CLas Infection Research Articles

Overexpressing a NPR1-like gene from Citrus paradisi enhanced Huanglongbing resistance in C. sinensis.

Overexpression of CiNPR4 enhanced resistance of transgenic citrus plants to Huanglongbing by perceiving the salicylic acid and jasmonic acid signals and up-regulating the transcriptional activities of plant-pathogen interaction genes. Developing transgenic citrus plants with enhanced immunity is an efficient strategy to control citrus Huanglongbing (HLB). Here, a nonexpressor of pathogenesis-related gene 1 (NPR1) like gene from HLB-tolerant 'Jackson' grapefruit (Citrus paradisi Macf.), CiNPR4, was introduced into 'Wanjincheng' orange (Citrus sinensis Obseck). CiNPR4 expression was determined in transgenic citrus plants using quantitative real-time PCR analyses. The Candidatus Liberibacter asiaticus (CLas) pathogen of HLB was successfully transmitted to transgenic citrus plants by grafting infected buds. HLB symptoms developed in transgenic and wild-type (WT) plants by 9months after inoculation. A CLas population analysis showed that 26.9% of transgenic lines exhibited significantly lower CLas titer levels compared with the CLas-infected WT plants at 21months after inoculation. Lower starch contents and anatomical aberration levels in the phloem were observed in transgenic lines having enhanced resistance compared with CLas-infected WT plants. CiNPR4 overexpression changed the jasmonic acid, but not salicylic acid, level. Additionally, the jasmonic acid and salicylic acid levels increased after CLas infection. Transcriptome analyses revealed that the enhanced resistance of transgenic plants to HLB resulted from the up-regulated transcriptional activities of plant-pathogen interaction-related genes.

Comparative analysis of bacterial and fungal endophytes responses to Candidatus Liberibacter asiaticus infection in leaf midribs of Citrus reticulata cv. Shatangju

The endophytic bacterial and fungal profiles were comparatively studied between Citrus Huanglongbing (HLB) infected and uninfected healthy leaves of a mandarin cultivar, Shatangju (Citrus reticulata Blanco), by using the next generation sequencing technology. A total of 53 endophytic bacterial orders in12 phyla and 24 endophytic fungal orders in 2 phyla were detected in healthy, asymptomatic and symptomatic leaf midribs. It was found that the predominant endophytic bacteria belonged to Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes and the predominant endophytic fungi belonged to Ascomycota and Basidiomycota. Operational taxonomic unit (OTU) analysis showed that OTU_2 and OTU_6 were the core endophytic fungal OTUs, and OTU_10, OTU_4 and OTU_141 were the core endophytic bacterial OTUs. The richness and diversity of the endophytic microbes were affected by infection of the HLB bacterium, Candidatus Liberibacter asiaticus (CLas). The most significant change was the reduction in endophytic fungal OTUs following CLas infection. Eleven endophytic fungal OTUs and 24 endophytic bacterial OTUs were found to be associated with tree healthy status in citrus. Some of the healthy and HLB disease specific OTUs were discussed, with emphasis on their possible roles in HLB pathogenesis.

Rootstock Influences on Health and Growth Following Candidatus Liberibacter asiaticus Infection in Young Sweet Orange Trees

Health, growth, fruit production, and fruit quality of citrus crops are severely affected by tree infection with Candidatus Liberibacter asiaticus (CLas) and subsequent development of the disease huanglongbing (HLB). The use of HLB-tolerant rootstocks is one strategy that is used to ameliorate the effects of HLB in commercial production. Although there is a clear long-term rootstock effect to improve tree performance, such field trials take many years for establishment and evaluation, and this long time-period is very limiting to expeditious evaluation of new rootstocks. In this study, we have conducted a 50-week greenhouse experiment to evaluate rootstock influences on Valencia sweet orange tree response to CLas infection. The infection of trees with CLas reduced scion and rootstock growth, increased leaf yellowing, and reduced the number of leaves per tree and leaf area, regardless of rootstock. There were clear rootstock influences on some traits during the 50-week study. In general, infected trees on US-942 rootstock had lower CLas root titers, less reduction of the number of leaves, less reduction of leaf area, and less leaf yellowing, as compared with some of the other rootstocks. The 50-week greenhouse evaluation method provided results that corresponded well with results from long-term field testing, indicating this may be a useful tool to accelerate evaluation and selection of new rootstocks, as well as in testing other HLB management strategies.

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

Huanglongbing (HLB) is the most devastating and widespread citrus disease. All commercial citrus varieties are susceptible to the HLB-associated bacterium, Candidatus Liberibacter asiaticus (CLas), which resides in the phloem. The phloem is part of the plant vascular system and is involved in sugar transport. To investigate the plant response to CLas, we enriched for proteins surrounding the phloem in an HLB susceptible sweet orange variety, Washington navel (Citrus sinensis (L) Osbeck). Quantitative proteomics revealed global changes in the citrus proteome after CLas inoculation. Plant metabolism and translation were suppressed, whereas defense-related proteins such as peroxidases, proteases and protease inhibitors were induced in the vasculature. Transcript accumulation and enzymatic activity of plant peroxidases in CLas infected sweet orange varieties under greenhouse and field conditions were assessed. Although peroxidase transcript accumulation was induced in CLas infected sweet orange varieties, peroxidase enzymatic activity varied. Specific serine proteases were up-regulated in Washington navel in the presence of CLas based on quantitative proteomics. Subsequent activity-based protein profiling revealed increased activity of two serine proteases, and reduced activity of one protease in two C. sinensis sweet orange varieties under greenhouse and field conditions. The observations in the current study highlight global reprogramming of the citrus vascular proteome and differential regulation of enzyme classes in response to CLas infection. These results open an avenue for further investigation of diverse responses to HLB across different environmental conditions and citrus genotypes.

'Candidatus Liberibacter Asiaticus' SDE1 Effector Induces Huanglongbing Chlorosis by Downregulating Host DDX3 Gene.

‘Candidatus Liberibacter asiaticus’ (CLas) is the pathogenic bacterium that causes the disease Huanglongbing (HLB) in citrus and some model plants, such as Nicotiana benthamiana. After infection, CLas releases a set of effectors to modulate host responses. One of these critical effectors is Sec-delivered effector 1 (SDE1), which induces chlorosis and cell death in N. benthamiana. In this study, we revealed the DEAD-box RNA helicase (DDX3) interacts with SDE1. Gene silencing study revealed that knockdown of the NbDDX3 gene triggers leaf chlorosis, mimicking the primary symptom of CLas infection in N. benthamiana. The interactions between SDE1 and NbDDX3 were localized in the cell membrane. Overexpression of SDE1 resulted in suppression of NbDDX3 gene expression in N. benthamiana, which suggests a critical role of SDE1 in modulating NbDDX3 expression. Furthermore, we verified the interaction of SDE1 with citrus DDX3 (CsDDX3), and demonstrated that the expression of the CsDDX3 gene was significantly reduced in HLB-affected yellowing and mottled leaves of citrus. Thus, we provide molecular evidence that the downregulation of the host DDX3 gene is a crucial mechanism of leaf chlorosis in HLB-affected plants. The identification of CsDDX3 as a critical target of SDE1 and its association with HLB symptom development indicates that the DDX3 gene is an important target for gene editing, to interrupt the interaction between DDX3 and SDE1, and therefore interfere host susceptibility.

Development on Citrus medica infected with 'Candidatus Liberibacter asiaticus' has sex-specific and -nonspecific impacts on adult Diaphorina citri and its endosymbionts.

Huanglongbing (HLB) is a deadly, incurable citrus disease putatively caused by the unculturable bacterium, ‘Candidatus Liberibacter asiaticus’ (CLas), and transmitted by Diaphorina citri. Prior studies suggest D. citri transmits CLas in a circulative and propagative manner; however, the precise interactions necessary for CLas transmission remain unknown, and the impact of insect sex on D. citri-CLas interactions is poorly understood despite reports of sex-dependent susceptibilities to CLas. We analyzed the transcriptome, proteome, metabolome, and microbiome of male and female adult D. citri reared on healthy or CLas-infected Citrus medica to determine shared and sex-specific responses of D. citri and its endosymbionts to CLas exposure. More sex-specific than shared D. citri responses to CLas were observed, despite there being no difference between males and females in CLas density or relative abundance. CLas exposure altered the abundance of proteins involved in immunity and cellular and oxidative stress in a sex-dependent manner. CLas exposure impacted cuticular proteins and enzymes involved in chitin degradation, as well as energy metabolism and abundance of the endosymbiont ‘Candidatus Profftella armatura’ in both sexes similarly. Notably, diaphorin, a toxic Profftella-derived metabolite, was more abundant in both sexes with CLas exposure. The responses reported here resulted from a combination of CLas colonization of D. citri as well as the effect of CLas infection on C. medica. Elucidating these impacts on D. citri and their endosymbionts contributes to our understanding of the HLB pathosystem and identifies the responses potentially critical to limiting or promoting CLas acquisition and propagation in both sexes.

A Transcriptomic and Proteomic Analysis of the Diaphorina citri Salivary Glands Reveals Genes Responding to Candidatus Liberibacter asiaticus.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the principal vector of the Candidatus Liberibacter asiaticus (CLas) bacterium that causes Huanglongbing (HLB) disease. The D. citri salivary glands (SG) is an important barrier to the transmission of CLas. Despite its importance, the transcriptome and proteome of SG defense against CLas are unstudied in D. citri. In the present study, we generated a comparative transcriptome dataset of the SG in infected and uninfected D. citri using an Illumina RNA-Seq technology. We obtained 407 differentially expressed genes (DEGs), including 159 upregulated DEGs and 248 downregulated DEGs. Functional categories showed that many DEGs were associated with the ribosome, the insecticide resistance, the immune response and the digestion in comparison with CLas-infected SG and CLas-free SG. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases confirmed that metabolism and immunity were important functions in the SG. Among the DEGs, 68 genes (35 upregulated and 33 downregulated) encoding putative-secreted proteins were obtained with a signal peptide, suggesting that these genes may play important roles in CLas infection. A total of 673 SG proteins were identified in uninfected D. citri by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, and 30 DEGs (15 upregulated and 15 downregulated) were found using the local tBLASTP programs. Among the 30 DEGs, many DEGs mainly involved in the metabolism and cellular processes pathways. This study provides basic transcriptome and proteome information for the SG in D. citri, and helps illuminate the molecular interactions between CLas and D. citri.

Peptide conjugated morpholinos for management of the huanglongbing pathosystem.

'Candidatus Liberibacter asiaticus' (CLas) is the causal agent of the devastating citrus disease Huanglongbing (HLB) and is transmitted by the insect vector Diaphorina citri (Hemiptera: Liviidae). A potential approach for treating CLas infection is the use of synthetic nucleic acid-like oligomers to silence bacterial gene expression. Peptide conjugated morpholinos (PPMOs) targeting essential genes in CLas and the psyllid vector's endosymbiotic bacteria, Wolbachia (-Diaphorina, wDi), were evaluated using in vitro and in vivo assays. Expression of the wDi gyrA gene was significantly reduced following incubation of wDi cells with PPMOs. In addition, the viability of isolated wDi cells was greatly reduced when treated with PPMOs as compared to untreated cells. Feeding D. citri adults with a complementary PPMO (CLgyrA-14) showed significantly reduced (70% lower) expression of the CLas gyrA gene. CLas relative density was significantly lower in the psyllids fed with CLgyrA-14, when compared to untreated insects. Psyllids that were treated with CLgyrA-14 were less successful in transmitting the pathogen into uninfected plants, compared to untreated insects. The expression of essential genes in the D. citri symbiont, wDi and the HLB pathogen were suppressed in response to PPMO treatments. This study demonstrates the potential of PPMOs as a novel strategy for management of bacterial pathogens of fruit trees, such as HLB. © 2020 Society of Chemical Industry.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analysis of Citrus limon Response to Graft Inoculation by Candidatus Liberibacter asiaticus.

Presymptomatic detection of citrus trees infected with Candidatus Liberibacter asiaticus (CLas), the bacterial pathogen associated with Huanglongbing (HLB; citrus greening disease), is critical to controlling the spread of the disease. To test whether infected citrus trees produce systemic signals that may be used for indirect disease detection, lemon (Citrus limon) plants were graft-inoculated with either CLas-infected or control (CLas-) budwood, and leaf samples were longitudinally collected over 46 weeks and analyzed for plant changes associated with CLas infection. RNA, protein, and metabolite samples extracted from leaves were analyzed using RNA-Seq, mass spectrometry, and 1H NMR spectroscopy, respectively. Significant differences in specific transcripts, proteins, and metabolites were observed between CLas-infected and control plants as early as 2 weeks post graft (wpg). The most dramatic differences between the transcriptome and proteome of CLas-infected and control plants were observed at 10 wpg, including coordinated increases in transcripts and proteins of citrus orthologs of known plant defense genes. This integrated approach to quantifying plant molecular changes in leaves of CLas-infected plants supports the development of diagnostic technology for presymptomatic or early disease detection as part of efforts to control the spread of HLB into uninfected citrus groves.

Gene Expression Profile of Mexican Lime (Citrus aurantifolia) Trees in Response to Huanglongbing Disease caused by Candidatus Liberibacter asiaticus.

Nowadays, Huanglongbing (HLB) disease, associated with Candidatus Liberibacter asiaticus (CLas), seriously affects citriculture worldwide, and no cure is currently available. Transcriptomic analysis of host–pathogen interaction is the first step to understand the molecular landscape of a disease. Previous works have reported the transcriptome profiling in response to HLB in different susceptible citrus species; however, similar studies in tolerant citrus species, including Mexican lime, are limited. In this work, we have obtained an RNA-seq-based differential expression profile of Mexican lime plants challenged against CLas infection, at both asymptomatic and symptomatic stages. Typical HLB-responsive differentially expressed genes (DEGs) are involved in photosynthesis, secondary metabolism, and phytohormone homeostasis. Enrichment of DEGs associated with biotic response showed that genes related to cell wall, secondary metabolism, transcription factors, signaling, and redox reactions could play a role in the tolerance of Mexican lime against CLas infection. Interestingly, despite some concordance observed between transcriptional responses of different tolerant citrus species, a subset of DEGs appeared to be species-specific. Our data highlights the importance of studying the host response during HLB disease using as model tolerant citrus species, in order to design new and opportune diagnostic and management methods.

Transcriptome Analyses of Diaphorina citri Midgut Responses to Candidatus Liberibacter Asiaticus Infection.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important transmission vector of the citrus greening disease Candidatus Liberibacter asiaticus (CLas). The D. citri midgut exhibits an important tissue barrier against CLas infection. However, the molecular mechanism of the midgut response to CLas infection has not been comprehensively elucidated. In this study, we identified 778 differentially expressed genes (DEGs) in the midgut upon CLas infection, by comparative transcriptome analyses, including 499 upregulated DEGs and 279 downregulated DEGs. Functional annotation analysis showed that these DEGs were associated with ubiquitination, the immune response, the ribosome, endocytosis, the cytoskeleton and insecticide resistance. KEGG enrichment analysis revealed that most of the DEGs were primarily involved in endocytosis and the ribosome. A total of fourteen DEG functions were further validated by reverse transcription quantitative PCR (RT-qPCR). This study will contribute to our understanding of the molecular interaction between CLas and D. citri.

Coordination of multiple regulation pathways contributes to the tolerance of a wild citrus species (Citrus ichangensis ‘2586’) against Huanglongbing

Huanglongbing (HLB), a highly destructive disease of citrus production worldwide, is presumably caused by ‘Candidatus Liberibacter asiaticus’ (CLas) because Koch's postulates have not been fulfilled. No HLB resistant genotypes have been so far identified in commercial citrus species, hindering the development of resistance breeding. China is an important center of citrus germplasm resources with abundant wild citrus species. However, studies on the susceptibility or resistance of these wild citrus and their response mechanisms to CLas infection have been limited. In this study, we evaluated the symptom development and transcriptional differences among two wild citrus species, Citrus ichangensis ‘2586’ and C. daoxianensis, and the highly susceptible C. sinensis. No typical symptoms was observed and much less CLas accumulated in C. ichangensis, compared with C. daoxianensis and C. sinensis. Much greater number of differentially expressed genes were identified and pathways were perturbed in diseased C. sinensis, followed by diseased C. daoxianensis then C. sinensis. Many pathways including cell wall, secondary metabolism, stress responses, RNA transcriptional regulation and protein modification were differentially regulated in three diseased citrus species but with different preferences. Particularly, genes associated with carbohydrate metabolism, callose deposition and photosynthesis process were significantly regulated in CLas-infected C. daoxianensis and C. sinensis but not in CLas-infected C. ichangensis. Taken together, it seem to indicate that the relatively successful defenses of C. ichangensis against multiplication of CLas and its symptom development may involve a novel mechanism in which the carbohydrate metabolism and photosynthesis process was balanced with the normal physiology of the plant, and coordinated with other regulation pathways. Evaluation of wild citrus resources to CLas infection is promising for citrus HLB resistance breeding in the future.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analyses of Citrus sinensis (L.) Osbeck Graft-Inoculated with "Candidatus Liberibacter asiaticus".

"Candidatus Liberibacter asiaticus" (CLas) is the bacterium associated with the citrus disease Huanglongbing (HLB). Current CLas detection methods are unreliable during presymptomatic infection, and understanding CLas pathogenicity to help develop new detection techniques is challenging because CLas has yet to be isolated in pure culture. To understand how CLas affects citrus metabolism and whether infected plants produce systemic signals that can be used to develop improved detection techniques, leaves from Washington Navel orange (Citrus sinensis (L.) Osbeck) plants were graft-inoculated with CLas and longitudinally studied using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry), and metabolomics (proton nuclear magnetic resonance). Photosynthesis gene expression and protein levels were lower in infected plants compared to controls during late infection, and lower levels of photosynthesis proteins were identified as early as 8 weeks post-grafting. These changes coordinated with higher sugar concentrations, which have been shown to accumulate during HLB. Cell wall modification and degradation gene expression and proteins were higher in infected plants during late infection. Changes in gene expression and proteins related to plant defense were observed in infected plants as early as 8 weeks post-grafting. These results reveal coordinated changes in greenhouse navel leaves during CLas infection at the transcript, protein, and metabolite levels, which can inform of biomarkers of early infection.

Enhancing PCR Capacity To Detect 'Candidatus Liberibacter asiaticus' Utilizing Whole Genome Sequence Information.

'Candidatus Liberibacter asiaticus' (CLas) is an unculturable α-proteobacterium associated with citrus Huanglongbing (HLB; yellow shoot disease). PCR procedures that accurately confirm or exclude CLas infection in citrus tissue/Asian citrus psyllid (ACP) samples are critical for HLB management. When CLas was described in 1994, a 23-bp signature oligonucleotide sequence (OI1) in the 16S rRNA gene (rrs, three genomic copies) was identified based on Sanger sequencing. OI1 contains single nucleotide polymorphisms (SNPs) distinguishing CLas from non-CLas species. The SNPs were used to design the primer HLBas, a key primer for a commonly used TaqMan PCR system (HLBas-PCR) for CLas detection. Recent developments in next-generation sequencing technology have led to the identification of 15 CLas whole genome sequence strains (WGSs). Analyses of CLas WGSs have generated a significant amount of biological information that could help to improve CLas detection. Utilizing the WGS information, this study re-evaluated the sequence integrity of OI1/HLBas and identified and/or confirmed a missing nucleotide G in the two primers. Replacement primers for OI1 and HLBas are proposed. At low cycle threshold (Ct) values (e.g., <30), HLBas-PCR remained reliable in CLas determination. At high Ct values (e.g., >30), HLBas-PCR alone was unreliable in differentiating whether samples contain low CLas titers or whether CLas is not present. The availability of ribonucleotide reductase (RNR)-PCR derived from the five-copy nrdB gene helped to resolve this problem. To further enhance low CLas titer detection, a 4CP-PCR system, based on a four-copy genomic locus, was developed. Evaluation of 107 HLB samples (94 citrus and 13 ACP) showed that 4CP-PCR was more sensitive than HLBas-PCR and shared similar sensitivity with RNR-PCR.

Metabolome and Microbiome Signatures in the Roots of Citrus Affected by Huanglongbing.

Huanglongbing (HLB) is a severe, incurable citrus disease caused by the bacterium 'Candidatus Liberibacter asiaticus' (CLas). Although citrus leaves serve as the site of initial infection, CLas is known to migrate to and colonize the root system; however, little is known about the impact of CLas infection on root metabolism and resident microbial communities. Scions of 'Lisbon' lemon and 'Washington Navel' orange grafted onto 'Carrizo' rootstock were grafted with either CLas-infected citrus budwood or uninfected budwood. Roots were obtained from trees 46 weeks after grafting and analyzed via 1H nuclear magnetic resonance spectroscopy to identify water-soluble root metabolites and high-throughput sequencing of 16S rRNA and ITS gene amplicons to determine the relative abundance of bacterial and fungal taxa in the root rhizosphere and endosphere. In both citrus varieties, 27 metabolites were identified, of which several were significantly different between CLas(+) and control plants. CLas infection also appeared to alter the microbial community structure near and inside the roots of citrus plants. Nonmetric multidimensional scaling (NMDS) and a principal coordinate analysis (PCoA) revealed distinct metabolite and microbial profiles, demonstrating that CLas impacts the root metabolome and microbiome in a manner that is variety-specific.

Reciprocal influences of rootstock and scion citrus cultivars challenged with Ca. Liberibacter asiaticus

The devastating citrus disease Huanglongbing (HLB), associated with bacterial pathogens of the genus Candidatus Liberibacter, has spread across many citrus production areas worldwide causing devastating economic losses. In Florida, infection rates in most commercial citrus orchards approach 100 percent by the time trees are 3–4 years old. Most scion cultivars are highly susceptible to Ca. Liberibacter asiaticus (CLas), the pathogen prevalent in Florida, but significant tolerance has been identified within the species Poncirus trifoliata (trifoliate orange) and some of its hybrids that are commonly used as rootstocks. In this study we investigated the relative influence of rootstock and scion in citrus plants composed of susceptible and tolerant cultivars in both the scion and the rootstock position on HLB disease progression and expression of stress and disease-related genes. Experiments were conducted in the greenhouse using the susceptible cultivars ‘Valencia’ orange (Citrus sinensis) and ‘Cleopatra’ mandarin (C. reticulata), and the tolerant trifoliate hybrid cultivars ‘US-802’ (C. grandis × P. trifoliata), ‘US-897’ (C. reticulata × P. trifoliata), and ‘US-942’ (C. reticulata × P. trifoliata). Plants were either mock-inoculated or graft-inoculated with CLas. The incidence of CLas infection at 6 and 12 months after inoculation and the severity of the disease was different among scion/rootstock combinations. Typically, trees with a tolerant cultivar in the scion position contained a lower number of bacteria and were less damaged by infection than trees with a susceptible cultivar in the scion position, regardless of rootstock. Previous observations of significant influence from rootstock on field tree tolerance to CLas may be partially the result of differential rootstock ability to tolerate other stresses, or respond favourably to therapeutic treatments, under the weakened condition caused by the infection. Differential expression of defense-related and other genes found largest fold-differences between non-infected and infected plants when a susceptible cultivar was in the scion position. Fewer genes responded in roots than in leaves, and genes associated with starch metabolism responded strongly and in an opposite direction when comparing roots and leaves, probably indicative of carbon depletion occurring in roots of infected plants.

Detection of ‘Candidatus Liberibacter asiaticus’ in citrus by concurrent tissue print‐based qPCR and immunoassay

‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the most destructive disease of citrus, huanglongbing (HLB). The most widely used methods for detection of CLas are PCR‐based and require purification of DNA from plant samples. Elution of DNA from tissue prints made on nitrocellulose membranes followed by qPCR (TPE‐qPCR) was compared to DNA extraction of plant tissue followed by PCR (X‐PCR) by testing the same tissue samples. The former estimated a higher CLas population in tissue prints than the latter (t‐test; P = 0.009). All extracts prepared for TPE‐qPCR throughout the experiment were also tested by conventional PCR and 80.8% were identified as positive. A similar set of stem and petiole tissue samples was tested by TPE‐qPCR and immunoassay. Although the detection rate by TPE‐qPCR was higher than by immunoassay, about 6% of tissue prints were positive by immunoassay but not by TPE‐qPCR. Thus, a higher detection rate would be achieved by combining TPE‐qPCR with immunoassay. Significant differences were observed in the performance of nitrocellulose membranes from different manufacturers in these assays. Immunotissue prints showed that the spatial distribution pattern of CLas infection varied widely from one sample to another, but the patterns were highly correlated among serial sections from the same sample, suggesting that CLas preferentially colonizes adjacent phloem cells in a vertical rather than horizontal direction.

Temporal Decline in Pathogen-Mediated Release of Methyl Salicylate Associated With Decreasing Vector Preference for Infected Over Uninfected Plants

Asian citrus psyllid Diaphorina citri Kuwayama is the vector of the phytopathogenic bacteria Candidatus Liberibacter asiaticus (CLas). D. citri is attracted to methyl salicylate (MeSA) that is emitted by CLas-infected citrus. MeSA is catalyzed from salicylic acid (SA) by salicylic acid methyl transferase (SAMT). In addition, salicylate hydroxylase, an enzyme that degrades SA and suppresses host plant defense, is unregulated in CLas-infected as compared with uninfected citrus. Based on these findings, we hypothesized that CLas-induced plant volatile emissions and SAMT expression may decrease over time following CLas infection. We first identified the putative Citrus sinensis SAMT (CsSAMT) that methylates SA to MeSA. Thereafter we compared MeSA emission and SAMT expression between uninfected citrus, citrus recently ( 1year) CLas-infected citrus. We found that MeSA emission increased in citrus recently infected with CLas, whereas both MeSA emissions and SAMT expression decreased in long-infected citrus. In behavioral assays, D. citri preferred settling in asymptomatic citrus recently infected with CLas rather than on uninfected citrus trees or long-infected trees. We conducted olfactometer assays that showed that the odor of citrus recently infected with CLas was more attractive to psyllid than the odor of uninfected citrus of long-infected trees. We linked the preference for citrus odor recently infected with CLas to the increase of MeSA emission following CLas infection. Overall, our study demonstrated pulsed changes in citrus odor following CLas infection. These changes affected the behavior of the CLas vector. Our study confirms previous reports demonstrating that plant defenses boosted following phytopatogen infection tend to decrease over time. Implications on the CLas epidemic are discussed.

A Targeted Mass Spectrometry-Based Metabolomics Approach toward the Understanding of Host Responses to Huanglongbing Disease.

Candidatus Liberibacter asiaticus (CLas) is the major culprit of Huanglongbing (HLB), the most destructive citrus disease worldwide. The polymerase chain reaction (PCR) is the most common method for detecting the presence of CLas in the tree. However, due to the uneven distribution of bacteria and a minimum bacterial titer requirement, an infected tree may test false negative. Thus, our current study profiled primary and secondary metabolites of CLas-free leaves harvested from a citrus undercover protection system (CUPS) to prevent a misjudgment of CLas infection. Functional enrichment analysis revealed several metabolic pathways significantly affected by CLas infection, mainly biosynthesis of amino acids and secondary metabolites. Comparisons of CLas-infected metabolite alterations among oranges, mandarins, and grapefruits revealed that host responses to CLas were different. The metabolite signature highlighted in this study will provide a fuller understanding of how CLas bacteria affect the biosynthesis of primary and secondary metabolites in different hosts.

Host Plant-Mediated Interactions Between 'Candidatus Liberibacter asiaticus' and Its Vector Diaphorina citri Kuwayama (Hemiptera: Liviidae).

The Diaphorina citri Kuwayama (Hemiptera: Liviidae) also known as Asian citrus psyllid transmits 'Candidatus Liberibacter asiaticus' (CLas) associated with the citrus Huanglongbing (HLB). Minimizing Asian citrus psyllid populations is one of the most important methods for HLB control. The development and survival of Asian citrus psyllid were evaluated on CLas+ and CLas- plants of Citrus reticulata Blanco. cv. Mashuiju (Sapindales: Rutaceae) or Citrus sunki Hort. ex Tanaka (Sapindales: Rutaceae) to assess the effects of CLas infection on vector populations. The development times were unaffected between CLas+ and CLas- plants for eggs and first to third instar nymphs but decreased for fourth instars, fifth instars, and total nymphal life on CLas+ plants. The survival rates of eggs and first and second instar nymphs were significantly lower, while those of third to fifth instar nymphs were significantly higher on CLas+ plants. However, overall nymphal survival did not differ between CLas+ and CLas- plants. The pre-oviposition period of Asian citrus psyllid on CLas+ plants decreased, while oviposition period increased, resulting in significantly higher fecundity and population trend index compared with those on CLas- plants, illustrating an overall fitness benefit of CLas infection to Asian citrus psyllid. On the other hand, the longevity and resistance to starvation and lower temperatures of adults from CLas+ plants were significantly lower than those from CLas- plants suggesting that there may be a fitness cost for Asian citrus psyllid on CLas+ plants. The development time of eggs and nymphs and adult longevity were always shorter on C. reticulata regardless of infection status. This knowledge of host plant, pathogen, and vector interactions is useful for developing Asian citrus psyllid-HLB management programs.