A great majority of the citrus growers with their orchards located above 1000 meters had inter-cropped citrus with large cardamom . Although, steady progress has been made in terms of infrastructure development to sustain citrus industry in Bhutan, much remains to do in terms of enhancing human capacity of stakeholders with respect to HLB at various levels. While citrus still remains as a main cash crop in Tsirang and Dagana districts, citrus farming system varies with location of the orchards and its altitude range.In our previous study, transcriptome data of HLB susceptible sweet orange and HLB-tolerant rough lemon were compared to identify differentially expressed genes. However, alteration in gene coexpression relationships was not studied. In this study, six coexpression networks were constructed separately for six RNA-seq datasets from two treatments [mock-inoculated and CLas-inoculated of SO and RL. In total, 2781 differentially associated pairs were identified by comparing the CK and HLB networks, 1141 decreased and 1640 increased coexpression interactions under HLB. Gene ontology analysis showed that programmed cell death, defense response, cell growth and differentiation were accompanied with increased association under HLB. However, GO terms such as root morphogenesis, protein translation and carbohydrate metabolic process were over-represented with decreased association under HLB. Huanglongbing is a devastating bacterial disease of citrus worldwide due to its intracellular and systemic infection. Various HLB symptoms are observed on different species/varieties of citrus plants: from yellow shoots to blotchy mottle on the leaves,hydroponic growing supplies from vein yellowing/vein corky to mosaic/green islands similar to zinc deficiency on the leaves, from whitish discoloration to stunted green leaves, etc.

These variations of symptoms, which result from a combination of biotic and abiotic stresses, are not only present on individual plants from a variety but also exist on individual branches of an infected plant. Our results indicated that the adaptation of the bacterial populations, such as the dynamics of ‘Candidatus Liberibacter asiaticus’ , plays an important role in the induction of various symptoms and that Las mutations as well as the number and recombination events of Las prophages/phages affect this phenomenon. In addition, the selection of the host plants for the bacterial populations is also critical for symptom expression during disease progression. Based on severity, we divided HLB symptoms into four grades. It is worth noting that the grades of HLB symptom severity show a positive correlation with our newly identified biomarkers from host plants, and that gene expression profiling of different grades of infected leaves rationalized the differentiation based on the dynamics of these biomarkers. Because of these findings, we propose new approaches that allow for rapid selection of variant citrus plants, including bud sports with greater HLB resistance/tolerance. Most commercially cultivated citrus cultivars are vulnerable to CLas and develop disease symptoms within a few years of infection. While improved production techniques are able to keep an infected tree alive, the only promising long term strategy to manage this disease is to utilize HLB resistant trees that can thrive in an endemic HLB environment. Genetically engineered citrus plants are an important component in the citrus improvement toolkit and offers promise of durable HLB resistance. Incorporation of gene via genetic engineering can potentially confer resistance in susceptible cultivars, while maintaining the varietal fidelity. We have tested several antimicrobial peptide gene constructs , Pathogenesis-related genes and the SAR inducing genes NPR1 and SABP2 in commercial sweet orange cultivars with varying degree of success.

Field trials in an HLB endemic environment as well as greenhouse based insect vector transmission trials have identified a few genes that can potentially confer tolerance to HLB. We have evidence that an engineered root stock can potentially protect the non-engineered scion. We will report on our current citrus improvement strategies to provide durable long term resistance to both scion and root stock cultivars. In addition strategies to develop consumer-friendly engineered trees will be detailed. We have developed a low-cost, hand-held, smartphone based spectrophotometer platform for detection and early diagnosis of Huanglongbing as well as differentiating between other underlying health conditions of citrus. The spectrophotometer clips onto a leaf, and nondestructively interrogates the internal physiology of a leaf using broadband diffuse reflectance spectroscopy in the near infrared wavelength , and records the resulting spectrum on a smartphone device. Diseases and other plant health conditions modify the physiological state of the leaf, thereby changing the optical scattering and absorption properties and creating a DRS spectrum uniquely correlated with each health condition. The integration of the optical sensor with a smartphone and cloud based computing enables recorded DRS spectrums to be analyzed by deep learning analytical algorithms and affords real-time diagnosis of the plant’s health with a high-degree of accuracy. The resulting platform can be an economical and geographically tailored solution for early stage detection of HLB, which requires collective community efforts and timely response for managing the spread of the disease. Results demonstrating differentiation among healthy, HLB, citrus stubborn disease , and citrus tristeza virus graft infected rough lemon and sweet orange greenhouse trees are obtained with greater than 99% accuracy. Progress with on-going longitudinal studies for early stage disease detection will also be discussed. In the age of HLB, the use of anti-microbials and other foliar agrochemicals has emerged as one of the few effective treatments against HLB infected citrus trees.

Aerial sprays are preferred as a means to deliver agrichemicals over large areas given that leaves are the most readily accessible tissues and represent a significant proportion of the total plant body. The main obstacle in using foliar applications of aqueous solutions on citrus trees is that penetration into the leaves is severely hindered by the presence of the protective waxy cuticle. Penetration into leaves is only possible through the stomata openings . Nevertheless, the collective surface area of stomatal openings that would allow for penetration of externally supplied solutions into the leaf is minimal, even under optimum circumstances, since stomata often close under a variety of biotic and abiotic conditions. To increase penetration of externally supplied substances across the leaf cuticle, and subsequently into the phloem, we examined the use of laser light as a tool to increase the permeability of the cuticle without extensive damage to the underlying leaf tissue. Our investigation demonstrated the effectiveness of laser light technology in drastically enhancing the penetration of antimicrobials and double stranded RNA into citrus leavtractes. Vector management has failed to contain the spread of Liberibacter. Although approved for use for the control of Liberibacter, the broadacting antibiotics Oxytetracycline and Streptomycin are far from ideal and likely to receive significant push back from consumers. An ideal antimicrobial system would be 1) highly effective against the target organism 2) with a narrow range and 3) minimal risk of resistance development. Toward this end, our group is exploring the recently elucidated prokaryotic immune system, CRISPR-Cas for its engineering potential as antimicrobial for the control of Liberibacter pathogens. CRISPR-Cas targets DNA in a sequence dependent manner making it highly specific and easily programmable. We have demonstrated that the CRISPR-Cas system will induce cell death when a bacterial cell is compelled to target its own genome and have achieved selective removal of individual strains from a bacterial community. Furthermore,grow table we are engineering a broad-host range bacteriophage to deliver this antimicrobial system to the target bacteria. The availability of multiple genomes of pathogenic Liberibacter sp. facilitated a construct design that selectively removes specified strains but will leave the remaining bacterial community undisturbed. Initially, single genomic regions of the pathogen have been targeted; however, the system can accommodate multiple genome-targeting spacers to diminish the chance of resistance and/or encompass diverse strains. We envision a construct ultimately able to target multiple regions within the bacterial genome, multiple pathogens within the same host, and the endosymbionts of the insect vectors; leading to comprehensive, engineered citrus crop protection.Huanglongbing has emerged as the most severe disease worldwide. HLB-symptomatic trees must be eradicated, causing a very high socio-economic impact on the citrus industry.

In Brazil, Asian citrus psyllid, Diaphorina citri Kuwayama is the insect vector. Currently, HLB management depends on the sanity of seedlings, plant eradication and efficient vector control. This research was carried out aiming to identify root stock citrus genotypes regarding to the attractiveness and host selection to D. citri. A completely randomized design experiment was used to evaluate 24 genotypes under greenhouse conditions with five replicates for each genotype. There were two experiments in September 2015 and in June 2016. For infestation, D. citri adults were evenly distributed inside the greenhouse at a proportion of 20 adults per plant for the first experiment and 56 adults per plant for the second experiment . The number of adults, eggs and nymphs were counted. For the first experiment, the number of eggs was assessed through a scale from 0 to 3 . Data were subjected to analysis of variance and the means were grouped by the Scott-Knott test . The variables number of adults and number of nymphs were transformed to root square . One of the major challenges in the fight against Asiatic huanglongbing , caused by ‘Candidatus Liberibacter asiaticus’ , is that current diagnosis methods are unreliable. Detection of the pathogen relies on qPCR analysis of DNA extracted from citrus leaves, often chosen based on the presence of symptoms. However, due to the extended incubation period observed in CLas-infected plants, the nonspecific nature of disease symptoms, and the uneven distribution of CLas in the tree, this method often results in false negative diagnostic results. Furthermore, recent research indicates that newly infected, young citrus leaves can become infectious after receiving an inoculum of the bacterium from an adult psyllid in just 10-15 days. Therefore, detection of psyllids harboring CLas may provide a more reliable assay for growers to use from the initial detection of invasion and throughout the asymptomatic period. The peptidome is comprised of a dynamic set of small polypeptides that are continuously produced by proteolysis and other cellular processes, and has been a source of biomarkers for cancers and neurodegenerative diseases in humans. This study uses a similar workflow to screen the peptidome profiles of psyllids reared on CLasinfected or uninfected citrus for biomarkers unique to infected or CLasfree psyllids. To that end, proteins were extracted from sets of ~1000 infected or uninfected adult psyllids collected within one week of molting. Native peptides were separated by centrifugal ultrafiltration and analyzed by high resolution mass spectrometry. We have identified candidate peptides that predict infection state of psyllids, which will be validated by testing a diverse collection of psyllid samples from Florida and California and by using an array of sample preparation methods to minimize any geographical or genotypic biases in the psyllid peptidome phenotype. As the peptidome reflects the physiological state of an organism, biomarkers of infection are expected accumulate long before bacterial titers exceed the threshold for detection by qPCR and symptoms appear. The information obtained from this work will be used to engineer field-deployable diagnostic tools using synthetic biology approaches. These simple-to-use tools will aid ongoing HLB and epidemiological studies on the spread of HLB as well as rapid detection and response to the appearance of CLas-positive insects in a grove. Huanglongbing , considered to be the most devastating of all citrus diseases, involves citrus host trees, the Asian citrus psyllid insect and a phloem restricted, bacterial pathogen Candidatus Liberibacter asiaticus . We have designed a web portal with information for consumers and growers with genomics and bio-informatics resources for the host citrus , the vector Asian citrus psyllid and multiple pathogens including CLas. We have implemented JBrowse to provide the context for expression data and features annotated on the genome. We have also used Biocyc Pathway Tools databases to model biochemical pathways within each organism and which can be used to explore the entire disease complex.Micro-CT analysis of the ACP will be combined with transcriptomics data from different tissues, life stages, conditions and sexes to create a 3D atlas that will reveal the internal anatomy of ACP overlaid with the expression profile of different tissues across major life stages. All tools like JBrowse, Biocyc, Blast and the Atlas connect to a central database containing gene models for citrus, ACP and multiple Candidatus Liberibacter pathogens.