Candidatus Liberibacter Research Articles

Endophytes and Plant Extracts as Potential Antimicrobial Agents against Candidatus Liberibacter Asiaticus, Causal Agent of Huanglongbing.

Huanglongbing (HLB), also known as citrus greening, is an insidious disease in citrus and has become a threat to the sustainability of the citrus industry worldwide. In the U.S., Candidatus Liberibacter asiaticus (CLas) is the pathogen that is associated with HLB, an unculturable, phloem-limited bacteria, vectored by the Asian Citrus Psyllid (ACP, Diaphorina citri). There is no known cure nor treatment to effectively control HLB, and current control methods are primarily based on the use of insecticides and antibiotics, where effectiveness is limited and may have negative impacts on beneficial and non-target organisms. Thus, there is an urgent need for the development of effective and sustainable treatment options to reduce or eliminate CLas from infected trees. In the present study, we screened citrus-derived endophytes, their cell-free culture supernatants (CFCS), and crude plant extracts for antimicrobial activity against two culturable surrogates of CLas, Sinorhizobium meliloti and Liberibacter crescens. Candidates considered high-potential antimicrobial agents were assessed directly against CLas in vitro, using a propidium monoazide-based assay. As compared to the negative controls, statistically significant reductions of viable CLas cells were observed for each of the five bacterial CFCS. Subsequent 16S rRNA gene sequencing revealed that each of the five bacterial isolates were most closely related to Bacillus amyloliquefaciens, a species dominating the market of biological control products. As such, the aboveground endosphere of asymptomatic survivor citrus trees, grown in an organic orchard, were found to host bacterial endophytes capable of effectively disrupting CLas cell membranes. These results concur with the theory that native members of the citrus microbiome play a role in the development of HLB. Here, we identify five strains of Bacillus amyloliquefaciens demonstrating notable potential to be used as sources of novel antimicrobials for the sustainable management of HLB.

Real-time on-site detection of the three 'Candidatus Liberibacter' species associated with HLB disease: a rapid and validated method.

Huanglongbing (HLB) is a devastating disease that affects all commercial citrus species worldwide. The disease is associated with bacteria of three species of the genus 'Candidatus Liberibacter' transmitted by psyllid vectors. To date, HLB has no cure, so preventing its introduction into HLB-free areas is the best strategy to control its spread. For that, the use of accurate, sensitive, specific, and reliable detection methods is critical for good integrated management of this serious disease. This study presents a new real-time recombinase polymerase amplification (RPA) protocol able to detect the three 'Ca. Liberibacter' species associated with HLB in both plant and insect samples, validated according to European and Mediterranean Plant Protection Organization (EPPO) guidelines and tested on 365 samples from nine different geographic origins. This new protocol does not require nucleic acid purification or specialized equipment, making it ideal to be used under field conditions. It is based on specific primers and probe targeting a region of fusA gene, which shows a specificity of 94%-100%, both in silico and in vitro, for the 'Ca. Liberibacter' species associated with HLB. The analytical sensitivity of the new protocol is excellent, with a reliable detection limit in the order of 101 copies per microliter in HLB-infected plant and insect material. The repeatability and reproducibility of the new methods showed consistent results. Diagnostic parameters of the new RPA protocol were calculated and compared with the gold standard technique, a quantitative real-time PCR, in both crude extracts of citrus plants and insect vectors. The agreement between the two techniques was almost perfect according to the estimated Cohen's kappa index, with a diagnostic sensitivity and specificity of 83.89% and 100%, respectively, and a relative accuracy of 91.59%. Moreover, the results are obtained in less than 35min. All these results indicate the potential of this new RPA protocol to be implemented as a reliable on-site detection kit for HLB due to its simplicity, speed, and portability.

Uptake of micronutrients in severely HLB-affected grapefruit trees grown on Florida Indian River flatwood soils

Citrus greening (HLB, huanglongbing) is caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus and vectored by the Asian citrus psyllid. To combat HLB-induced nutrient uptake issues, growers use higher fertilizer application rates to prolong the producing life of affected trees. However, current nutrient recommendations in Florida were created in pre-HLB times and must be adjusted. Therefore, the objective of this study was to compare granular and liquid fertilization to identify best application methods for uptake of micronutrients into both belowground and aboveground components of HLB-affected grapefruit grown on flatwood soils. A large-scale field study consisting of 6-year-old HLB-affected “Ruby Red” grapefruit trees grafted on “sour orange” rootstock was conducted in the Florida Indian River District. Eight different treatments consisting of four liquid and four granular fertilizers with 1×, 2×, and 4× the current University of Florida, Institute of Food and Agricultural Sciences recommended rates of boron, zinc, manganese, and iron were applied three times a year (granular), biweekly (liquid), or weekly (liquid), for 3 years. Analyses of nutrients in soil, root, and leaf were conducted every 6 months throughout the study. Higher rates of boron, zinc, and manganese were observed in the soil of plots treated with liquid fertilizers with 2× and 4× the micronutrient recommendations. Root and leaf micronutrient analysis revealed significant differences in boron, zinc, manganese, and iron among treatments which varied between seasons. This study demonstrated that ground-applied micronutrient overdoses and application methods impacted nutrient uptake, however, no patterns with soil concentrations and tree uptake were established.

Direct Infusion Device for Molecule Delivery in Plants.

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed. To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant's trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed. Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants fortesting and facilitate research and screening purposes.

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Cost Analysis of Using Cover Crops in Citrus Production

Florida has a long association with citrus (Citrus sp.) production. However, the citrus industry in Florida has been devastated by Huanglongbing (HLB) or citrus greening disease (Candidatus Liberibacter asiaticus). HLB affects the citrus tree phloem and eventually causes tree death. Cover crops, or noncash crops, have traditionally been used in row crop production to improve soil organic matter, for nitrogen fixing, and for weed control. Citrus growers may benefit from adopting cover crops because healthier soils could improve yields and fruit quality of citrus trees. However, growers are uncertain about the costs and benefits associated with cover crop investments. The objective of our study was to analyze whether cover crops represent an economically feasible option for Florida citrus growers. We calculated the break-even prices for ‘Valencia’ and non-‘Valencia’ oranges (Citrus sinensis) in terms of price per box (equivalent to 90 lb of oranges in Florida) and price per pound solids per box (amount of soluble solids per box of oranges) by considering additional costs and short-term savings from using cover crops across various yield and quality scenarios based on the past 10 years of data. Considering the short-term savings from adopting cover crops, the per-acre cost of production increased by $107.3/acre or by 5.73% and constituted 5.42% of the total production cost during the first year of adoption. After the 2018–19 peak, the yield and quality for both ‘Valencia’ and non-‘Valencia’ oranges have decreased steadily. Adopting cover crops in the current yield–quality scenario will not be profitable for either ‘Valencia’ or non-‘Valencia’ oranges. However, for ‘Valencia’ oranges, at the median yield and quality levels of 193.5 boxes/acre and 6.08 lb solids/box, respectively, cover crop adoption would be profitable because the break-even price of $2.25/lb solids would be comparable to the market prices of the past 5 years.

Development of a Loop-Mediated Isothermal Amplification (LAMP) Method to Detect the Potato Zebra Chip Pathogen 'Candidatus Liberibacter solanacearum' (Lso) and Differentiate Haplotypes A and B.

'Candidatus Liberibacter solanacearum' (Lso) is the causal agent of zebra chip of potato (Solanum tuberosum), which can significantly reduce potato yield. In this study, a loop-mediated isothermal amplification (LAMP) method for the detection of Lso haplotypes A and B was developed and evaluated. Two sets of LAMP primers named LAMP-A and LAMP-B were designed and tested for specificity and sensitivity. Both LAMP-A and LAMP-B were specific to Lso in in silico analysis using the Primer-Blast tool. The LAMP-A and LAMP-B could only produce positive signals from DNA mixtures of Lso-infected tomato but not from the genomic DNA of 37 nontarget plant pathogens. The sensitivity of LAMP-A and LAMP-B on Lso haplotypes A and B were tested on gBlocks and genomic DNA from Lso-infected tomato. On the genomic DNA for LAMP-A, the lowest amount of template DNA for a positive LAMP reaction was 2 to 20 ng on four haplotype A strains and 20 to 80 ng on four haplotype B strains; for LAMP-B, the lowest amount of template DNA for a positive LAMP reaction was 0.02 to 2 ng on four haplotype B strains and 20 ng to no amplification on four haplotype A strains. On gBlocks for LAMP-A, the lowest number of copies for a positive LAMP reaction was 60 on haplotype A and 600 on haplotype B; for LAMP-B, the lowest number of copies for a positive LAMP reaction was 60 on haplotype B and 600 on haplotype A. Therefore, considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, the LAMP-A and LAMP-B primers can be used together to test the probable Lso-infected plant or psyllid samples to rapidly, accurately, and directly differentiate haplotypes A and B. We highly recommend this LAMP system to plant pathology practitioners and diagnostic labs for routine detection of Lso and confirmation of zebra chip disease on potato or tomato.

Trunk Injection of Oxytetracycline for Huanglongbing Management in Mature Grapefruit and Sweet Orange Trees.

Huanglongbing (HLB) is a devastating bacterial disease associated with 'Candidatus Liberibacter asiaticus'. The location of the pathogen within the vasculature of the tree has left growers with limited options for the effective management of the disease. Trunk injection is a crop protection technique that applies therapeutics directly into the xylem of woody tree species and allows for their systemic uptake and transport, which may provide more effective management of vascular diseases such as HLB. In this study, mature 'Valencia' and 'Hamlin' sweet orange (Citrus sinensis) and 'Duncan' grapefruit (C. paradisi) trees were injected with oxytetracycline (OTC) in the spring and/or fall to evaluate the effects of injection timing and response to injection. In addition to seasonal evaluations of tree health and bacterial titer, preharvest fruit drop, yield, and fruit quality were measured at harvest to determine the effects of OTC injection. The benefits associated with injection included a reduction in fruit drop, an increase in fruit yield and fruit size, and improvements in juice quality. However, results varied due to the timing of injection and were not consistent across all three varieties. Residue analysis at different time points after injection suggests that trunk injection effectively delivers therapeutics to mature citrus trees. This study provides fundamental information on the short-term benefits associated with trunk injection of OTC for HLB management in citrus groves. The potential for use of trunk injection at the commercial scale and the possible risks are discussed.

Novel bacterial plasmid produces small interfering RNAs (siRNAs) that induce effective gene silencing in the Asian citrus psyllid Diaphorina citri

BackgroundThe Asian citrus psyllid, Diaphorina citri, is a vector of the plant pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing disease. HLB represents the main threat to the citrus industry around the world due to its fast spreading, high infectivity, and incurability. An alternative for the control of this plague in Rutaceae is the implementation of RNA interference (RNAi). Here, we propose a novel method to produce small interfering RNA (siRNA) in Escherichia coli. The method includes a reporter system to visually demonstrate dsRNA expression. We produced and tested siRNAs against three D. citri genes: Abnormal Wing Disk (AWD), Superoxide dismutase 1 (SOD), and Wingless (WNT). These genes play key roles in psyllid development and maturity, and thus, represent promising targets for potential vector control. ResultsThe blue coloration in bacterial cultures was easily observable, and it corroborated the generation of the dsRNA that is in the same transcript. We evaluated the dosage efficiency of siRNA using the AWD siRNA. The RNAi treatment was evaluated, and all the siRNAs tested were able to induce silencing (-3.05 for AWD, -2.60 for SOD and -2.57 for WNT). ConclusionsThe novel bacterial plasmid effectively produces siRNAs, and the blue color reporter is visually facilitated to check the expression of each dsRNA. Treating D. citri with the produced siRNAs resulted in a decrease in gene expression. In addition, psyllid mortality was observed, being the highest when treated with WNT-siRNA. Our results suggest the potential of siRNA treatment as a method for controlling Huanglongbing.How to cite: Rueda-Silva JC, González-Campos LI, Durán-Armenta LF, et al. Novel bacterial plasmid produces small interfering RNAs (siRNAs) that induce effective gene silencing in the Asian citrus psyllid Diaphorina citri. Electron J Biotechnol 2023;64. https://doi.org/10.1016/j.ejbt.2023.03.006.

Microscopic and transcriptomic analyses of early events triggered by Candidatus Liberibacter asiaticus in young flushes of HLB-positive citrus trees.

Candidatus Liberibacter asiaticus (CLas) causes the devastating citrus disease Huanglongbing (HLB). Young flushes are the center of the HLB pathosystem due to their roles in the psyllid life cycle and in the acquisition and transmission of CLas. However, the early events of CLas infection and how CLas modulates young flush physiology remain poorly understood. Here, transmission electron microscopy (TEM) analysis showed the mean diameter of the sieve pores decreased in young leaves of HLB-positive trees after CLas infection, consistent with CLas-triggered callose deposition. RNA-Seq-based global expression analysis of young leaves of HLB-positive sweet orange with (CLas-Pos) and without (CLas-Neg) detectable CLas demonstrated a significant impact on gene expression in young leaves, including on the expression of genes involved in host immunity, stress response, and plant hormone biosynthesis and signaling. CLas-Pos and CLas-Neg expression data displayed distinct patterns. The number of upregulated genes was higher than that of the downregulated genes in CLas-Pos for the following categories: plant-pathogen interactions, glutathione metabolism, peroxisome, and calcium signaling that are commonly associated with pathogen infections compared to healthy control. On the contrary, the number of upregulated genes was lower than that of the downregulated genes in CLas-Neg for genes involved in plant-pathogen interactions and peroxisome. Additionally, qRT-PCR based expression analysis temporally visualized the induced expression of companion cell specific genes, phloem protein 2 (PP2) genes, and sucrose transport genes in young flush triggered by CLas. This study advances our understanding of early events during CLas infection of citrus young flushes.

Development and Statistical Validation of E-Probe Diagnostic Nucleic Acid Analysis (EDNA) Assays for the Detection of Citrus Pathogens from Raw High-Throughput Sequencing Data

The cost for high-throughput sequencing (HTS) has decreased significantly and has made it possible for the application of this technology for routine plant diagnostics. There are constraints to the use of HTS as a diagnostic tool, including the need for dedicated personnel with a bioinformatic background for data analysis and the lack of a standardized analysis pipeline that makes evaluating and validating results generated at different HTS laboratories difficult. E-probe diagnostic nucleic acid analysis (EDNA) is an in-silico bioinformatic tool that utilizes short curated electronic probes (e-probes) designed from pathogen-specific sequences that allow users to detect and identify single or multiple pathogens of interest in raw HTS data sets. This platform streamlines the bioinformatic data analysis into a graphical user interface as a plant diagnostic tool used by diagnosticians. In this study, we describe the process for the development, validation, and use of e-probes for detection and identification of a wide range of taxonomically unique citrus pathogens that include citrus exocortis viroid, citrus tristeza virus, ‘ Candidatus Liberibacter asiaticus’, and Spiroplasma citri. We demonstrate the process for evaluating the analytical and diagnostic sensitivity and specificity metrics of the in-silico EDNA assays. In addition, we show the importance of including background noise (internal controls) to generate variance in noninfected samples for a valid statistical test using the quadratic discriminant analysis. The fully validated EDNA assays from this study can be readily integrated into existing citrus testing programs that utilize HTS. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Physiological Variables Influenced by 'Candidatus Liberibacter asiaticus' Infection in Two Citrus Species.

'Candidatus Liberibacter asiaticus' is the bacterium associated with the citrus disease known as huanglongbing (HLB). This study evaluated the influence of 'Ca. L. asiaticus' infection on a number of key plant physiological variables concerning photosynthesis, cell integrity, reactive oxygen species scavengers' activity, and osmoregulation of two different species of citrus-the pomelo Citrus maxima and the mandarin C. reticulata 'Tankan'-relative to their measured 'Ca. L. asiaticus' infection load. Results indicated that all measured physiological variables except soluble sugar were affected by increased 'Ca. L. asiaticus' infection titers, wherein the variety C. maxima proved overall more resistant than C. reticulata. 'Ca. L. asiaticus' infection was linked in both plants to decrease in chlorophyll concentration, cell membrane permeability, and malondialdehyde, as well as increased free proline and starch contents. Chlorophyll fluorescence measurements taken 9 months after grafting the mandarin C. reticulata with 'Ca. L. asiaticus' scions revealed a significant decrease in the photosynthesis variables maximum photochemical quantum yield of photosystem II (PSII), effective photochemical quantum yield of PSII, and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae, whereas nonphotochemical fluorescence quenching increased significantly; C. maxima plants, on the other hand, did not show significant differences until the 12th month from infection exposure. The variables superoxide dismutase, catalase, peroxidase, and soluble protein initially increased and later decreased. In addition, progression of 'Ca. L. asiaticus' replication in both citrus species was accompanied by rapid changes in three reactive oxygen species scavenging enzymes in C. maxima, while the pattern was different in C. reticulata. We hypothesize that the observed interspecific differences in physiological change are related to their relative resistance against 'Ca. L. asiaticus' infection. These results provide a scaffold for better describing the pathogenesis, selecting the most resistant breeds, or even validating pertaining omics research; ultimately, these detailed observations can facilitate the diagnosis of 'Ca. L. asiaticus' infection.

Spatial Distribution and Temporal Dynamics of Candidatus Liberibacter Asiaticus in Different Stages of Embryos, Nymphs and Adults of Diaphorina citri.

Huanglongbing, a globally devastating citrus disease, is associated with Candidatus Liberibacter asiaticus (CLas) and is mainly transmitted by Diaphorina citri. Verification of the distribution and dynamics of CLas in D. citri is critical to understanding CLas transmitted by vectors in nature. Here, the distribution and titers of CLas in different sexes and tissues of D. citri adults were investigated by fluorescence in-situ hybridization (FISH) and quantitative real-time PCR (qRT-PCR). Results showed that CLas had widespread distribution in the brain, salivary glands, digestive system, and reproductive system of both females and males, indicating a systemic infection of CLas in D. citri. Moreover, CLas fluorescence intensity and titers were significantly increased in both the digestive system and the female reproductive system with development and there was a marked decreased in both the salivary glands and the male brain, but there was no significant change in the female brain or the male reproductive system. Furthermore, the distribution and dynamics of CLas in embryos and nymphs were investigated. CLas was observed in all laid eggs and subsequent first-second-instar nymphs, indicating that a high percentage of embryos and nymphs resulting from infected D. citri mothers were infected with CLas.

An effector of 'Candidatus Liberibacter asiaticus' manipulates autophagy to promote bacterial infection.

Autophagy functions in plant host immunity responses to pathogen infection. The molecular mechanisms and functions used by the citrus Huanglongbing (HLB)-associated intracellular bacterium 'Candidatus Liberibacter asiaticus' (CLas) to manipulate autophagy are unknown. We identified a CLas effector, SDE4405 (CLIBASIA_04405), which contributes to HLB progression. Transgenic SDE4405 in Wanjincheng orange (Citrus sinensis) promotes CLas proliferation and symptom expression via suppressing host immunity response. SDE4405 interacts with ATG8-family proteins (ATG8s) and their interactions activate autophagy in Nicotiana benthamiana. The occurrence of autophagy is also significantly enhanced in SDE4405-transgenic citrus plants. Interrupting NbATG8s-SDE4405 interaction by silencing NbATG8s reduces Pseudomonas syringae pv. tomato strain DC3000ΔhopQ1-1 (Pst DC3000ΔhopQ1-1) proliferation in N. benthamiana, and transient overexpression of CsATG8c and SDE4405 in citrus promotes Xanthomonas citri subsp. citri (Xcc) multiplication, suggesting SDE4405-ATG8s interaction negatively regulates plant defense. These data show the role of the CLas effector protein in manipulating autophagy and provide new insights into the molecular interaction between CLas and citrus.

Variation in microbial feature perception in the Rutaceae family with immune receptor conservation in citrus.

Although much is known about the responses of model plants to microbial features, we still lack an understanding of the extent of variation in immune perception across members of a plant family. In this work, we analyzed immune responses in Citrus and wild relatives, surveying 86 Rutaceae genotypes with differing leaf morphologies and disease resistances. We found that responses to microbial features vary both within and between members. Species in 2 subtribes, the Balsamocitrinae and Clauseninae, can recognize flagellin (flg22), cold shock protein (csp22), and chitin, including 1 feature from Candidatus Liberibacter species (csp22CLas), the bacterium associated with Huanglongbing. We investigated differences at the receptor level for the flagellin receptor FLAGELLIN SENSING 2 (FLS2) and the chitin receptor LYSIN MOTIF RECEPTOR KINASE 5 (LYK5) in citrus genotypes. We characterized 2 genetically linked FLS2 homologs from "Frost Lisbon" lemon (Citrus ×limon, responsive) and "Washington navel" orange (Citrus ×aurantium, nonresponsive). Surprisingly, FLS2 homologs from responsive and nonresponsive genotypes were expressed in Citrus and functional when transferred to a heterologous system. "Washington navel" orange weakly responded to chitin, whereas "Tango" mandarin (C. ×aurantium) exhibited a robust response. LYK5 alleles were identical or nearly identical between the 2 genotypes and complemented the Arabidopsis (Arabidopsis thaliana) lyk4/lyk5-2 mutant with respect to chitin perception. Collectively, our data indicate that differences in chitin and flg22 perception in these citrus genotypes are not the results of sequence polymorphisms at the receptor level. These findings shed light on the diversity of perception of microbial features and highlight genotypes capable of recognizing polymorphic pathogen features.

The Effect of Interstock on The Development of Huanglongbing Disease and Vegetative Growth of Three Commercial Citrus Varieties in Indonesia

Huanglongbing (HLB) disease, causes a 60-95% decline in Indonesia citrus production in almost all production centers. The use of suitable citrus interstocks can increase fruit production and quality, improve compatibility, and can also increase tolerance to HLB with tolerant interstocks. Potential interstock, providing an alternative for citrus farmers to be able to save their plants from the destruction of HLB. The purpose of this study was to determine the effect of several interstocks in inhibiting the development of HLB disease and the growth of citrus plants. Eight interstock combination treatments (JC, JC+Rough lemon, JC+Carrizo citrange, JC+citrumelo, JC+Poncirus trifoliata, JC+Volkamer lemon, JC+Troyer citrange and JC+ Kanci kumquat) on 3 commercial citrus varieties (C. reticulata var. Batu 55, C. nobilis var. Pontianak and C. aurantifolia var. Nimas Agrihorti). The combination of interstock affects plant tolerance to HLB. In the three upper limit varieties tested, the mildest symptoms of HLB and the healthiest was found in the combination of interstock JC+P.trifoliata, and Candidatus Liberibacter asiaticus were detected after inoculation using conventional PCR amplification in the second test. Based on the PCR index and the severity of the symptoms, the combination of interstock JC+P. trifoliata was classified as mild severity and the healthiest against CLas infection in the 3 scion varieties tested (C. reticulata var. Batu 55, C. nobilis var. Pontianak and C. aurantifolia var. Nimas Agrihorti). Vegetative growth was found to be varied, and almost all of them could grow well except shoot growth which was stagnant at 5-6 months after inoculation.

Genetic transformation of 'Hamlin' and 'Valencia' sweet orange plants expressing the cry11A gene of Bacillus thuringiensis as another tool to the management of Diaphorina citri (Hemiptera: Liviidae).

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Liviidae) is the vector of Candidatus Liberibacter spp., the bacteria associated with huanglongbing (HLB), the most devastating disease of citrus worldwide. HLB management has heavily counted on insecticide applications to control the ACP, although there are efforts towards more sustainable alternatives. In previous work, our group assessed the potential bioactivity of different strains of Bacillus thuringiensis (Eubacteriales: Bacillaceae) (Bt) containing cry/cyt genes as feasible tools to control ACP nymphs. Here, we report an attempt to use the cry11A gene from Bt to produce transgenic sweet orange plants using two promoters. For the genetic transformation, 'Hamlin' and 'Valencia' sweet orange seedlings were used as sources of explants. Transgenic plants were detected by polymerase chain reaction (PCR) with specific primers, and the transgene copy number was confirmed by Southern blot analyses. Transcript expression levels were determined by qPCR. Mortality assays of D. citri nymphs were carried out in a greenhouse, and the effect of the events tested ranged from 22 to 43% at the end of the five-day exposure period. To our knowledge, this is the first manuscript reporting the production of citrus plants expressing the Bt cry11A gene for the management of D. citri nymphs.

Effect of Systemic Insecticides Applied via Drench on the Mortality of Diaphorina citri on Curry Leaf

Huanglongbing (HLB), the most serious disease in citriculture, is caused by the bacteria Candidatus Liberibacter spp., which is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. HLB is mainly controlled with insecticides, necessitating the development of alternative methods, e.g., the use of trap plants such as curry leaf Bergera koenigii, which is highly attractive to the ACP. We evaluated the effects of the main systemic insecticides used by citrus growers, applied via drench to adults of D. citri on the curry leaf tree. We tested the persistence of three pesticides: thiamethoxam, thiamethoxam + chlorantraniliprole, and imidacloprid in protected cultivation and the field condition at 7, 14, 28, 42, 56, 70, 98, and 154 days after the application. Different concentrations of insecticides containing the active ingredient thiamethoxam were tested on adults to determine the LC10 and LC50. Finally, we assessed the sublethal effects on the oviposition and development of D. citri. The insecticides controlled the adults for long periods. However, in the field experiment, from 42 days after application there was a decrease in mortality caused by pesticides applied via drench, while in the protected cultivation, mortality did not decline until the last day of evaluation. The median lethal concentration (LC50) for thiamethoxam was 0.031 g of active ingredient per plant, and for thiamethoxam in a mixture, the LC50 was 0.028 g a.i. per plant. In the experiment with sublethal doses, D. citri did not oviposit on the treated plants. Our findings suggest that the attract-and-kill system using the curry leaf tree and systemic insecticides is effective for the control of D. citri and contributes to the integrated management of HLB.

Detection of "Candidatus Liberibacter asiaticus" Using Conventional PCR and Nested-PCR and Identification of Primer Sensitivity through Q-PCR in HLB Infected Mandarin Orange and Acid Lime Plants in Tamil Nadu

One of the most destructive citrus diseases in the world is called citrus Huanglongbing (HLB). For effective HLB associated ‘Candidatus liberibacter spp.’ prevention, sensitive and precise diagnostics are essential. Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter africanus (CLaf), and Candidatus Liberibacter americanus (CLam) are among the ‘Candidatus liberibacter spp.’ that infect citrus. The most common of these is the species known as Las. To assess the impact of citrus greening disease in the citrus orchard, leaf samples exhibiting greening symptoms were picked up from 210 mandarin orange trees from nine localities in four districts and among them 189 plants showed HLB positive and leaf samples are picked up from 216 acid lime trees from nine localities in six districts of Tamil Nadu where citrus is cultivated in a larger area and among them 202 plants showed HLB positive. A “conventional singleplex polymerase chain reaction (PCR) Nested PCR and Q-PCR (Quantitative PCR)” was performed on the extracted DNA samples to detect Ca. Liberibacter asiaticus. Among all the mandarin orange growing localities from “Kanalkadu and Thandikudi in Dindigul district” and among the acid lime fields, the samples from “Sankarankoil from Tirunelveli district and Kallar from Nilgiris district” showed consistent amplification of 1160 base pairs fragment by using specific primers OI1 and OI2C and also 703 base pairs fragment with gene-specific primers A2 and J5 targeting beta-operon (rpiKAJL-rpoBC) gene which is a ribosomal protein gene of Ca. Liberibacter asiaticus, these results showed that these districts are exclusively positive to Clas . The nested PCR method significantly increased the sensitivity to identify Las up to 10 times and 100 times, respectively, compared to qPCR and conventional PCR in this study. A set of nested PCR primer pairs were examined to diagnose Las. Nested PCR was used to analyse 14 samples from 2 different citrus cultivars in 2 different districts. Among those again kanalkadu orchard for mandarin orange and Sankankoil orchard for acid lime showed more disease incidence and consistent amplification. The findings indicate that all the samples tested positive for HLB; the samples of blotched and chlorotic leaves from the acid lime field at Sankarankoil (thirunelveli district) and mandarin orange field at kanlakaadu (Dindigul district) had the highest positive detection rate (100%). The outcomes show that the nested PCR primer pairs are capable of detecting Las in a variety of symptomatic tissues, citrus cultivars, and geographical locations. The set of nested PCR primers used in the current work will be a very helpful addition to the existing methods for Las detection.

Comparative transcriptomic and proteomic analyses of midrib and root of Wanjincheng orange (Citrus sinensis Osbeck) in response to the early invasion of Candidatus Liberibacter asiaticus

Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is the most destructive disease of citrus plant. HLB can harm almost all citrus varieties and no cure is currently available. Our previous study reported that Wanjincheng orange (Citrus sinensis Osbeck) leaf and root exhibit significant difference in response to CLas infection using leaf-disk grafting. In this study, we used a multiomic approach (proteomics and transcriptomics) to identify nodes that can help in explaining these differences and ultimately contribute to the understanding of the response of midrib and root in the early invasion of CLas. The results of comparative transcriptomic analysis displayed that 2,782 differentially expressed genes (DEGs) were upregulated and 2,807 were downregulated. Meanwhile, the results of comparative proteomic analysis displayed that 226 and 182 differentially expressed proteins (DEPs) were up- and downregulated by CLas, respectively. Transcriptomic and proteomic association analyses revealed 2 downregulated and 6 upregulated genes in midrib and root. The analysis of DEGs and DEPs revealed that secondary metabolites, and detoxification pathways played important roles in response to the early invasion of CLas. Moreover, nitrogen metabolism in midrib and root was influenced by CLas. This study revealed the transcriptomic and proteomic differences between midrib and root in Wanjincheng orange during early invasion of CLas and provided some potential genes and proteins for breeding HLB-tolerant citrus in future.