Causal Agent Of Citrus Huanglongbing Research Articles

Integrated bacterial transcriptome and host metabolome analysis reveals insights into "Candidatus Liberibacter asiaticus" population dynamics in the fruit pith of three citrus cultivars with different tolerance.

"Candidatus Liberibacter asiaticus" (CLas), the causal agent of citrus Huanglongbing (HLB), is able to multiply to a high abundance in citrus fruit pith. However, little is known about the biological processes and phytochemical substances that are vital for CLas colonization and growth in fruit pith. In this study, CLas-infected fruit pith of three citrus cultivars ("Shatangju" mandarin, "Guanxi" pomelo, and "Shatian" pomelo) exhibiting different tolerance to CLas were collected and used for dual RNA-Seq and untargeted metabolome analysis. Comparative transcriptome analysis found that the activation of the CLas noncyclic TCA pathway and pathogenic-related effectors could contribute to the colonization and growth of CLas in fruit pith. The pre-established Type 2 prophage in the CLas genome and the induction of its CRISPR/cas system could enhance the phage resistance of CLas and, in turn, facilitate CLas population growth in fruit pith. CLas infection caused the accumulation of amino acids that were correlated with tolerance to CLas. The accumulation of most sugars and organic acids in CLas-infected fruit pith, which could be due to the phloem blockage caused by CLas infection, was thought to be beneficial for CLas growth in localized phloem tissue. The higher levels of flavonoids and terpenoids in the fruit pith of CLas-tolerant cultivars, particularly those known for their antimicrobial properties, could hinder the growth of CLas. This study advances our understanding of CLas multiplication in fruit pith and offers novel insight into metabolites that could be responsible for tolerance to CLas or essential to CLas population growth.IMPORTANCECitrus Huanglongbing (HLB, also called citrus greening disease) is a highly destructive disease currently threatening citrus production worldwide. HLB is caused by an unculturable bacterial pathogen, "Candidatus Liberibacter asiaticus" (CLas). However, the mechanism of CLas colonization and growth in citrus hosts is poorly understood. In this study, we utilized the fruit pith tissue, which was able to maintain the CLas at a high abundance, as the materials for dual RNA-Seq and untargeted metabolome analysis, aiming to reveal the biological processes and phytochemical substances that are vital for CLas colonization and growth. We provided a genome-wide CLas transcriptome landscape in the fruit pith of three citrus cultivars with different tolerance and identified the important genes/pathways that contribute to CLas colonization and growth in the fruit pith. Metabolome profiling identified the key metabolites, which were mainly affected by CLas infection and influenced the population dynamic of CLas in fruit pith.

Association of Non-host Crop Plants with Mandarin in Host Location and Survival of Diaphorina citri Kuwayama (Hemiptera: Psyllidae).

Research efforts have been made to develop novel tactics, such as those targeting behavioral control, for management of the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae), vector of the causal agent of citrus Huanglongbing. Here, we investigated whether association of "Ponkan" mandarin (Citrus reticulata) with volatiles from non-host crops: avocado, passion fruit or coffee, alters host location by the Asian citrus psyllid; and whether they can be temporary hosts for the Asian citrus psyllid. In wind tunnel assays, we found that the association of mandarin seedling with avocado plant volatiles reduced in 30% the number of psyllids sitting on host plants compared to the mandarin alone. In contrast, passion fruit plant volatiles facilitated host location by psyllids, which found mandarin seedlings faster than when exposed to mandarin alone. The association with coffee volatiles did not alter the attractiveness of mandarin to the Asian citrus psyllid. Survival and half-lethal time (LT50) of D. citri fed on non-host plants were longer than those insects with water only, but shorter than those fed on mandarin. Among the non-host plants, D. citri performed better in coffee, followed by avocado and passion fruit plants. Our results indicate that the association of mandarin with avocado plant can be beneficial for Asian citrus psyllid management.

Antennae-abundant expression of candidate cytochrome P450 genes associated with odorant degradation in the asian citrus psyllid, Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, is a notorious pest that is an efficient vector for Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus huanglongbing (HLB). The olfactory system of insects is crucial for foraging and mating behavior. Antennae-abundant odorant degrading enzymes (ODEs), including cytochrome P450 (CYPs), are important in degrading redundant odorant molecules to recover the insect olfactory. In this study, to isolate the antennal CYP genes of D. citri, we generated four transcriptomes from female/male antennae and body through deep sequencing of RNA libraries. Seven DcCYP genes preferentially expressed in antennae were first identified by comparing the antennal and body transcriptomes. Phylogenetic analysis grouped four DcCYPs (DcCYP6a13, DcCYP6j1, DcCYP6k1, and DcCYP6a2) into the CYP3 class, whereas DcCYP4d2, DcCYP4c62, and DcCYP4d8 were clustered in the CYP4 clade. qRT-PCR analyses across developmental stages and tissues showed they were antennae-abundant in both genders and constantly expressed from the first instar nymph to the adult. The results presented here highlight the isolation and expression of CYP genes in D. citri antennae, providing valuable insights into their putative role in odorant degradation.

Biological Features and In Planta Transcriptomic Analyses of a Microviridae Phage (CLasMV1) in "Candidatus Liberibacter asiaticus".

“Candidatus Liberibacter asiaticus” (CLas) is the causal agent of citrus Huanglongbing (HLB, also called citrus greening disease), a highly destructive disease threatening citrus production worldwide. A novel Microviridae phage (named CLasMV1) has been found to infect CLas, providing a potential therapeutic strategy for CLas/HLB control. However, little is known about the CLasMV1 biology. In this study, we analyzed the population dynamics of CLasMV1 between the insect vector of CLas, the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) and the holoparasitic dodder plant (Cuscuta campestris Yunck.); both acquired CLasMV1-infected CLas from an HLB citrus. All CLas-positive dodder samples were CLasMV1-positive, whereas only 32% of CLas-positive ACP samples were identified as CLasMV1-positive. Quantitative analyses showed a similar distribution pattern of CLasMV1 phage and CLas among eight citrus cultivars by presenting at highest abundance in the fruit pith and/or the center axis of the fruit. Transcriptome analyses revealed the possible lytic activity of CLasMV1 on CLas in fruit pith as evidenced by high-level expressions of CLasMV1 genes, and CLas genes related to cell wall biogenesis and remodeling to maintain the CLas cell envelope integrity. The up-regulation of CLas genes were involved in restriction–modification system that could involve possible phage resistance for CLas during CLasMV1 infection. In addition, the regulation of CLas genes involved in cell surface components and Sec pathway by CLasMV1 phage could be beneficial for phage infection. This study expanded our knowledge of CLasMV1 phage that will benefit further CLas phage research and HLB control.

Novel insight into the distribution and dissemination of Candidatus Liberibacter asiaticus, the causal agent of citrus Huanglongbing.

Huanglongbing (HLB), also known as citrus greening, is the most severe pandemics in citrus in more than 50 countries in Asia, Africa and America, causing serious economic losses worldwide (Gottwald, 2020; Wang, 2019). The disease is associated with a phloem-limited and fastidious member of the α-proteobacteriacea. 'Candidatus Liberibacter asiaticus' (CLas) is the most prevalent strain. It was frequently detected in leaves, stems and roots based on PCR detection which recognized both living and dead cells.

Genetic Diversity of Tamarixia radiata Populations and Their Associated Endosymbiont Wolbachia Species from China

Tamarixia radiata is one of the established biocontrol pests against the major Asian citrus psyllid, Diaphorina citri, a vector of Candidatus Liberibacter that is a causal agent of citrus Huanglongbing (HLB) disease. Updated information and regional exploration on biocontrol pests are important elements for effective disease management strategies. In this study, the diversity and parasitism rate of T. radiata populations were evaluated. Due to the importance of the host–parasitoid relationship, the presence of Wolbachia as an endosymbiont was also investigated. The parasitism rate of various T. radiata populations from Ecuador and China ranged between 57.27% and 66.32%, respectively, with a non-significant emergence rate and a statistically similar sex ratio. Sequence analysis of ITS and COI from T. radiata populations was consistent with the morphological hypothesis that the collections represent a single species, whereas phylogeny of the wsp gene confirmed the presence of Wolbachia pipientis as an endosymbiont within T. radiata populations. Based on partial COI sequences, the maximum genetic diversity such as total haplotype diversity (Hd = 0.788), nucleotide, diversity (π = 0.2439), and average nucleotide difference (k = 171.844) was also estimated for different T. radiata populations. Furthermore, neutrality tests based on COI sequences indicated an overall contraction in T. radiata populations, whereas an expansion trend was observed in associated W. pipientis strains. This study clearly demonstrated the presence of genetically diverse T. radiata populations that were able to parasitize D. citri effectively, and these can be further explored as promising biocontrol candidates in integrated pest management strategies to solve citriculture economic loss caused by D. citri.

The Total Population Size of 'Candidatus Liberibacter asiaticus' Inside the Phloem of Citrus Trees and the Corresponding Metabolic Burden Related to Huanglongbing Disease Development.

'Candidatus Liberibacter asiaticus' (CLas) is the predominant causal agent of citrus huanglongbing, the most devastating citrus disease worldwide. CLas colonizes phloem tissue and causes phloem dysfunction. The pathogen population size in local tissues and in the whole plant is critical for the development of disease symptoms by determining the load of pathogenicity factors and metabolic burden to the host. However, the total population size of CLas in a whole plant and the ratio of CLas to citrus cells in local tissues have not been addressed previously. The total CLas population size for 2.5-year-old 'Valencia' sweet orange on 'Kuharske' citrange rootstock trees was quantified using quantitative PCR to be approximately 1.74 × 109 cells/tree, whereas 7- and 20-year-old sweet orange trees were estimated to be 4.3 × 1010 cells/tree, and 6.0 × 1010 cells/tree, respectively. The majority of CLas cells were distributed in leaf tissues (55.58%), followed by those in branch (36.78%), feeder root (4.75%), trunk (2.39%), and structural root (0.51%) tissues. The ratios of citrus cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples were within approximately 39 to 79, 44 to 124, 153 to 1,355, 191 to 1,054, and 561 to 3,760, respectively, representing the metabolic burden of CLas in different organs. It was estimated that the ratios of phloem cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples are approximately 0.39 to 0.79, 0.44 to 1.24, 1.53 to 13.55, 1.91 to 10.54, and 5.61 to 37.60, respectively. Approximately 0.01% of the total citrus phloem volume was estimated to be occupied by CLas, explaining the difficulty to observe CLas in most tissues under transmission electron microscopy. The CLas titer inside the leaf was estimated to be approximately 1.64 × 106 cells/leaf or 9.2 × 104 cells cm-2 in leaves, approximately 104 times less than that of typical apoplastic bacterial pathogens. This study provides quantitative estimates of phloem colonization by bacterial pathogens and furthers the understanding of the biology and virulence mechanisms of CLas.

Spatiotemporal Dynamics of 'Candidatus Liberibacter asiaticus' Colonization Inside Citrus Plant and Huanglongbing Disease Development.

'Candidatus Liberibacter asiaticus' (CLas), the causal agent of citrus huanglongbing (HLB), colonizes inside the phloem and is naturally transmitted by the Asian citrus psyllid (ACP). Here, we investigated spatiotemporal CLas colonization in different tissues after ACP transmission. Of the nine plants successfully infected via ACP transmission, CLas was detected in the roots of all trees at 75 days postremoval of ACPs (DPR) but in the mature leaf of only one tree; this finding is consistent with the model that CLas moves passively from source to sink tissues. At 75 and 365 DPR, CLas was detected in 11.1 and 43.1% of mature leaves not fed on by ACPs during transmission, respectively, unveiling active movement to the source tissue. The difference in colonization timing of sink and source tissues indicates that CLas is capable of both passive and active movement, with passive movement being dominant. At 225 DPR, leaves fed on by ACPs during the young stage showed the highest ratio of HLB symptomatic leaves and the highest CLas titer, followed by leaves that emerged after ACP removal and mature leaves not fed on by ACPs. Importantly, our data showed that ACPs were unable to transmit CLas via feeding on mature leaves. It is estimated that it takes 3 years at most for CLas to infect the whole tree. Overall, spatiotemporal detection of CLas in different tissues after ACP transmission helps visualize the infection process of CLas in planta and subsequent HLB symptom development and provides evidence showing that young leaves should be the focus of HLB management.

A Sec-Dependent Secretory Protein of the Huanglongbing-Associated Pathogen Suppresses Hypersensitive Cell Death in Nicotiana benthamiana.

“Candidatus Liberibacter asiaticus” (CLas) is a phloem-restricted Gram-negative bacterium that is the causal agent of citrus huanglongbing (HLB). In this study, we identified a CLas-encoded Sec-dependent secretory protein CLIBASIA_04405 that could contribute to the pathogenicity of this bacterium. The gene expression level of CLIBASIA_04405 was significantly higher in citrus than in psyllids. Transient overexpression of the mature CLIBASIA_04405 protein (m4405) in Nicotiana benthamiana leaves could suppress hypersensitive response (HR)-based cell death and H2O2 accumulation triggered by the mouse BAX and the Phytophthora infestans INF1. An alanine-substitution mutagenesis assay revealed the essential of amino acid clusters EKR45–47 and DE64–65 in cell death suppression. Challenge inoculation of the transgenic N. benthamiana-expressing m4405 with Pseudomonas syringae DC3000ΔhopQ1-1 demonstrated the greatly reduced bacterial proliferation. Remarkably, transcriptome profiling and RT-qPCR analysis disclosed that the gene expression of six small heat shock proteins (sHSPs), a set of plant defense regulators, were significantly elevated in the transgenic m4405 lines compared with those in wild-type N. benthamiana. In addition, the transgenic m4405 lines displayed phenotypes of dwarfism and leaf deformation. Altogether, these data indicated that m4405 was a virulence factor of CLas.

Development on Infected Citrus over Generations Increases Vector Infection by 'Candidatus Liberibacter Asiaticus in Diaphorina citri'.

‘Candidatus Liberibacter asiaticus’ (CLas) is a major causal agent of citrus Huanglongbing (HLB), which is transmitted by Asian citrus psyllid (ACP), Diaphorina citri, causing severe losses in various regions of the world. Vector efficiency is higher when acquisition occurs by ACP immature stages and over longer feeding periods. In this context, our goal was to evaluate the progression of CLas population and infection rate over four ACP generations that continuously developed on infected citrus plants. We showed that the frequency of CLas-positive adult samples increased from 42% in the parental generation to 100% in the fourth generation developing on CLas-infected citrus. The bacterial population in the vector also increased over generations. This information reinforces the importance of HLB management strategies, such as vector control and eradication of diseased citrus trees, to avoid the development of CLas-infected ACP generations with higher bacterial loads and, likely, a higher probability of spreading the pathogen in citrus orchards.

Friend or foe? Relationship between ‘Candidatus Liberibacter asiaticus’ and Diaphorina citri

Interactions between insects and plant pathogens have been more enthusiastically studied in the recent decade, especially those relationships which takes the insects as vectors. The spectrum of these interactions ranges from mutualistic to pathogenic. The length of the co-evolutionary process will determine whether a microorganism shares a friend or a foe relationship with its host, and a friendship connection is frequently observed if the coexistence is longer. This review updates knowledge about the morphological, physiological and genetic mechanisms that drive the interaction between ‘Candidatus Liberibacter asiaticus’ (Las) and its vector, the Asian citrus psyllid, Diaphorina citri. Las is the predominant causal agent of citrus huanglongbing (HLB) disease, the major constrain to citrus production worldwide. This bacterium is transmitted by D. citri, in a propagative-circulative manner during its feeding from plant host. Understanding of the interactions among vector, plant pathogen and host plant are important for the management of this vector-borne disease complex.

The in Planta Effective Concentration of Oxytetracycline Against 'Candidatus Liberibacter asiaticus' for Suppression of Citrus Huanglongbing.

Huanglongbing (HLB) or greening currently is the most devastating citrus disease worldwide. The fastidious phloem-colonizing bacterium 'Candidatus Liberibacter asiaticus' (CLas) is the causal agent of citrus HLB in Florida. Bactericides containing the active ingredient oxytetracycline (OTC) have been used in foliar spray to control citrus HLB in Florida since 2016. However, the minimum concentration of OTC required to suppress CLas in planta remains unknown. We developed a new method for evaluating the effects of OTC treatment on CLas titers in infected plants and determined the relationship between OTC residue levels and control levels achieved for CLas using mathematical modeling in greenhouse and field experiments. In both greenhouse and field, OTC spray did not reduce the titers of CLas, and it produced undetectable or mild levels of OTC residue in leaves within 7 days post-application (DPA). In greenhouse, OTC injection at 0.05 g per tree decreased CLas titers to an undetectable level (cycle threshold value ≥ 36.0) from 7 to 30 DPA and produced a residue level of OTC at 0.68 to 0.73 µg/g of fresh tissue over this period. In the field, OTC injection at 0.50 g per tree resulted in the decline of CLas titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27 to 0.33 µg/g of fresh tissue. In both trials, a first-order compart model of OTC residue dynamics in leaves of trunk-injected trees was specified for estimating the retention of effective concentrations. Furthermore, nonlinear modeling revealed significant positive correlations between OTC residue levels in leaves and the control levels for CLas achieved. The results suggested that the minimum concentrations of OTC required to suppress CLas populations in planta to below the detection limit are 0.68 and 0.86 µg/g and that the minimum concentrations of OTC required for initial inhibition of CLas growth in planta are ∼0.17 and ∼0.215 µg/g in leaf tissues under greenhouse and field conditions, respectively. This finding highlights that a minimum concentration of OTC should be guaranteed to be delivered to target CLas in infected plants for effective control of citrus HLB.

The secreted salivary proteome of Asian citrus psyllid Diaphorina citri

AbstractThe Asian citrus psyllid Diaphorina citri Kuwayama is a sap‐sucking hemipteran that serves as a vector for Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus huanglongbing (HLB). Salivary secretions play critical roles in insect–host plant interactions and are responsible for the establishment of insect herbivory. In the present study, we aim to identify the major proteins in D. citri saliva. Saliva proteins are collected from the artificial diet upon D. citri feeding. Collected proteins are concentrated, filtered, digested and then analyzed using liquid chromatography‐tandem spectrometry (MS/MS). The MS/MS data are then analyzed by searching the D. citri genome deposited in the NCBI. Eighty‐nine proteins are identified in D. citri saliva. Eighty‐six of these proteins are from D. citri, whereas three proteins are from Wolbachia endosymbiont. The proteins identified belong to several functional groups, including enzymes, cytoskeletal proteins, sheath proteins, receptor proteins, transporter proteins, chromatin‐, RNA‐ and DNA‐binding proteins, and other proteins. These proteins could be involved in many key functions, including detoxification of plant defencee compounds and suppression of plant defence, as well as facilitation of stylet penetration, sap sucking and digestion. The identification of these proteins is an essential step for the understanding of D. citri–plant interactions. The identification of key enzymes in the saliva of D. citri could lead to a discovery of proper inhibitors that disrupt its feeding, consequently preventing the spread of the HLB pathogen.

Temperature and host age effects on the life history of Tamarixia radiata, a dominant parasitoid of citrus psyllid Diaphorina citri

The Asian citrus psyllid, Diaphorina citri, is the primary vector for the bacterium Candidatus Liberibacter asiaticus, the causal agent of Citrus Huanglongbing. In this study, we investigated the effects of temperature and psyllid host age on the biology of Tamarixia radiata, the species-specific dominant nymphal parasitoid of D. citri in South China. Both temperature and host age significantly affected the fecundity, parasitism, and development of T. radiata. For the temperature effects, the averaged fecundity and parasitism of T. radiata was significantly increased with temperatures between 18 and 26 °C. The highest averaged fecundity (15.6 eggs/female) and parasitism (78%) were recorded at 26 °C. The developmental time of T. radiata on the 4th and 5th instars nymphs averaged 19.6 days at 18 °C, 13.4 days at 26 °C, and 10.2 days at 34 °C. The optimal temperatures for the development and survival of T. radiata was in the range of 26–30 °C. For the age effects, the development time from egg to adult parasitoid of T. radiata increased with the age of host nymph, 11.5 days in the 3rd instar, 12.3 days in the 4th instar, and 13.4 days in the 5th instar at 26 °C. Moreover, citrus psyllid host age also affected the sex ratio of T. radiata, with 42.78%, 60.66%, and 76.56% females developing from 3rd, 4th and 5th instar nymphs, respectively. These findings may prove to be helpful toward the development of biological control strategies targeting D. citri.

The Asian Citrus Psyllid Host Murraya koenigii Is Immune to Citrus Huanglongbing Pathogen ‘Candidatus Liberibacter asiaticus’

The Asian citrus psyllid (ACP) Diaphorina citri, vector of 'Candidatus Liberibacter asiaticus' (CLas), the putative causal agent of citrus Huanglongbing (HLB), is controlled by application of insecticides, which, although effective, has resulted in serious biological imbalances. New management tools are needed, and the technique known as "trap crop" has been attracting attention. A potential plant for use as a trap crop in the management of the ACP is Murraya koenigii (curry leaf). However, for this plant to be used in the field, it needs to be attractive for the vector and must not harbor CLas. To verify the potential of curry leaf as trap crop for the management of HLB, we investigated the ability of D. citri to transmit CLas to M. koenigii, and to other test plants, including M. paniculata (orange jasmine) and cultivar Valencia sweet-orange seedlings. For the tests, the insects were reared on a symptomatic CLas-infected plant and allowed to feed on the three test plant species. The overall maximum transmission rate for the citrus seedlings was 83.3%, and for orange jasmine was 33.3%. Successful transmission of CLas by ACP to the curry-leaf seedlings was not observed, and it was treated as immune to CLas. Supported by the previous results that M. koenigii is attractive for ACP, these results indicate that curry leaf is an excellent candidate for use as a trap crop, to improve the management of the insect vector and consequently of HLB.

Effect of parental RNA interference of a transformer‐2 homologue on female reproduction and offspring sex determination in Asian citrus psyllid

AbstractAsian citrus psyllid Diaphorina citri Kuwayama is the vector of Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus huanglongbing (HLB). A comprehensive understanding of molecular components that govern key physiological traits in D. citri would promote the development of novel genetic control strategies to fight this pest. In the present study, a transformer 2 homologue (Dctra‐2) implicated in embryonic sex differentiation in dipteran insects is targeted to interfere with the offspring sex ratio of D. citri. The results of the present study show that Dctra‐2 is constitutively expressed at all developmental stages and in both females and males during the adult stage. Quantitative reverse transcriptase‐polymerase chain reaction analysis confirms the double‐stranded RNA‐mediated RNA interference (RNAi) of the Dctra‐2. RNAi effects in D. citri female adults can be transferred in a trans‐generational manner to their eggs and progeny, which are commonly referred as parental RNAi. RNAi females lay fewer eggs, with a mean of 12.44, and the hatching rate of laid eggs is reduced approximately by 29.86% compared with the control. However, parental RNAi of Dctra‐2 do not produce male‐biased progeny. The results suggest that Dctra‐2 is an effective RNAi target for use in the management of D. citri. In addition, the parental RNAi would provide an effective tool for studying the function of early embryonic genes in D. citri.

'Candidatus Liberibacter asiaticus', Causal Agent of Citrus Huanglongbing, Is Reduced by Treatment with Brassinosteroids.

Huanglongbing (HLB) constitutes the most destructive disease of citrus worldwide, yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, ‘Candidatus Liberibacter asiaticus’. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced in leaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified. The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB.

Development of single chain variable fragment (scFv) antibodies against surface proteins of ‘Ca. Liberibacter asiaticus’

‘Candidatus Liberibacter asiaticus’ is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of ‘Ca. Liberibacter asiaticus’. The primary library has more than 107 unique antibodies and the genes that encode them. We have screened this library for antibodies that bind to specifically-chosen proteins that are present on the surface of ‘Ca. Liberibacter asiaticus’. These proteins were used as targets for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule protein KpsF; a protein component of the type IV pilus (CapF); and, two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and ‘Ca. Liberibacter asiaticus’ infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for the production of scFvs. Screening of these scFvs was more efficient when phage-bound, rather than soluble scFvs, were used. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by ‘Ca. Liberibacter asiaticus’. Applications could include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications.

Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

‘Candidatus Liberibacter asiaticus’ (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.

Comparison between single and duplex conventional PCR for detection of Candidatus Liberibacter asiaticus, the causal agent of citrus Huanglongbing disease in Thailand

Detection of Candidatus Liberibacter asiaticus (Las), the causal agent of citrus Huanglongbing (HLB) disease, was carried out using a newly developed duplex conventional PCR and was proven with a single conventional PCR for rapid and accurate diagnosis of HLB in Thailand. Symptomatic and non-symptomatic leaves of Citrus spp. were collected from commercial citrus grower groves and private back yard gardens throughout Thailand. For comparison, known HLB samples were received from international sources. Total DNA of citrus leaf mid veins was extracted following standard CTAB methods. Single conventional PCR using outer membrane protein (omp) gene with forward primer OMP/TH1 and reverse primer OMP/TH2 and DNA polymerase (phpol2) gene with forward primer PhPol2F and reverse primer PhPol2R resulted in a 981 bp and 710 bp product, respectively. Duplex conventional PCR gave two bands of 981 bp and 710 bp at the same size as single conventional PCR. Detection accuracy of omp and phpol2 genes by single and duplex conventional PCR techniques was 100% and 85.71%, respectively when compared with rplJ gene as a standard method. Detection of 61 citrus samples by single conventional PCR of omp and phpol2 genes and duplex conventional PCR gave Las positive at 72.13%, 80.33% and 78.69%, respectively. However, duplex conventional PCR provided a better detection than single conventional PCR because it gave positive detection for both genes together or each gene alone. Sensitivity detection of citrus total DNA with omp and phpol2 genes by single conventional PCR was 10−2 (1.6 × 10−2 μg) dilution and 10−6 (2 × 10−6 μg) dilution, respectively. The use of two genes in a duplex conventional PCR was more reliable and was less costly than two separate single conventional PCR reactions. Furthermore, the developed duplex conventional PCR will be useful for Las detection and identification for plant health certification and control program.