisms orchestrated by signaling pathways regulated by plant hormones [Salicylic acid (SA) and Jasmonic acid (JA)]. Investigations of plant immune response to phytopathogens and phloem-feeders have revealed that SA plays a critical role in reprogramming of the activity and/or localization of transcriptional regulators via post-translational modifications. We explored the contributing effects of herbivory by a phytopathogen vector [Asian citrus psyllid, Diaphorina citri] and pathogen [Candidatus Liberibacter asiaticus (CaLas)] infection on response of sweet orange [Citrus sinensis (L.) Osbeck] using manipulative treatments designed to mimic the types of infestations/infections that citrus growers experience when cultivating citrus in the face of Huanglongbing (HLB) disease. Results: A one-time (7 days) inoculation access period with CaLas-infected vectors caused SA-associated upregulation of PR-1, stimulating defense response after a long period of infection without herbivory (270 and 360 days). In contrast, while repeated (monthly) 'pulses' of 7 day feeding injury by psyllids stimulated immunity in CaLas-infected citrus by increasing SA in leaves initially (up to 120 days), long-term (270 and 360 days) repeated herbivory caused SA to decrease coincident with upregulation of genes associated with SA metabolism (BMST and DMR6). Similarly, transcriptional responses and metabolite (SA and its analytes) accumulation in citrus leaves exposed to a continuously reproducing population of D. citri exhibited a transitory upregulation of genes associated with SA signaling at 120 days and a posterior downregulation after long-term psyllid (adults and nymphs) feeding (270 and 360 days). Conclusions: Herbivory played an important role in regulation of SA accumulation in mature leaves of C. sinensis, whether or not those trees were coincidentally infected with CaLas. Our results indicate that prevention of feeding injury inflicted by D. citri from the tritrophic interaction may allow citrus plants to better cope with the consequences of CaLas infection, highlighting the importance of vector suppression as a component of managing this cosmopolitan disease. [ABSTRACT FROM AUTHOR]

Citrus greening, or huanglongbing (HLB), currently is the most destructive disease of citrus. HLB disease is putatively caused by the phloem-restricted α-proteobacterium ‘Candidatus Liberibacter asiaticus’. This bacterium is transmitted primarily by the Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae). Most animal pathogens are considered pathogenic to their insect vectors, whereas the relationships between plant pathogens and their insect vectors are variable. Lately, the relationship of ‘Ca. L. asiaticus’ with its insect vector, D. citri, has been well investigated at the molecular, biochemical, and biological levels in many studies. Herein, the findings concerning this relationship are discussed and molecular features of the acquisition of ‘Ca. L. asiaticus’ from the plant host and its growth and circulation within D. citri, as well as its transmission to plants, are presented. In addition, the effects of ‘Ca. L. asiaticus’ on the energy metabolism (respiration, tricarboxylic acid cycle, and adenosine triphosphate production), metabolic pathways, immune system, endosymbionts, and detoxification enzymes of D. citri are discussed together with other impacts such as shorter lifespan, altered feeding behavior, and higher fecundity. Overall, although ‘Ca. L. asiaticus’ has significant negative effects on its insect vector, it increases its vector fitness, indicating that it develops a mutualistic relationship with its vector. This review will help in understanding the specific interactions between ‘Ca. L. asiaticus’ and its psyllid vector in order to design innovative management strategies. [ABSTRACT FROM AUTHOR]

Background Huanglongbing, a devastating disease of citrus, is caused by the obligate, intracellular bacterium "Candidatus Liberibacter asiaticus" (C Las). C Las is transmitted by Diaphorina citri , the Asian citrus psyllid. Development of transmission-blocking strategies to manage huanglongbing relies on knowledge of C Las and D. citri interactions at the molecular level. Prior transcriptome analyses of D. citri point to changes in psyllid biology due to C Las infection but have been hampered by incomplete versions of the D. citri genome, proper host plant controls, and/or a lack of a uniform data analysis approach. In this work, we present lessons learned from a quantitative transcriptome analysis of excised heads, salivary glands, midguts, and bacteriomes from C Las-positive and C Las-negative D. citri using the chromosomal length D. citri genome assembly. Results Each organ had a unique transcriptome profile and response to C Las infection. Though most psyllids were infected with the bacterium, C Las-derived transcripts were not detected in all organs. By analyzing the midgut dataset using both the Diaci_v1.1 and v3.0 D. citri genomes, we showed that improved genome assembly led to significant and quantifiable differences in RNA-sequencing data interpretation. Conclusions Our results support the hypothesis that future transcriptome studies on circulative, vector-borne pathogens should be conducted at the tissue-specific level using complete, chromosomal-length genome assemblies for the most accurate understanding of pathogen-induced changes in vector gene expression. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, transmits ‘Candidatus Liberibacter asiaticus’ (CLas), a phloem-limited bacterium associated with the severe Asian form of huanglongbing (HLB), and the most destructive disease of citrus. The pathogen and the psyllid, both of South Asian origin, are now widespread in citrus regions of Asia and the Americas. There is no cure for the disease. Application of synthetic pesticides, in some instances more frequently than fortnightly, to minimise incidence of ACP in citrus orchards, has not prevented inevitable impacts of the disease in regions of Asia where CLas is present. Despite the inevitable spread of the disease, significant progress has been made in Sarawak since the mid-1990s towards effectively implementing integrated pest management (IPM) programs for stemming the impact of the disease and detrimental consequences of over-reliance on synthetic pesticides. Growers are encouraged to plant pathogen-free trees, remove diseased trees, monitor incidence of the psyllid, and to use pesticides judiciously to reduce their detrimental impacts on natural enemies. Knowledge has been enhanced through research on seasonal incidence of the psyllid, use of mineral oils, development of protocols and iodine–starch test kits for detecting infected trees, PCR for confirming the presence of CLas in symptomatic leaves, methods for monitoring incidence the psyllid, and training extension staff and growers. However, major impediments to increasing the average longevity of trees beyond 12 years in well-managed orchards, based on pathogen-free trees, still need to be addressed. These include grower knowledge, marcotting, aggressive marketing of synthetic pesticides, high prices of mineral oils, spray application procedures, and better reliance on natural enemies of the psyllid.

Huanglongbing (HLB) is a devastating citrus disease worldwide. A three-pronged approach to controlling HLB has been suggested, namely, removal of HLB-symptomatic trees, psyllid control, and replacement with HLB-free trees. However, such a strategy did not lead to successful HLB control in many citrus-producing regions, such as Florida. We hypothesize that this is because of the small-scale or incomprehensive implementation of the program; conversely, a comprehensive implementation of such a strategy at the regional level can successfully control HLB. To test our hypothesis, we investigated the effects of region-wide comprehensive implementation of this scheme to control HLB in Gannan region, China, with a total planted citrus acreage of over 110,000 ha from 2013 to 2019. With the region-wide implementation of comprehensive HLB management, the overall HLB incidence in Gannan decreased from 19.71% in 2014 to 3.86% in 2019. A partial implementation of such a program (without a comprehensive inoculum removal) at the regional level in Brazil resulted in HLB incidence increasing from 1.89% in 2010 to 19.02% in 2019. Using dynamic regression model analyses with data from both Brazil and China, we constructed a model to predict HLB incidence when all three components were applied at 100%. It was predicated that in a region-wide comprehensive implementation of such a program, HLB incidence would be controlled to a level of less than 1%. We conducted economic feasibility analyses and showed that average net profits were positive for groves that implemented the comprehensive strategy, but groves that did not implement it had negative net profits over a 10-year period. Overall, the key for the three-pronged program to successfully control HLB is the large scale (region-wide) and comprehensiveness in implementation. This study provides valuable information to control HLB and other economically important endemic diseases worldwide. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid, Diaphorina citri is the main vector of citrus greening disease, also known as Huanglongbing (HLB). Currently, mitigating HLB depends on the control of D. citri using insecticides. To design innovative control strategies, we should investigate various biological aspects of D. citri at the molecular level. Herein we explored the Toll signaling system-related proteins in D. citri using in silico analyzes. Additionally, the transcripts of the identified genes were determined in all life stages from eggs to adults. Our findings reveal that D. citri genome possesses Toll signaling pathway-related genes similar to the insect model, Drosophila melanogaster, with slight differences. These genes include cact, TI, Myd88, Dif/DI, pll, tub, and spz encoding Cactus, Toll, Myeloid differentiation factor 88, Dorsal related immunity factor/Dorsal, Pelle, Tube, and Spaetzle, respectively. Unlike D. melanogaster, in D. citri Dorsal, immunity factor and Dorsal are the same protein. In addition, in D. citri, Pelle protein possesses a kinase domain, which is absent in Pelle of D. melanogaster. Gene expression analysis showed the transcript for cact, TI, Myd88, pll, tub, and spz are maximum in adults, suggesting the immunity increases with maturity. Instead, Dif/DI transcripts were maximal in eggs and adults and minimal in nymphal stages, indicating its role in embryonic development. The overall findings will help in designing pioneering control strategies of D. citri based on repressing its immunity by RNAi or CRISPR and combining that with biological control.

Asian Citrus Psyllid (ACP), Diaphorina citri, is a key vector transmitting the causative agent of Huanglongbing (HLB) disease. Population growth of ACP is evident after feeding on plant flush shoots, as they only oviposit here. However, the underlying mechanism as to why flush shoots govern oviposition is unclear. This study compares the fecundity and ovarian morphology of ACP between young flush and mature leaves. Furthermore, the transcriptome of mated females infesting Murraya paniculata was analysed. Finally, the gene of the key Target of Rapamycin (TOR) signalling pathway was silenced by RNAi. Results indicated that flush shoot feeding activated the development of the psyllids ovary and therefore induced oviposition. A total of 126 and 2794 differentially expressed genes were detected at 1 and 5 days, respectively, after pest infestation of flush shoots compared to mature leaves. Many genes are involved in protein metabolism, Mitogen‐Activated Protein Kinase (MAPK) signalling pathway, hormone synthesis, and TOR signalling pathway: all thought to activate reproduction. Silencing of the positive regulator gene DcRheb in the TOR pathway resulted in lower levels of ecdysone and juvenile hormone and decreased vitellogenin synthesis, further disrupting reproductive ability. This study enhances understanding of the molecular mechanism underlying ACP's reproductive strategy. [ABSTRACT FROM AUTHOR]

The insecticides of microbial origin may be promising biocontrol agents to control pests. In this study, a major metabolite was extracted and purified from Serratia marcescens KH-001 and identified as prodigiosin with a molecule weight of 324.2094, and our primary aim was to evaluate the effects of prodigiosin against Diaphorina citri Kuwayama (Hemiptera: Liviidae) in the laboratory and in the field. Toxicity of purified prodigiosin to nymphs increased with increasing temperature from 25 to 35°C. The laboratory experiment results observed inhibition effects of purified prodigiosin at LC50 on oviposition and egg hatch of D. citri at 30°C. Furthermore, adults that fed on citrus leaves treated with LC20 and LC50 solution of purified prodigiosin at 30°C excreted less honeydew (28–35%) compared with controls, suggesting moderate antifeedant activity of prodigiosin. The results of field experiment showed that 10% prodigiosin emulsifiable concentrate exhibited better control efficacy (70–100%) at three concentrations (100, 200, and 500 mg/liter) in July and August than in October. These data indicate that prodigiosin will be a highly effective compound for the control of D. citri depending on the prevailing temperature conditions and may become a commercially available product to be widely used in citrus orchards. [ABSTRACT FROM AUTHOR]

Abstract Background Asian citrus psyllid (ACP) is the primary vector responsible for the transmission of the phloem-limited bacteria Candidatus Liberibacter spp., associated with huanglongbing (HLB), which causes great loss to the citrus industry. Although the roles of leaf color and volatile compounds in the orientation of ACP have been proven, the quantification of color and allelochemicals in the host plant are kept unclear, especially in wild citrus germplasms. Results Chongyi wild mandarin significantly attracted more ACP than wild Hong Kong kumquat, ‘Gannan zao’ navel orange and orange jasmine did in the four-choice and olfactometer assays. The color parameters of the tender leaves from Chongyi wild mandarin and ‘Gannan zao’ were similar. The yellow color in both of them was less saturated than that of the other two plants species, but Chongyi wild mandarin had significant lower carotenoid content (P < 0.05). Notably metabolic profiling differences were observed among the healthy tender shoots from the four tested plants via UPLC-QQQ-MS and GC-MS analyses. Comparing with the other three plant species, 66 and 50 metabolites with significantly different contents in Chongyi wild mandarin were selected as UPLC-identified and GC-identified metabolites of interest (P < 0.05), respectively. Flavonoids accounted for a large group of secondary metabolites of interest, which may function as stimulants or repellents of ACP. Higher content of salicylic acid o-hexoside and lower content of (+)-jasmonic acid in Chongyi wild mandarin may lead to higher amount of methyl salicylate (an ACP attractant) and lower amount of trans-ocimene (an attractant to herbivores’ natural enemies) as well as the suppression of JA-mediated wounding response. This kind of synergistic or antagonistic effect among the metabolites differentially accumulated in Chongyi wild mandarin made it a more attractive host plant to ACP. Conclusions Less saturated yellow color, high amount of attractants, low amount of repellents and insensitivity of JA-mediated wounding response are the four possible reasons why Chongyi wild mandarin attracted more ACP. This work may shed light on the olfactory and visual response of ACP to wild citrus germplasm hosts, and suggest the feasibility of developing ACP attractants or repellents patterned on potential metabolites.

Huanglongbing in citrus is caused by a phloem-limited, uncultivable, gram-negative α-proteobacterium, Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by the phloem-sucking insect, Diaphorina citri (Hemiptera: Liviidae), in a persistent, circulative, and propagative manner. In this study, we investigated the metabolomic and respiration rates changes in D. citri upon infection with CLas using gas chromatography-mass spectrometry (GC-MS) and gas exchange analysis. The level of glycine, L-serine, L-threonine, and gamma-amino butyric acid were higher in CLas-infected D. citri, while L-proline, L-aspartic acid, and L-pyroglutamic acid were lower in CLas-infected D. citri compared with the control. Citric acid was increased in CLas-infected D. citri, whereas malic and succinic acids were reduced. Interestingly, most of the reduced metabolites such as malate, succinate, aspartate, and L-proline are required for the growth of CLas. The increase in citric acid, serine, and glycine indicated that CLas induced glycolysis and the tricarboxylic acid cycle (TCA) in its vector. In agreement with the GC-MS results, the gene expression results also indicated that glycolysis and TCA were induced in CLas-infected D. citri and this was accompanied with an increases in respiration rate. Phosphoric acid and most of the sugar alcohols were higher in CLas-infected D. citri, indicating a response to the biotic stress or cell damage. Only slight increases in the levels of few sugars were observed in CLas-infected D. citri, which indicated that sugars are tightly regulated by D. citri. Our results indicated that CLas induces nutrient and energetic stress in its host insect. This study may provide some insights into the mechanism of colonization of CLas in its vector.

Background: The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an insect pest species responsible for damages of citrus fruit quality and tree health. This insect is a vector of bacteria 'Candidatus Liberibacter' spp. a putative causal agent of citrus greening disease known as Huanglongbing (HLB), considered one of the most destructive diseases of citrus orchards worldwide. Disease management is mainly based on vector control using pesticides which can affect natural enemies that play an important role in pest control. The entomopathogenic fungi (EPF) Beauveria bassiana (2067 and 2121) and Metarhizium anisopliae (2411) were achieved by applying a suspension of 1 × 107 spores against D. citri nymphs and artificially infected the citrus seedlings under controlled and semi-field conditions. Also, the activity of these EPFs on the coccinellid and lacewing predators was evaluated. The effects of the main chemicals used in citrus plantations on the selected fungi for possible combined use was also investigated. Results: The obtained results showed that under controlled conditions, the percentage of insect mortality produced by EPF varied between 82.8 and 85.9%. Under semi-field conditions, the infection and mortality of D. citri caused by the M. anisopliae 2411 strain was significantly higher than that of B. bassiana 2067, 78.9 and 51%, respectively. Non-significant effect of EPF on the natural enemies of D. citri was observed, suggesting that EPF and predators had compatible effects. Conclusion: This study clearly demonstrated that EPF were able to kill D. citri and can be explored as a promissory biocontrol candidate. Simultaneous use in an integrated pest management program could be possible by applying EPF combined with pesticides. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The African citrus triozid (ACT) Trioza erytreae (Del Guercio) (Hemiptera: Triozidae) and the Asian citrus psyllid (ACP) Diaphorina citri (Kuwayama) (Hemiptera: Liviidae) are primary vectors of the 'Candidatus' Liberibacter spp. ACT is associated with 'Candidatus Liberibacter africanus' (CLaf) causal agent of the African citrus greening disease (ACGD), whereas ACP vectors 'Candidatus Liberibacter asiaticus' (CLas) and 'Candidatus Liberibacter americanus' (CLam), the Asian and the American strains, respectively, associated with huanglongbing.A preliminary survey in Kenyan citrus groves after the invasion of ACP had revealed that lime‐green sticky traps (Asacp) were not effective in detecting and monitoring ACT and ACP. Therefore, this study compared eight differently coloured double‐sided sticky traps to evaluate, which colour was most effective for detecting ACT and ACP, particularly at low densities. The traps were coded Red, Blue, Asgreen1, GLMgreen, Asacp, Asyellow, White, and Black.Asyellow and GLMgreen traps captured more ACTs and ACPs than any other trap type. However, since there was no clear difference in the efficiency of Asyellow and GLMgreen in trapping ACP, any of the two trap types would be useful in the presence of both pests.Among the eight traps, Asyellow and GLMgreen traps are potentially promising options for monitoring and detecting the pests in areas where they coexist.Our findings will guide biosecurity agencies in decision‐making and designing ecologically friendly integrated pest management (IPM) strategies for citrus greening vectors in Africa as well as serve as an early warning to safeguard against pests' invasion into unaffected areas. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is a devastating citrus disease associated with Candidatus Liberibacter asiaticus (CLas) and is transmitted by the psyllid Diaphorina citri Kuwayama. Diaphorina citri belongs to Hemiptera order, which has cysteine peptidases as the most abundant proteolytic enzymes present in digestive tract. As cysteine peptidases are involved in different insect development processes, this class of enzymes has acquired biotechnological importance. In this context, we identified a cathepsin L-like (DCcathL1) from the Diaphorina citri transcriptome database and expressed the enzyme in E. coli. Quantitative real-time RT-PCR was conducted to determine DCcathL1 gene expression in different parts and developmental phases of the insect. We observed that DCcathL1 expression in the gut was 2.59 and 2.87-fold higher than in the head and carcass, respectively. Furthermore, DCcathL1 expression was greater in eggs than in nymphs and adults, suggesting a putative role of the enzyme in the embryonic development. In addition, enzymatic inhibitory activity using four recombinant Citrus cystatins were performed. Among them, Csin CPI-2 was the strongest DCcathL1 inhibitor with a K i value of 0.005 nM. Our results may contribute in the development of strategies for D. citri control, such as silencing the DCcathL1 gene and the use of transgenic plants that overexpress peptidase inhibitors. [ABSTRACT FROM AUTHOR]

Diaphorina citri Kuwayama, also known as the Asian citrus psyllid, is a pest of citrus known for its transmission of Candidatus Liberibacter asiaticus (Ca. L. asiaticus), the causal bacterium of Huanglongbing. The African citrus triozid Trioza erytreae (Del Guercio) (Hemiptera: Triozidae) has been the putative vector of Candidatus Liberibacter africanus (Ca. L. africanus) which causes the African citrus greening disease, until the recent detection of D. citri on the continent. Following reports of D. citri in Kenya and Tanzania, we surveyed citrus plants to establish the presence/absence of D. citri in Ethiopia in citrus-growing regions ranging from 900 to 2,460 m above sea level (masl). Diaphorina citri adults were detected in five of the surveyed sites in Ethiopia. Adult insects encountered were collected using an aspirator and stored in 97% ethanol. The mitochondrial cytochrome oxidase 1 (mt COI) gene of the collected insects was amplified using LepF1/LepR1 primers, and sequences obtained showed low variation, which fell within the acceptable range of species. BLAST was used to query the sequences obtained, and all the sequences linked to D. citri accessions that are available in GenBank. The analysis of the sequences revealed a new haplotype of the species that differs from haplotypes previously reported. Phylogenetic relationships of our samples and other D. citri reference sequences was inferred using the Maximum-likelihood method. Monophyly was observed between the samples and the publicly available sequences from global accessions. This is the first report of the presence of D. citri in Ethiopia. [ABSTRACT FROM AUTHOR]

Background: Phloem-feeding insects are known to modulate the salicylic acid (SA) signaling pathway in various plant-insect interaction models. Diaphorina citri is a phloem feeding vector of the deadly phytopathogens, Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, and the interactions of D. citri with its host that may modulate plant defenses are not well understood. The objectives of this study were to investigate the molecular mechanisms involved in transcriptional regulation of SA modification and activation of defense-associated responses in sweet orange (Citrus sinensis) exposed to various durations (7-, 14- and 150- days) of continuous feeding by D. citri. Results: We quantified expression of genes involved in SA pathway activation and subsequent modification, as well as, associated SA metabolites (SA methyl ester, 2,3-DHBA, and SA 2-O-β-D-glucoside). NPR1 and PR-1 expression was upregulated in plants exposed to continuous feeding by D. citri for 14 days. Expression of BSMT-like, MES1-like and DMR6-like oxygenase, as well as, accumulation of their respective SA metabolites (SA methyl ester, 2,3-DHBA) was significantly higher in plants exposed to continuous feeding by D. citri for 150 days than in those without D. citri infestation. Concomitantly, expression of UGT74F2-like was significantly downregulated and its metabolite, SA 2-β-D-glucoside, was highly accumulated in trees exposed to 150 d of feeding compared to control trees without D. citri. Conclusions: D. citri herbivory differentially regulated transcription and SA-metabolite accumulation in citrus leaves, depending on duration of insect feeding. Our results suggest that prolonged and uninterrupted exposure (150 d) of citrus to D. citri feeding suppressed plant immunity and inhibited growth, which may highlight the importance of vector suppression as part of huanglongbing (HLB) management in citrus. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is an economic insect pest in most citrus-growing regions and the vector of 'Candidatus Liberibacter asiaticus' (CLas), one of at least three known bacteria associated with Huanglongbing (HLB or citrus greening disease). D. citri harbors bacterial endosymbionts, including Wolbachia pipientis (strain Wolbachia wDi), 'Candidatus Carsonella ruddii,' and 'Candidatus Profftella armatura.' Many important functions of these bacteria can be inferred from their genome sequences, but their interactions with each other, CLas, and their D. citri host are poorly understood. In the present study, the titers of the endosymbionts in different tissues, in each sex, and in insects reared on healthy citrus (referred to as unexposed) and CLas-infected citrus (referred to as CLas-exposed) D. citri were investigated using real-time, quantitative PCR (qPCR) using two different quantitative approaches. Wolbachia and CLas were detected in all insect tissues. The titer of Wolbachia was higher in heads of CLas-exposed males as compared to unexposed males. In males and females, Wolbachia titer was highest in the Malpighian tubules. The highest titer of CLas was observed in the gut. Profftella and Carsonella titers were significantly reduced in the bacteriome of CLas-exposed males compared with that of unexposed males, but this effect was not observed in females. In ovaries of CLas-exposed females, the Profftella and Carsonella titers were increased as compared to non-exposed females. CLas appeared to influence the overall levels of the symbionts but did not drastically perturb the overall microbial community structure. In all the assessed tissues, CLas titer in males was significantly higher than that of females using absolute quantification. These data provide a better understanding of multi-trophic interactions regulating symbiont dynamics in the HLB pathosystem. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the most detrimental insect pest of citrus crops due to its role as a vector of Candidatus Liberibacter asiaticus (Las), the bacterial causal agent of huanglongbing, also known as citrus greening disease. Trees infected with Las decline rapidly and fruit production decreases until eventual tree death. Few treatment options for infected trees are available for disease management. A technique called "thermotherapy" is under development to reduce bacterial titers in infected trees; however, the effect of these treatments on the transmission cycle of Las is not known. Field and laboratory assays were conducted to determine whether thermotherapy treatment reduced Las acquisition by D. citri. Trees in the field were treated with a mobile heat treatment system. Potted trees in the laboratory were treated in a steam chamber. We monitored acquisition rates in D. citri following thermal treatment of Las-positive Citrus sinensis (L.) (Rutaceae). Psyllid acquisition and Las titer in thermotherapy-treated trees were compared with untreated Las-positive and untreated Las-negative trees. Our results confirmed the efficacy of whole-tree thermotherapy on Las in potted citrus trees. In contrast, thermotherapy did not significantly reduce plant Las titers or acquisition of Las by D. citri under field conditions. These results suggest that further development of field application methods is needed to determine the utility of thermotherapy as a tool for huanglongbing management. [ABSTRACT FROM AUTHOR]

Asiatic huanglongbing (HLB), also known as citrus greening or yellow shoot disease, is a serious disease putatively caused by the bacterium ' Candidatus Liberibacter asiaticus' Jagoueix et al. (Rhizobiales: Rhizobiaceae) (CLas) transmitted by the Asian citrus psyllid (Diaphorina citri Kuwayama (Hemiptera: Liviidae)). Plant resistance to CLas holds promise as a strategy for reducing the economic impact of HLB; thus, citrus breeders are developing new varieties and cultivars and evaluating these for resistance/tolerance to CLas. A high-throughput inoculation program was established to expedite testing of germplasm. Colonies of Asian citrus psyllid maintained on CLas-infected plants are used for the inoculations. The primary inoculation step is to cage a healthy citrus seedling with new leaf growth for a 2-wk inoculation feeding period by 20 adult Asian citrus psyllids (no regard to sex or age) from these colonies. The adults are removed after the inoculation period, and the plants are then treated with a pesticide to eliminate any adults missed or infestation of immatures. The objective here was to evaluate inoculation rates under this procedure. Ten sets of 20 healthy citrus seedlings were subjected to the procedure, one set a month, and the percentage of seedlings successfully inoculated based on testing by quantitative polymerase chain reaction were determined 6 mo after each 2-wk inoculation period. The results indicated a 77% mean transmission rate (percent infected seedlings), with rates ranging from 40 to 100%. Transmission rates were positively correlated with the percentage of Asian citrus psyllid that tested positive for CLas but not correlated with titers of CLas in these psyllids. Possible reasons for variability in transmission rates are discussed. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB), formerly known as citrus greening disease, is one of the most devastating bacterial diseases in citrus worldwide. HLB is caused by ‘Candidatus Liberibacter asiaticus’ bacterium and transmitted by Diaphorina citri. Both ‘Ca. L. asiaticus’ and its vector manipulate the host metabolism to fulfill their nutritional needs and/or to neutralize the host defense responses. Herein, we discuss the history of HLB and the complexity of its pathosystem as well as the geographical distribution of its pathogens and vectors. Recently, our recognition of physiological events associated with ‘Ca. L. asiaticus’ infection and/or D. citri-infestation has greatly improved. However, the roles of citrus metabolites in the development of HLB symptoms are still unclear. We believe that symptom development of HLB disease is a complicated process and relies on a multilayered metabolic network which is mainly regulated by phytohormones. Citrus metabolites play vital roles in the development of HLB symptoms through the modulation of carbohydrate metabolism, phytohormone homeostasis, antioxidant pathways, or via the interaction with other metabolic pathways, particularly involving amino acids, leaf pigments, and polyamines. Understanding how ‘Ca. L. asiaticus’ and its vector, D. citri, affect the metabolic pathways of their host is critical for developing novel, sustainable strategies for HLB management.

A study was conducted to evaluate mosquito repellents against Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) and to expand our knowledge of the potential of essential plant oils as deterrents of ACP infestations. The psyllid vectors a bacterium, ‘ Candidatus Liberibacter asiaticus,’ presumed causal agent of a serious disease of citrus, huanglongbing (also known as citrus greening disease). Twenty-two candidate deterrents were tested as 20% solutions in methanol using a laboratory assay. Adults were free to enter and settle in one of three vials containing orange jasmine flush, one vial treated with a candidate deterrent, one vial treated with water or one treated with methanol. Seven mosquito repellents (CisPMD, DEET, DHS220, Icaridin, IR-3535, S-220, and TransPMD) and four essential oils (citronella, lavender, lemon eucalyptus, and a commercial mixture of oils from orange, clove, cinnamon, eucalyptus and rosemary) reduced ACP infestations on flush in the vials. Following a 24-h assay period, an average of 75% (range 58–88%) fewer psyllids were found in vials treated with these deterrents as compared to the combined number found in the control vials. Greenhouse evaluations were conducted on phytotoxicity of 5 or 25% solutions of eight deterrents and on ACP colonization of citrus seedlings sprayed with these deterrents. Few ACP colonized seedlings sprayed with 25% solutions, but this was attributed to phytotoxicity. Infestations were reduced on seedlings treated with 5% solutions, but these reductions also were largely attributed to phytotoxicity. Although unsprayed seedlings were in close proximity, adults found these plants and laid relatively large numbers of eggs. While some of the mosquito repellents and essential plant oils deterred infestations, none was completely effective and 5–25% solutions were too phytotoxic to be feasible as direct plant sprays. These results indicated the deterrents as tested hold little potential as a major tactic for reducing ACP infestations in citrus. Alternative application approaches to avoid phytotoxicity could be explored, and individual constituents of the more promising essential plant oils could be investigated. [ABSTRACT FROM AUTHOR]

Huanglongbing in citrus is caused by a phloem-limited, uncultivable, gram-negative α-proteobacterium, Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by the phloem-sucking insect, Diaphorina citri (Hemiptera: Liviidae), in a persistent, circulative, and propagative manner. In this study, we investigated the metabolomic and respiration rates changes in D. citri upon infection with CLas using gas chromatography-mass spectrometry (GC-MS) and gas exchange analysis. The level of glycine, L-serine, L-threonine, and gamma-amino butyric acid were higher in CLas-infected D. citri, while L-proline, L-aspartic acid, and L-pyroglutamic acid were lower in CLas-infected D. citri compared with the control. Citric acid was increased in CLas-infected D. citri, whereas malic and succinic acids were reduced. Interestingly, most of the reduced metabolites such as malate, succinate, aspartate, and L-proline are required for the growth of CLas. The increase in citric acid, serine, and glycine indicated that CLas induced glycolysis and the tricarboxylic acid cycle (TCA) in its vector. In agreement with the GC-MS results, the gene expression results also indicated that glycolysis and TCA were induced in CLas-infected D. citri and this was accompanied with an increases in respiration rate. Phosphoric acid and most of the sugar alcohols were higher in CLas-infected D. citri, indicating a response to the biotic stress or cell damage. Only slight increases in the levels of few sugars were observed in CLas-infected D. citri, which indicated that sugars are tightly regulated by D. citri. Our results indicated that CLas induces nutrient and energetic stress in its host insect. This study may provide some insights into the mechanism of colonization of CLas in its vector. [ABSTRACT FROM AUTHOR]

BACKGROUND Control of the Asian citrus psyllid Diaphorina citri Kuwayama, the most important pest of citrus worldwide, is based on the use of insecticides, though unsatisfactory results have recently been reported. In this study, insecticide resistance of D. citri to three insecticides (bifenthrin, malathion, and chlorpyrifos) was examined. RESULTS Three populations (designated Dci-CParácuaro, Dci-El Junco, and Dci-Antúnez) of both adults and fourth-instar D. citri individuals were collected in 2014 at two different times and on one occasion, respectively, from three locations (Crucero de Parácuaro, El Junco, and Antúnez). These locations represent the major commercial Mexican lemon production areas in the Apatzingán Valley in the state of Michoacán, Mexico. The three populations of D. citri adults and fourth-instar nymphs at the different collection times showed low levels of resistance (≤7-fold) to bifenthrin, but were very resistant to malathion (≤345- and ≤432-fold for adults and fourth instars, respectively) and chlorpyrifos (≤2435- and ≤1424-fold for adults and fourth instars, respectively). CONCLUSION Resistance levels to the tested insecticides were highly variable but homogeneous among seasons and localities. Resistance management programmes that include crop sanitation, use of biological and cultural control practices, and rotation of insecticide classes should be established, particularly in areas where D. citri has developed resistance to malathion and chlorpyrifos. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Asian citrus psyllid Diaphorina citri is the vector of the citrus Huanglongbing ( HLB) associated bacterial agent ' Candidatus Liberibacter asiaticus' ( C Las). The molecular interactions between C Las and D. citri remain unclear. In the present study, protein profiles of mitochondrial, microsomal and cytosolic fractions from uninfected and CLas-infected adult D. citri are investigated using two-dimensional gel electrophoresis. The comparative analysis reveals a total of 18, 24 and 20 protein spots that are unique or differentially expressed in mitochondrial, microsomal and cytosolic proteins fractions respectively. These proteins are successfully identified by mass spectrometry. Among the 62 identified proteins, 30 are up-regulated, whereas 32 are down-regulated. These proteins include important components in energy metabolism such as ATP synthase, ATPase, ATP/ ADP carrier protein, etc.; host stress responses such as heat shock proteins; host detoxification processes (i.e., cytochrome P450 and glutathione S-transferase); and the cytoskeleton (such as actin, tubulin, myosin and tropomyosin). These data suggest that, after C Las infection, several proteins of D. citri, especially energy metabolism and protein biosynthesis, are altered, and extensive host defence responses are induced. In conclusion, the present study reports proteomic information that is helpful in understanding the vector-pathogen relationship between C Las and D. citri, and could be used to identify potential targets for limiting the spread of C Las, as well as to provide new insights into HLB management. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a damaging pest of citrus globally and has recently been detected in Tanzania. Although direct damage by the pest is seldom of economic importance, the insect is more notorious for its ability to vector the fastidious phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), the putative causal bacterium of Huanglongbing or Asian citrus greening disease. For many years, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae) was known to be the main vector of the African citrus greening disease caused by Candidatus Liberibacter africanus (CLaf), but the recent arrival of D. citri on the continent adds to the dynamics of infection and spread of both diseases on mainland Africa. Following the recent report of the presence of D. citri in Tanzania, an additional delimiting survey was carried out in the region, focusing on Kenya, mainland Tanzania and Zanzibar to detect the presence and ascertain the extent of spread of D. citri. We employed molecular tools based on the use of DNA barcoding to confirm the identity of D. citri. In addition to D. citri, the occurrence of T. erytreae in the same sampling locations is also reported. Adults and nymphs of either D. citri or T. erytreae were collected from citrus at many of the surveyed sites ranging from 19 to 668 m above sea level (masl) in Tanzania, 20-1666 masl in Kenya, and 42-48 masl in Zanzibar. Diaphorina citri was sympatric with T. erytreae at the mid to higher elevations of 1375-1666 masl and no T. erytreae or its open-gall symptoms were detected below 523 masl. Sequences obtained were queried via BLAST and all linked to D. citri of different accession numbers already available on GenBank. This is the first report of the presence of D. citri in Kenya and Zanzibar. The potential implication of the detection and spread of the two pathogens, CLaf and CLas to the citrus industry in East Africa and movement of suitable host plants is discussed. [ABSTRACT FROM AUTHOR]

Huanglongbing is causing economic devastation to the citrus industry in Florida, and threatens the industry everywhere the bacterial pathogens in the Candidatus Liberibacter genus and their insect vectors are found. Bacteria in the genus cannot be cultured and no durable strategy is available for growers to control plant infection or pathogen transmission. However, scientists and grape growers were once in a comparable situation after the emergence of Pierce’s disease, which is caused by Xylella fastidiosa and spread by its hemipteran insect vector. Proactive quarantine and vector control measures coupled with interdisciplinary data-driven science established control of this devastating disease and pushed the frontiers of knowledge in the plant pathology and vector biology fields. Our review highlights the successful strategies used to understand and control X. fastidiosa and their potential applicability to the liberibacters associated with citrus greening, with a focus on the interactions between bacterial pathogen and insect vector. By placing the study of Candidatus Liberibacter spp. within the current and historical context of another fastidious emergent plant pathogen, future basic and applied research to develop control strategies can be prioritized.