Microscopic observations of leaf midrib cross-sections: The damage to the phloem observed in cross sections of midrib tissue show the phloem cell wall thickening, collapsed cambium, and phloem cell plugging in Las infected leaves with normal and high B fertilization rates. Only a single leaf sample from the boron deficient plants was both infected with Las and not suspected to have a recent change in fertilization. There is little of note of the Las infected boron deficient leaves. However, noninfected boron deficient leaves showed similar microscopic symptoms to HLB infection. Boron deficiency also results in phloem cell wall thickening, cambium collapse, and starch accumulation. Although less frequent, some phloem plugging can also be observed. Unlike the leaf midribs with HLB damage under normal fertilization in this experiment, there has been substantial phloem multiplication leading to a thickened phloem layer in the B deficient noninfected leaves. However, phloem multiplication is a common symptom of HLB in leaf midribs. In most cases the B deficiency symptoms resemble the damage observed in the early stages of HLB symptom development (personal observations). However, this damage would be uniform throughout the tree in B deficiency compared to the noncontinuous symptoms observed in HLB affected trees. It has previously been subjected that Las in damaged phloem tissue is mostly dead (Folimonova et al., 2010). This damage caused by B deficiency is most likely responsible for the loss of detection of Las in B deficient trees. The poor rate of infection could also be due to phloem damage limiting the spread of Las from the infected budwood. This would fit with the hypothesis that the active Las population exists in presymptomatic leaves. After symptoms develop and phloem damage occurs the bacteria appear to become trapped and die. At present there is little evidence of a significant effect of B fertilization on HLB with a lack of effect on leaf chlorophyll content, trunk caliper, or leaf nutrient status. However, Las population data suggest that phloem degeneration could be reduced at high B fertilization rates although this is not readily observed in leaf midrib cross sections. The most conclusive results will come from analysis of a B fertilization and HLB interaction of biomass measures taken at the end of the experiment. However, microscopy analysis does show similarities in phloem damage occurring from HLB and B deficiency. Although increased B fertilization does not appear to alleviate phloem damage, this does suggest that the damage is caused by similar mechanisms. B deficiency is known to inhibit proper cell wall formation resulting in physiological responses due to plant cell sensing of weakened cell wall structure. It is possible that as Las inhabits and grows in phloem tissue it causes damage to cell wall integrity, through an unknown mechanism and triggers a similar physiological response to that of B deficiency. Please refer to the final comprehensive report for final biomass analyses, with no page limitations and including figures and tables which better illustrate the microscopy results.
The effects of B nutrient status on Las populations in leaf midribs was somewhat unexpected. Because Las populations in leaf midribs has a bimodal distribution a permutation based ANOVA (PERMANOVA) was used for statistical analysis. PERMANOVA does not make assumptions about normality of distributions, providing added statistical power with bimodal distributions. When all population data from trees with successful infection was considered together with repeated measures, no significant differences were detected. The mean Las titer beginning at 10 months post inoculation (mpi), after most infected trees tested positive by qPCR, are presented (Table 1). A dramatic reduction in mean Las titer is observed at 14 mpi. When analyzed alone there is a weak significance of B status on Las titer with lower Las populations as the concentration of B in the trees declines (Table 1). This reduction in Las titer as the B concentration in the tree declines appears to be due to a shift in the bimodal distribution towards low population leaves. A similar and highly significant trend is seen when the rate of successful inoculation is analyzed (Table 1). The reduced infection rate and Las titer at deficient B concentrations is suspected to be caused by less efficient phloem generation after graft inoculation and reduced phloem function caused by B deficiency. The infection rate is based on leaf sampling. It is possible that a higher infection rate occurred, but B deficiency induced phloem disfunction prevented spread of sufficient Las from the inoculation site on the stem to the tree canopy. Table 1. Las titer and infection success rate in leaf midribs from 3-4 pooled leaf samples at multiple months post inoculation (mpi) Log Las genomes/g midrib tissue* Presence of Las* B Treatment 10 mpi 12 mpi 14 mpi Deficient 4.1 ‘ 2.4 5.8 ‘ 2.3 2.7 ‘ 4.6a 3/10a Normal 3.9 ‘ 3.6 5.1 ‘ 3.0 6.3 ‘ 2.3ab 8/10b High 4.3 ‘ 2.9 7.0 ‘ 1.6 7.6 ‘ 1.4b 7/10b Pseudo-F p-value p-value** Pseudo-F p-value B level 1.61 0.21 0.0313 9.52 0.0003 MPI 2.39 0.102 0.16 0.77 B level x MPI 1.34 0.27 0.78 0.53 *Values with the same letter are not significantly different **p-values for analysis of only 14 mpi Samples of leaf midrib have been collected, fixed, and embedded. Sectioning and staining of the samples for light microscopy is currently underway. The general functionality of phloem and starch accumulation resulting from HLB infection will be visible using light microscopy. To attempt to observe effects of treatments on Las cells, especially in B deficient plants sectioning and observation by TEM will be done on representative samples for each treatment group identified by light microscopy.
Boron (B) deficiency causes phloem plugging and collapse and excess callose formation. These phloem disfunctions are similar to those caused by Candidatus Liberibacter asiaticus (Las) during the development of Huanglongbing (HLB). Degradation of phloem tissue in B deficient plants is attributed to the lack of sufficient B to participate in its essential role in proper cell wall formation. The purpose of this study was to compare the effects of B deficiency and Las on phloem tissue to determine if cell wall damage is a likely cause of symptoms in HLB and to determine if supplemental B would reduce symptoms or Las infection, phloem damage, or improve health and vigor of Las infected trees. Changes in these factors are monitored by 1) qPCR of Las DNA in leaf midribs, 2) microscopy of leaf midrib tissue, and 3) classic measurements of growth and health including caliper, SPAD, biomass, and nutrient analysis. Due to the extremely slow development of B deficiency in the trees, achieved only after 12 months, we requested a 9-month extension to complete the remaining measurements for this study. Tree nutrient status showed that leaf B concentrations had reached desired levels in each treatment, specifically: B-deficient, HLB-inoculated: 5.8 ‘ 0.8 ppm B-deficient, uninoculated: 7.2 ‘ 4.3 ppm Normal B, HLB-inoculated: 90.6 ‘ 42.8 ppm Normal B, uninoculated: 128.5 ‘ 27.0 ppm High-B, HLB-inoculated: 254.6 ‘ 120.5 ppm High-B, uninoculated: 361.2 ‘ 30.2 ppm Results were highly significant for boron fertilization, HLB inoculation, and there was also a significant BxHLB interaction. The BxHLB interaction was likely due to a greater suppression of leaf B concentrations by HLB when amounts were either high or normal, than when amounts were deficient. This may in part be due to the lower success of HLB inoculation by bud grafting on the B-deficient trees. The BxHLB interaction was the only significant leaf nutrient interaction. This lack of other nutrient interaction suggests that supplemental B fertilization is not reducing the effects of Las on nutrient distribution in the plant. Therefore, there is no evidence that B is altering the effects of Las on nutrient uptake or distribution in citrus trees. It is noteworthy that changes in concentrations of Ca in the leaf mirrored those of B, but that HLB inoculation in particular caused a reduction in leaf Ca concentrations. For most other nutrients the leaf concentrations are at optimum concentrations or above. Mg remains below optimum, which was likely caused by an imidacloprid application. The leaf Mg concentrations are the same among all treatments, so confounding effects should be minimized. Changes in B fertilization did also cause significant changes in leaf concentrations of N, P, K, and Fe.
J.B. Beavers first suggested in 1982 that male and female Diaprepes were attracted to frass of the opposite sex. We have identified,synthesized and confirmed the activity of a novel compound, methyl (E)-3-(2-hydroxyethyl)-4-methyl-2-pentenoate produced in the gut of males that elicit antennal responses and are attractive to both males and females in olfactometer assays. We also demonstrated that the compound carvacrol, a fairly common plant volatile but absent in citrus, is also produced in the gut of males and females, elicits antennal responses and may act synergistically as an attractant of Diaprepes. In the fall of 2012, we ran further olfactometer trials to determine if there is a female-produced compound that may be involved in aggregation and/or mating in this species. We collected wild Diaprepes from a field location near Fort Pierce and used these in olfactometer trials because wild weevils tend to be more active than colony-reared individuals. We demonstrated that prior exposure to male-produced pheromone influences the response of females in olfactometer trials. Females held in cages with synthetic pheromone or with citrus flush previously fed upon by males and containing male frass were significantly less likely to move toward male pheromone in a two-choice olfactometer assay. Therefore, we now cage weevils separately or 24 hours prior to their use in bioassays. Both males and females moved toward citrus flush previously fed upon by males (mFUF) when given a choice between mFUF and filtered, humidified air. It is probable that we previously failed to detect male orientation to male pheromone due to the conditions experienced by males in our colony prior to testing. Males but not females moved toward citrus flush previously fed upon by females (fFUF) when given the choice between fFUF and air. Both male and female weevils preferred the odor of the opposite sex when given the choice between mFUF and fFUF. Neither males nor females moved toward carvacrol vs. air with 50% of the weevils failing to respond. More weevils of both sexes responded to linalool vs. air but failed to choose between the two. A blend of mFUF, carvacrol and linalool was more attractive to both males and females compared with mFUF alone. Similarly, a blend of fFUF, carvacrol and linalool was significantly more attractive to males that fFUF alone. Females responded in numerically greater but statistically insignificant numbers to fFUF, carvacrol and linalool compared with fFUF alone. This response merits further study. To summarize, we have improved the resolution of our olfactometer studies by isolating weevils from the opposite sex and their odors for 24 h prior to their use in bioassays. Our results demonstrate the presence of a female odor that elicits male response, and a synergistic interaction between pheromones (male and female) and plant odors (kairomones). We will continue these studies to further develop an attractant for Diaprepes abbreviatus.
Recent activities have focused on the expansion of genome browser capability on the CG-HLB Genome Resources Website (http://citrusgreening.org/). In addition to Ca. Liberibacter genome sequences, users can now search and view genome sequence data for chromosomes and plasmids of other bacterial pathogens of citrus including Xanthomonas axonopodis citri, Xanthomonas axonopodis citrumelo, Xylella fastidiosa 9a5c, Xanthomonas fuscans aurantifolii, and Spiroplasma citri. In addition, the draft genome sequence of Wolbachia wDi, completed at the end of the last grant cycle, is including owing to general interest in using information in the sequence data of this psyllid endosymbiont for development of psyllids that do not transmit greening. Addition of these sequences to the genome browser makes the CG-HLB website a central hub for genome resources on bacterial citrus pathogens and provides a foundation for genome comparison and identification of features contributing to host and tissue specificity, particularly between phloem limited pathogens such as Liberibacter and Spiroplasma citri. Genomes which have not been sequenced to completion, such as Wolbachia wDi and Spiroplasma citri have been unified into ‘pseudomolecules’ so that genome characterization data for all sequence contigs can be accessed from a single menu item. Characterization presently available for genomes newly added to the browser includes links to the NCBI protein record and to a pre-generated list of similar proteins. The viewer for X. fuscans aurantifolii additionally draws from annotations generated at the PATRIC database. Insertion of additional feature tracks is ongoing. The sequence and annotation of the Wolbachia wDi contigs was released this month by NCBI (http://www.ncbi.nlm.nih.gov/nuccore/AMZJ00000000) allowing the implementation of links to individual gene pages in the NCBI database. Tracks are also being added to the Wolbachia wDi browser to reflect functional characterization and localization data for individual proteins. Features of this genome and how to access it on the genome viewer will be described at the upcoming IRCHLB III meeting in Orlando FL. A manuscript entitled ‘Characterization of the Asian citrus psyllid transcriptome’ has been submitted for publication to PLoS One. Our contributions to the transcriptome characterization include repeat characterization and development of repeat finding computational pipelines. These tools are applicable to ongoing sequence analysis and correct assembly of the Asian citrus psyllid genome.
The purpose of this project is to determine methods to effectively 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 the last quarter from Riverside, we have forwarded plant materials infected with viroids, citrus tatterleaf virus, phytoplasmas, citrus stubborn, citrus psorosis, and Citrus tristeza virus to Ft. Collins, CO for treatment by cryotherapy. From Florida, three clones infected with HLB were forwarded to Ft. Collins, CO. All of these materials have been cryotherapied using the current protocol. Surviving plants following treatment are held for 12 weeks, and then leaves are removed, nucleic acid extracted, and forwarded to riverside for laboratory testing of the target pathogens. Personnel from Ft. Collins, CO traveled to Riverside for training on shoot tip grafting and also to train Riverside personnel on recently developed protocols for cryotherapy. Jointly they conducted cooperative research on modification of the cryotherapy protocols was in order to more efficiently eliminate graft transmissible pathogens. Following the training in shoot tip grafting, a comparison of effectiveness of elimination of graft transmissible pathogens by cryotherapy and shoot tip grafting will be done. Research is continuing in Riverside on the use of very young plants as indicator plants for biological indexing, and a comparison of results will be made with the results from biological indexing by the conventional protocol.
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. We have received the sequencing result and are currently analyzing the data. The publication of Sweet orange genome significantly helps our analysis. Lately, Dr. Brlansky joined this project as a co-PI. The addition of expertise will help us in sample collection and improve our research capacity in characterizing the involvement of RNA virus as potential pathogen.
During the first year of this proposal we cloned, purified and performed small molecule screens on CLIBASIA_01180 and CLIBASIA_01510 transcription factors. The effects of these ligands were further confirmed in vitro performing thermal denaturation experiments at different concentrations. During this quarter we focused on the optimization of secondary assays (in vitro and in vivo) to prove the specificity of the chemicals identified. The choice of the secondary assay was tailored to each of the proteins and was based on its biological activity. We then focus on the identification and determination of ligand binding pockets on these proteins by building a structural model of each protein. For CLIBASIA_01510, seventeen chemicals were identified that increased its thermal denaturation in a concentration dependent manner. CLIBASIA_01510 is a homolog to proteins involved in gene regulation through direct interaction with the beta subunit of the RNA polymerase, not by direct binding to DNA. Based on these observations the effect of the chemicals identified in the screen were tested in vivo using a two hybrid system. We further determined the optimal growth media and growth phase to obtain tight protein:protein interactions. Then, the effect of the ligands was tested. It was observed that two chemicals identified on the chemical screens were able to disrupt the CLIBASIA_01510:RpoB interactions. The structural model of this protein indicated several hydrophobic regions that may be involved in protein:protein interactions. In order to determine the location and specificity of the ligand interactions we are currently performing site directed mutagenesis on the residues identified. These mutants will be tested for its capacity to interact with the RNA polymerase and the chemicals identified on the screens. A similar approach is being used for the location of the ligand binding site in CLIBASIA_01180.
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. We are evaluating different solvents and adjuvants for the five compounds. Their antimicrobial activities against Liberibacter crescens, which is closely related Ca. L. asiaticus and could be cultured, are being tested. 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 . 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 was tested using citrus (grapefruit) seedlings in greenhouse. Three isolates could promote growth of grapefruit seedlings. Those isolates are being used to evaluate their potential to prevent citrus seedlings from Las infection vectored by psyllids in a greenhouse. Several isolates also exhibit inhibitory activity again the citrus canker pathogen Xanthomonas citri ssp. citri.
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 three times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We are continuing to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove. We are characterizing the two putative virulence genes sndA and stbA of Las, e.g. subcellular localization and host proteins interacting with them.
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. The sensitivity of biofilm and planktonic cells of Xac 306 to copper (copper sulfate) were evaluated by measuring the MICs. Biofilms are less susceptible to copper than planktonic cells. Effect of the selected compounds on sensitivity of Xac planktonic cells and biofilm cells to copper sulfate was also investigated. Two compounds could significantly increase the sensitivity of Xac planktonic cells to copper sulfate. In a in vitro biofilm system test, the combined use of copper sulfate and the compounds individual or both resulted in significantly increased killing compared to killing by copper sulfate alone. Currently, we are further evaluating the compounds combining with copper in control citrus canker.
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. Characterization of the mutants on LPS, capsule and biofilm formation are complete. Currently, 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 plant RNA is extracted and is being subjected to qRT-PCR and microarray analysis. Anatomical analysis of the inoculated tissues is being conducted usign transmission electron microscopy.
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care costs tend to be higher than commercial groves due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. During the recently completed quarter (October 1 to December 31, 2012), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ The Sugar Belle’ harvest was completed with fruit utilized for fresh market packing. ‘ Plant Improvement Team: o The G-3 Eucalyptus clone was identified to be a good candidate as a windbreak plant due to plant growth habit. Per consultation of Dr. Rockwood, a harvest of one of two rows of the trial suggested for near future. o Rootstock is being grown in the ‘Arboretum’ for budding scions for HLB evaluation o Sprouting and additional horticultural care completed on Dr. Gmitter’s Hybrid evaluation o Rootstocks for Dr. Gmitter’s HLB tolerance evaluation established in ‘New Block’ o Another cultivar has been added to the New Varieties Development and Management Corporation area ‘ Dr. Futch is evaluating trifoliate rootstocks for HLB tolerance. ‘ Applications continued in the ‘commercial scale’ nutritional trial. ‘ Topping of 6 year old Hamlin orange is having good rejuvinative response ‘ Commercial Trials: o BASF has observed Hamlin size improvement in programs evaluating applications of Headline’ o Eurofins area planted o Evaluations of Agri Quest Citrus Root Health Improvement Project continue o ‘ Drs. Stelinski and Rogers have continued evaluations of Asian citrus psyllid and citrus leafminer management in their areas. ‘ Drs. Albrigo and Wong have continued to evaluate antibiotics to manage HLB o Hedging and toping improved overall tree condition in trial area
The Blood orange and the Cara cara orange derive their unique colors, flavors and health benefits from the expression of anthocyanins and lycopenes in the ripening fruit, respectively. Activation of the anthocyanin pathway has become well characterized in the past ten years. A MybA transcriptional activation gene has been shown to up-regulate the expression of anthocyanin biosynthetic genes in many species, including citrus (Butelli et al., 2012). In fact, the native citrus MybA1 gene (aka. CsRuby; GenBank JN402334) was recently isolated from the blood orange variety Moro (Butelli et al., 2012). Lycopene production requires a minimal metabolic pathway of three genes for most plants that generate and ripen fruit. Up-regulation of lycopene production in citrus has produced the flavorful Cara cara cultivar. The lycopene pathway is well understood and three genes sufficient for its biosynthesis (crtE AAA64977.1, crtI AAA64981.1 and crtB AAA64982.1) have been characterized and tested (Yoon et al., 2007). To date the MybA gene from the blood orange ‘Moro’ has been determined. The gene has been synthesized and cloned into an expression construct. Currently the gene is scheduled for testing in Tomato and Citrus tissues. Backup MybA gene’s have also been obtained from Plum, Grape and Arabidopsis. The genes for the lycopene metabolic pathway have been determined and those necessary for over-expression needed to produce a Cara-cara like fruit are currently being synthesized. Backup lycopene gene’s from Tomato and Arabidopsis have also been determined. Five promoters providing fruit specific expression have been identified within the Citrus genome. We are currently ordering primers for amplification from the genome. Backup promoters from the Tomato genome have also been determined. Finally, post-doctoral researchers are currently being interviewed to fill this position.
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