During July and August of 2012, the citrus agents were actively involved in the continued education of citrus growers in the identification of HLB and practices to suppress and/or control psyllids. Educational activities included: grower programs (Citrus Expo and local and regional grower meetings), newsletters, consultations (phone and email), and grove visits. In some parts of the industry, smaller growers are still struggling with the proper identification of HLB, even with the disease being present in Florida since 2006. While no part of the state is HLB free, the incidences of HLB continues to increase impacting all locations where citrus is produced both commercially and in the urban landscape with greater frequency. Agent’s involvement also includes continued support of citrus health management areas (CHMAs) and activities to broaden participation in area sprays by both large and small growers. The successes obtained by CHMAs are directly related to getting as many growers to participate in the coordinated sprays as possible targeting psyllids. To increase grower participation, agents must keep HLB and psyllid information constantly in front of all citrus growers, both small and large. To document the progress of HLB in symptomatic trees, the agents post photos from known positive HLB trees in their area on a monthly basis to the ‘citrus agents’ web site (http://citrusagents.ifas.ufl.edu/hlb_photos/index.htm). This site has monthly photos going back 2 years and easily tacks the progression of the disease in symptomatic trees. The surprising thing about this photo series is that some trees have not declined as earlier expected and still have acceptable crop loads at harvest time. Most of the sites where photos are collected are on some form of enhance nutritional programs to try to maintain tree health as long as possible. The education of homeowners that grow citrus in the urban landscape is also important. Many landscape citrus trees are now becoming HLB positive and citizens are asking for information on the protection of young citrus trees and/or options for mature trees. To broaden our ability to educate these clients, the agents assist with Master Gardener training to educate the new trainees as well as offer assistance to county extension service offices to support their education on identification and control options.
Transcription factors: In order to study the effects of the identified chemicals on CLIBASIA_01180 in a living system, Sinorhizobium meliloti has been utilized a surrogate host. To this end, a mini-transposon mutant of the homolog of CLIBASIA_01180 in S. meliloti was obtained. The transposon is inserted after the coding sequence of the MarR, which would still be functional. Thus, the mutant can be used as a reporter due to the presence of a gus gene in the transposon. Using this strain and the wild type, we did a toxicity test, and found the three of the chemicals previously described were highly toxic while the others were not even at high concentrations. Using lower and up to the highest non-lethal concentrations of chemicals supplemented in the growth media, Gus activity was measured. This activity will reflect the chemical’s ability to enter the cell as well as its ability to affect the DNA-binding activity of the MarR in vivo. Some small differences were found, but because so many of the chemicals will toxic at low concentrations, it was difficult to measure their effect on the activity of the MarR. Because our surrogate also infects plants, we wanted to examine if the homolog of CLIBASIA_01180 in S. meliloti plays a role in the infection of legumes. For this purpose, mini-transposon mutants that would generate an inactive MarR were utilized in comparison to the wild type in infections of Medicago sativa and Medicago truncatula. These mutants also contained a gus reporter gene, making it possible to see if the MarR is expressed in the nodule, as well. The results suggest that this MarR is not essential for nodule formation or function, as the wild type and mutants had similar patterns of nodule formation. However, the Gus staining does show that the MarR is expressed in the nodules, indicating that it can play an important role in plant-bacteria relationship. In order to better understand the effect disrupting CLIBASIA_01180 will have on Ca. L. asiaticus, we are attempting to identify its natural ligand(s). A KEGG small molecule thermal melting screen identified several putative hits that are plant signaling molecules, as well as other biologically relevant ligands. The next step is to validate these hits using gel shift assays. If this regulator is sensing a plant signaling molecule, it would provide a very interesting target to prevent the bacteria from responding to its environment during infection. ZnuA purification: ZnuA as well as its closest homologous (Clibasia_02120 ) were successfully purified. The ongoing assays are directed to enhance ZnuA solubility. We expect to improve the protein recovery by removing the amino terminal sequence of the protein. This highly hydrophobic amino terminal region (30 to 40 aminoacids) hooks the protein to the bacterial membrane. Working with D30ZnuA will allow us to reach the necessary concentration of soluble (functional) protein to perform the biochemical assays proposed.
We successfully synthesized the pheromone methyl (E)-3-(2-hydroxyethyl)-4-methyl-2-pentenoate at USHRL, Ft. Pierce for use in behavioral assays and field trials. Two-way and wind tunnel olfactometer trials were initiated to determine the response of male and female weevils of varying age and mating status to the male-produced pheromone. We confirmed positive female response in a two-way olfactometer to the pheromone compound and no male response. A trial was conducted to test possible effect of rearing conditions and prior exposure on female response to pheromone. We were concerned that continuous exposure of females to male odors in crowded rearing conditions modifies behavior in bioassays designed to test response to those same male odors. Field-collected female Diaprepes weevils were caged in isolation from males and male odors or caged with rubber septa loaded with methyl (E)-3-(2-hydroxyethyl)-4-methyl-2-pentenoate for 24 hours prior to being used in a 2-way olfactometer. Females caged in isolation from males or male odors responded by walking toward synthetic pheromone more often compared with females previously caged with rubber septa emitting the synthetic pheromone. This information will be used to improve experimental design of behavioral assays. Female response to carvacrol was demonstrated in an olfactometer. Carvacrol is a common plant compound that is absent from citrus but present in the gut of male and female Diaprepes adults and appears to be functioning as a pheromone in this species. A multvariate screen is being conducted to identify principal drivers in blends of pheromone and host plant volatiles. Pupae, teneral adults and adult males and females of varying age were dissected and extracts of their hind guts and mid guts were analyzed to determine the location and timing of pheromone and carvacrol production. Results are being collated and prepared for publication.
The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing and citrus canker. Using input from stakeholders and scientists, threatened germplasm has been identified, collected, and established at Ft. Pierce. Presently 76 accessions are being held at Ft. Pierce. Additional nucellar, old line, and unique accessions have been identified which represent germplasm not generally available or in protected collection, and we are adding these accessions to the recovery collection. The new accessions will be antibiotic treated and subjected to clean up. Additional testing is planned in collaboration with the Florida Citrus Germplasm Introduction Program. Twenty five accessions are presently being held in Riverside in various stages of clean up for release from quarantine status.
The purpose of this project is to determine methods to easily eliminate Candidatus Liberibacter asiaticus (Las), the bacterium associated with huanglongbing (HLB) in Florida, from citrus with emphasis on cryotherapy, and to determine the effectiveness of using young indicator plants for biological indexing to verify elimination of graft transmissible pathogens. In this quarter, we have forwarded material to Ft. Collins, CO for treatment by cryotherapy. The methodology for cryotherapy to remove pathogens is being developed. Following cryotherapy treatment, we wait about 12 weeks before testing the surviving buds to give time for any pathogen that may have survived to multiply to the point where it may be detected using laboratory diagnostic methods. In Riverside, the evaluation of the use of very young plants for biological indexing of Citrus tatterleaf virus, citrus vein enation, Citrus psorosis virus, Citrus concave gum disease, and citrus viroids is being continued with the results obtained from using young indicator plants being compared to the conventional method of biological indexing for these pathogens.
This year we have broadened our bioinformatics efforts to include not only Liberibacter genome analysis but also the bioinformatic characterization of other organisms relevant to citrus greening biology. Liberibacter: The CG-HLB Genome Resources Website (http://citrusgreening.org/) hosts a genome browser to aid users in searching and identifying genome properties of sequenced Liberibacter strains. Sequences for CLas psy62, CLso ZC-1 and the phage regions of CLas UF506 are presently available. We are assisting in genome comparisons with an additional CLas strain for inclusion upon public release. Submission of the genome sequence for a cultural Liberibacter, L. crescens BT-1, was reported at the recent Citrus Health Research Forum. Inclusion in the genome viewer will allow visualization of the regions of difference, highlighting genetic features related to citrus pathogenicity and to the ability to grow in culture. Asian Citrus Psyllid (ACP) endosymbionts: Biology of ACP and its bacterial endosymbionts is a focus of intense interest owing to growing interest in exploiting vector biology for control of citrus greening. To corroborate candidate endosymbionts previously identified by rDNA amplification, raw reads from the ACP metagenome sequence were mapped to reference genome sequences. Results of the read mapping provided the most support for Wolbachia and an enteric bacterium most similar to Salmonella. Wolbachia-derived reads were extracted using the complete genome sequences for four Wolbachia strains. Reads were assembled into a draft genome sequence, and the annotation assessed for the presence of features potentially involved in host interaction. Candidate host interaction factors encoded by the ACP Wolbachia genome include 54 ankyrin repeat-containing proteins, a Type IV secretion pathway, and a bacterioferritin gene linked to iron homeostasis in the host. Several metabolic capabilities were identified in the Wolbachia strain that are absent from CLas. Genome alignment with the complete sequences reveals membership of ACP Wolbachia in supergroup B, further supported by phylogenetic analysis of FtsZ. FtsZ and Wsp phylogenies additionally indicate that the Wolbachia strain in the Florida ACP isolate falls into a sub-clade of supergroup B, distinct from Wolbachia present in Chinese ACP isolates, supporting the hypothesis that the ACP introduced into Florida did not originate from China. A manuscript describing this work has recently been accepted for publication by PLoS ONE. The draft genome sequence for ACP Wolbachia has been submitted to Genbank under Bioproject PRJNA29451, and the annotated draft is currently being uploaded onto the CG-HLB Genome Resources genome viewer for easy visualization by researchers interested in exploiting Wolbachia for ACP control. Asian Citrus Psyllid (ACP): As part of a larger effort on ACP sequencing, we have characterized repeat sequences in the current ACP genome assembly and recently submitted transcriptome. Repeats play an important role in genome structure and regulation, and characterization is critical to accurate genome assembly. We have also mined the ACP transcriptome for osmoregulatory transporter genes on behalf of Angela Douglas (Cornell) to use in RNAi-based psyllid control.
Over the course of this funding period, the CG-HLB Genome Resources Website (http://citrusgreening.org/) was developed to serve as a hub for genome resources of Ca. Liberibacter and other organisms impacting the transmission and development of citrus greening. Initially hosting materials for viewing the Ca. L. asiaticus draft genome, the site now hosts a GBrowse based genome viewer on which genome properties of CLas psy62, CLso ZC-1 and phage regions of CLas UF506 can be viewed and searched, a major advantage of the GBrowse model being its ability to hyperlink to diverse outside resources including gene characterization at NCBI, 3rd party sites, and resources funded by CRDF such as the Grishin lab structural analyses. Multiple analyses have been conducted in house for CLas psy62 including screening the sequence for compositional variation suggestive of horizontal transfer and for repeated elements having potential application to diagnostics. Gene regulation is of particular interest given its importance in mediating adaptation of the organism to its host and vector, and predictions of regulatory motifs have also been generated using experimentally determined features from free-living Liberibacter relatives as models. These include rpoH, associated with induction of genes associated with survival at high temperatures, and rirA, a protein known to regulate uptake and metabolism of iron. Results of these analyses are available on the CG-HLB Genome Resources Website. Although the original grant emphasized genome resources for Liberibacter, the biology of the psyllid vector (ACP) and its bacterial endosymbionts became an increasing focus owing to the potential for exploitation of vector biology for disease control, and the apparent need for bioinformatically skilled analyses. To corroborate candidate endosymbionts previously identified by rDNA amplification, raw reads from the ACP metagenome sequence were mapped to reference genome sequences. Results of the read mapping provided support for Wolbachia and an enteric relative of Salmonella. Wolbachia-derived reads were extracted and assembled into a draft genome sequence. The annotation was assessed for features potentially involved in host interaction. Candidate host interaction factors include 54 ankyrin repeat-containing proteins, a Type IV secretion pathway, and a bacterioferritin gene linked to iron homeostasis in the host. Several metabolic capabilities were identified in the Wolbachia strain that are absent from CLas. Genome alignment with the complete sequences reveals membership of ACP Wolbachia in supergroup B, further supported by phylogenetic analysis of FtsZ. FtsZ and Wsp phylogenies additionally indicate that the Wolbachia strain in the Florida ACP isolate falls into a sub-clade of supergroup B, distinct from Wolbachia present in Chinese ACP isolates, supporting the hypothesis that the ACP introduced into Florida did not originate from China. A paper describing this work has been published in PLoS ONE. Contig sequences are available at NCBI, and the annotated draft genome sequence for ACP Wolbachia can be accessed in the supplemental information of the publication as well as in the CG-HLB Genome Resources genome viewer. Project personnel additionally participated in the characterization of repeat sequences in the ACP transcriptome. Repeats play an important role in genome structure and regulation, and their characterization is critical to accurate genome assembly. This work has been submitted for publication in PLoS ONE. Our work on Liberibacter and ACP was presented at, among others, the HLB/Zebra Chip meeting (November 2009), APS (August, 2010), HLB Conference (January 2011), and Citrus Health Research Conference (September,2012).
Citrus canker is an economically important bacterial disease of most commercial citrus cultivars resulting in significant losses worldwide. Spread of citrus canker has been a severe problem to the citrus industry of Florida. How bacteria escape from infected plants is underexplored. Understanding the molecular determinants of lesion rupture, how Xcc survives in the intercellular spaces, and how Xcc releases from lesions of host plants will provide many fundamental and practical benefits. Despite the tremendous effort to eradicate citrus canker, the pathogen has spread to most citrus production areas in Florida and continues to spread. Understanding the genetic mechanism of release of Xanthomonas axonopodis pv. citri (Xac) from citrus canker lesions will help develop effective control and containment strategies to stop citrus canker pathogen from spreading. The goal of the proposed research is to understand the genetic mechanism of release of Xac from citrus canker lesions. The specific objectives are to: 1. characterize critical genes involved in release of X. axonopodis pv. citri from citrus canker lesions; 2. understand the release mechanism by studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions. We have identified 12 EZ-Tn5 transposon mutants of Xac with reduced capacities of release from citrus canker lesions. The insertion sites of the 12 mutants have been identified with insertions in 11 different genes. Currently, complementation analysis of the mutants is underway. Bacterial growth assays of the mutants and the wild type strain in grapefruit ‘Duncan’and sweet orange ‘Valencia’ have been conducted. Three mutants were tested for affect in release from citrus canker lesions (dispersal assay) on grapefruit for 5, 7, 10 and 14 days as compared to wild type strain. The preliminary assay showed differences in growth rate (total count) and dispersal rates (surface count). The dispersal will thus be calculated according to percentage point difference to negate any effect due to growth changes. Pectate lyase assay, proteinase assay and motility tests, and EPS assay have been completed for all the mutants. Further characterization of the mutants on LPS, capsule and biofilm formation is in progress. Also we are investigating the release process by studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions.
The funds for this project were recently released and data was collection soon after during the week of 13 August, 2012. We have already improved the capabilities of laser induced breakdown spectroscopy (LIBS) system by adding multiple detectors in the range 200-1,000 nm. Meanwhile, at the Southwest Florida Research and Education Center, several citrus trees are being treated with multiple nutrient compositions to induce differences in the foliar nutrient concentrations. Leaves from these trees will be collected and analyzed using the LIBS system, followed by chemical analysis.
As a proof of concept, we initially tried to find out if Laser Induced Breakdown Spectroscopy (LIBS) technique can differentiate the healthy citrus leaves from symptomatic stressed leaves. We collected LIBS spectra from abiotic stressed leaves that included magnesium, manganese, iron and zinc deficient leaves, and biotic stressed leaves that included canker and HLB-infected leaves. The classification studies indicated that the nutrient deficient and diseased (symptomatic) leaves could be easily categorized from the healthy ones with more than 90% accuracy. The next step is to perform peak analysis to identify the peaks representing these variations. This step is currently under investigation. We also started the next step of the project in which we will try to evaluate the ability and accuracy of LIBS technique in quantifying the amount of nutrients in the leaves and compare that with currently used analytical approach. We have collected the first set of data. Five six-month-old leaves were sampled from individual trees in two citrus greening studies. The data collected from mature Valencia trees at the Southwest Florida Research and Education Center (SWFREC) and mature Valencia trees in a commercial grove north of Immokalee in Hendry County. The leaves were refrigerated prior to being shipped overnight to the Citrus Research and Education Center (CREC) for spectral analysis using LIBS system. A total number of LIBS spectra collected were about 1250 (about 125 samples with 10 replicates each). The LIBS spectra were collected based on the previously established procedure. After LIBS data collection, the leaves were returned to SWFREC where the samples were dried and ground. The ground samples were analyzed for total nitrogen by combustion using the NA2500 C/N Analyzer (Thermoquest CE Instruments) and for P, K, B, Ca, Cu, Fe, Mg, Mn and Zn by Inductively Coupled Plasma (ICP) after ash digestion. Currently, data analysis protocols are being developed to allow qualitative and quantitative analysis based on the nutrient concentrations.
Development of alternative or complementary approaches for effective management of citrus greening is highly desirable and will greatly help the citrus industry due to the difficulty to control the HLB disease. Considering the highly destructive nature of HLB disease and the lack of control measures, there is a huge potential to develop antimicrobial small molecules against the causal agent thus to suppress the population of Ca. L. asiaticus in planta and to reduce the innoculum for psyllid transmission. Treatment of Ca. L. asiaticus infected citrus could be pursued by applying antimicrobials to infected trees. The most common targets for antimicrobial agents development include receptors, proteins and enzymes, DNA, RNA and ribosomal targets. Among them, proteins have become the major target due to their druggable characteristics. In this study, we presented our research on screening small molecule inhibitors against SecA. SecA is one essential component of the Sec machinery which provides a major pathway of protein translocation from the cytosol across or into the cytoplasmic membrane. The Sec pathway was also shown to be required for virulence of Ca. L. asiaticus in our study. SecA is the protein translocase ATPase subunit, which involves in pre-protein translocation across and integration into the cellular membrane in bacteria. In our recent study, we further expanded our previous study in identifying lead antimicrobial compounds with higher activities by targeting SecA using various computational techniques like homology modeling, virtual screening, molecular docking & minimization. Due to the uncultivable nature of Ca. L. asiaticus, we tested the potential inhibitory effect of the selected compounds against Agrobacterium tumefaciens, which is phylogenetically related to Ca. L. asiaticus. Twenty compounds were selected for biological activity study against SecA of Ca. L. asiaticus and A. tumefaciens. Five compounds were found to inhibit the ATPase activity of SecA of Ca. L. asiaticus in nano molar concentrations and showed antimicrobial activities against A. tumefaciens with MBC ranging from 128 ug/ml to 256 ug/ml. These compounds appear to be suitable as lead compounds for further development of antimicrobial compounds against Ca. L. asiaticus. Those findings were published in the article entitled: Identification of small molecule inhibitors against SecA of Candidatus Liberibacter asiaticus by structure based design. on Eur J Med Chem (http://dx.doi.org/10.1016/j.ejmech.2012.05.035). To test the application potential of those compounds on plants, the phytotoxicity studies were performed on the five compounds against citrus. At higher concentrations (0.25mg/ml), all five compounds showed phytotoxicity. When the concentrations were diluted to 0.025mg/ml, the toxicity went down to mild to low. Currently, we are testing the inhibitory effects of the five compounds again Ca. L. asiaticus in planta. Currently, we are optimizing these five antimicrobial compounds to identify compounds higher antimicrobial activity. Utilizing similarity search methods on these five active structures, fourteen compounds were selected from commercially available compound database for antimicrobial activity study. The selected fourteen compounds are being tested for their inhibition against SecA ATPase activity.
Pathogenic microorganisms affecting plant health are major and chronic threat to food production and ecosystem stability worldwide. As agricultural production intensified over past few decades, producers became more and more dependent on agrochemicals as a relatively reliable method of crop production helping with economic stability of their operations. However, increasing use of chemical inputs causes several negative effects, e.g. development of pathogen resistance to the applied agents and their non target environmental impacts. Furthermore the growing cost of pesticides and consumer demand for pesticide free food has led to the search of substitute of these products. In addition, there are a number of fastidious diseases for which chemical solutions are few, ineffective or nonexistence. Biological control is thus being considered as an alternative or a supplemental way of reducing the use of chemicals for agriculture and to manage fastidious diseases. Citrus Huanglongbing (HLB, greening) is one of the most devastating diseases and presents an unprecedented threat to Florida citrus industry. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las) (Duan et al. 2009). Thus, alternative management approaches to manage HLB are necessary. The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. Currently, we are setting up the experiments to test different Microbe Products in management of HLB. We have developed a culture collection of approximately 400 bacteria initially isolated from the root and rhizosphere of citrus. These bacterial isolates have been screened for various beneficial traits related to P solubilization, siderophore production, nitrogen fixation, indole acetic acid (IAA) synthesis, production of antibiotic and lytic enzymes, induction of systemic resistance, production of ACC deaminase1-amino-cyclopropane-1-carboxylate deaminase and production of quorum sensing [N-acyl homoserine lactones] signals. We are also evaluating the antagonistic activity of these bacterial strains against some well-known plant pathogenic fungi. Especially we have screened a bacterial isolate designated as 43A which possess multiple plant growth promoting activity and is also able to antagonize different fungi. We are also testing the plant growth promoting activity of 24 isolates using seed germination pouch in greenhouse. We have also selected several Bacillus spp. possessing multiple beneficial traits to develop bacterial consortium which can be further developed as carrier based bioformulation. Assay for compatibility between isolates using antagonistic survival tests showed that all the selected beneficial bacteria are compatible with each other. Plant growth promoting activity of six selected isolates was evaluated using the model plant Arabidopsis grown in vitro. The results suggested that three isolates could promote plant growth. The plant growth promoting activity of these six isolates is being tested using citrus (grapefruit) seedlings in greenhouse.
Management of phloem-limited bacterial diseases is very challenging. These bacteria employ unusual and sometimes unique strategies by which to optimize their niche occupation and obtain their nourishment from the host plant. Their location within the living (sieve tubes) plant cells, rather than in the intercellular spaces, offers different challenges and opportunities for them to avoid the host plant’s defense system. Phloem is also difficult for any bactericides to reach to control the pathogen population. Among the phloem-limited bacterial diseases, citrus Huanglongbing (HLB, greening) is one of the most devastating diseases. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las). The goal of the proposed study is to develop HLB management strategies which boost plant defense to protect citrus from HLB by exploiting the interaction between Las and citrus and understanding how Las manipulates plant defense. As requested by CRDF and SAB, we have revised project and will focus on the following two objectives: to characterize how Las causes HLB disease symptoms and how Las manipulates plant defense response by investigating the roles of putative virulence factors; to test different compounds in controlling HLB and characterize their mechanisms in controlling HLB. Recently, we compared the gene expression of PR1, PR2 and PR5 in healthy trees and Las infected citrus plants. The expression of PR1, PR2 and PR5 was significantly reduced in HLB diseased grapefruit as compared to healthy grapefruit after inoculation with Xac AW. We also tested whether infection by Las can make citrus more susceptible to infection by Xanthomonas citri subsp. citri. We also sprayed two times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB. Currently, We are checking the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We will continue to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove.
The goal of this study is to understand the role of biofilm formation and quorum sensing (QS) in X. citri ssp. citri infection of citrus fruit and to prevent its infection by interfering with biofilm formation and QS. The hypotheses of the proposed research are (1) Biofilm formation and QS play important roles in X. citri ssp. citri infection of citrus fruit; (2) Control of citrus canker could be improved by interfering with biofilm formation and QS of X. citri ssp. citri. The hypotheses are based on previous studies and our preliminary studies. The specific objectives of this proposed research are as follows: Objective I: To understand the role of biofilm formation and QS in X. citri ssp. citri infection of citrus fruit. Objective II: To prevent X. citri ssp. citri infection of fruit by combining copper with inhibitors of biofilm formation and QS. Recently, we compared the attachment of the QS mutants on the citrus fruit surface. Compared with wild type stain Xac 306, the quorum sensing mutant ‘rpfF showed significantly reduced attachment to the fruit surface as revealed by CLSM (confocal laser scanning microscopy) observation with the GFP-labeled bacterial strains. We also evaluated the effect of nine compounds on Xac biofilm formation on abiotic surfaces using the crystal violet staining method. The data obtained showed that three compounds were active in inhibiting Xac biofilm formation in NB liquid medium at. Plant test in greenhouse showed that treatment with the three compounds prior to infection could reduce biofilm formation of Xac on leaf surface, reduce the formation of canker lesions on spray-inoculated grapefruit leaves with the wild-type strain. Effects of the three compounds on Xac on detached immature citrus fruit were also tested using spray inoculation. Preliminary results showed that these small molecules affected the infection of Xac 306 on unwounded and wounded citrus fruits at sub-inhibitory concentrations. We are currently testing the effect of those compounds in different combinations with copper based bactericides in controlling Xac infection of grapefruit plants in the greenhouse.
Per the requirements from CRDF, we have revised our goal of this proposal to focus it on the identification of the causal agent of citrus blight. Citrus blight has imposed consistent losses and challenges to citrus industry since the causal agent of the disease remains unknown. The present study would be instrumental in knowing the mysterious pathogen causing citrus blight and pave way for devising efficient management or control methods to help citrus industry to tackle citrus blight. We will characterize the microbiomes of the blight diseased and healthy citrus roots through metagenomic approaches. Recently, we have survey three groves at Lake Alfred, Auburndale, and Haines city. Citrus blight trees at different development stages and healthy trees are being confirmed based on symptoms, water injection, and P12 antibody that have been known as the diagnosis tools for citrus blight. We finalize the blight diseased and healthy citrus trees to be used for sampling. Root samples were collected from 24 trees. The first set of DNA and RNA samples have been purified and sent for deep sequencing to identify the microbes associated with blight diseased and healthy citrus.
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