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 four times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB in one grove. Multiple compounds showed control effect. To further test those compounds, we have selected two more groves to expand the field test. The disease index of the two groves have been investigated. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We also compared the SA levels in HLB infected and healthy Valencia citrus. We are continuing to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove. We have applied different compounds at three separate field trials. Following up study is ongoing. We are characterizing the two putative virulence genes sndA and stbA of Las, e.g. subcellular localization and host proteins interacting with them using yeast two hybrid system. The positive clones identified are being sequenced.
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. 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. Three compounds exhibited a significant reduction in biofilm formation both on polystyrene surface and in glass tubes compared to the untreated control, where the level of biofilm formation were reduced to 50% and 60% of control, respectively. 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 Xac 306 infection of unwounded and wounded citrus fruits at sub-inhibitory concentrations. We have completed 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. In the NB medium, planktonic cells exhibited a MIC of 0.50 mM CuSO4 without biofilm inhibitor. In the presence biofilm inhibitors at sub-MIC concentrations , the MICs of CuSO4 against Xac 306 planktonic cells were decreased to 0.25 mM. 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. The results have been accepted for publication consideration by Phytopathology. We also identified multiple new biofilm inhibitors. We tested the survival of both biofilm deficient and QS mutants on fruit surface. Effects of biofilm formation inhibitors on Xac infection on detached immature citrus fruit were tested using spray inoculation. The inhibitors affected the infection of Xac on both unwounded and wounded citrus fruits.
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. All the 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. We are repeating this assay to eliminate any false positives. 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 studying 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. Anatomical analysis of the inoculated tissues were conducted using transmission electron microscopy. The total RNA of grapefruit infected with wild-type Xac and mutant were extracted and microarray analysis was conducted. We are analyzing the data using Mapman and GO based approaches. Quantitative reverse transcription PCR was conducted to study expression of selected plant genes.
The objectives for this project are 1) to complete the Las genome sequence and conduct comparative genomics studies on the Liberibacter species; 2) to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development; 3) to confirm if Las bacteria are seed-transmissible and their role in HLB development. Complete genome sequence of Candidatus Liberibacter asiaticus (Las) was obtained using metagenomic approaches with shotgun sequencing and BAC clone libraries. In collaboration with Dr. Hong Lin at the USDA-ARS in Parlier, California, we have also obtained a complete genome sequence of Ca. L. solanacearum. Based on the variations within the Las prophages, FP1 (CP001677.5) and FP2 (JF773396.1), twelve (A to H) different populations (genotypes) have been identified. The genetic variations that distinguish type A and B are localized to FP1 and FP2 regions. Typing results revealed A, B and C as the three most abundant groups in libraries from psyllid, citrus and periwinkle, although psyllid contained much more type A sequence than either plant host. The various Las populations exist not only in different varieties, but also in different individuals of the same variety. Using Las-specific molecular markers, we are now able to differentiate the Las populations with different pathogenicity, and differentiate psyllid-transmissible and non-transmissble populations. Seed transmission of Las was tested in grapefruit, sweet orange, sour orange, lemon, and trifoliate orange. A very low titer of Las was detected from the embryos and seedlings using nested PCR and real-time PCR. Most, if not all the seedlings did not show typical HLB symptoms and contained a relatively low Las bacterial titer for HLB, even in the four to five year old seedlings. The results indicated that the seed-transmitted Las could not cause typical HLB disease by themselves. Psyllid transmission studies on the Las-positive seedlings were performed. A high percentage of psyllids acquired Las bacterium but did not have the same bacterial levels as those from HLB-affected citrus plants. However, it is the first time that a seed transmitted plant was confirmed by PCR using several Las-specific primer sets. Further study with graft transmission and electron microscopy confirmed the unique nature of seed transmission of HLB. Using PhyloChip’ G3, A total of 7,028 known OTUs (“species”) of bacteria were detected in citrus leaf midribs of antibiotics-treated and non-treated HLB-affected trees. These OTUs were from 58 phyla with the following five containing 100 or more OTUs: Proteobacteria (44.1%), Firmicutes (23.5%), Actinobacteria (12.4%), Bacteroidetes (6.6%) and Cyanobacteria (3.2%). The results illustrate that the low Las level seen was a combination of a seasonal fluctuation, part of the bacterial population dynamics, and a response to the antibiotic treatments. We have also conducted a few functional genomics studies of the Las bacteria using alternative expression systems to: 1) characterize the ATP translocase; 2) reveal only one of the two putative znu operons is responsible for zinc uptake; and 3) demonstrate that Las encoded a functional flagellin characteristic of a Pathogen-Associated Molecular Patterns (PAMP). More details can be seen in publications: 1) MPMI 22:1011-1020, 2009, 2) PLoS ONE 6(4): e1913, 2011, 3) J. Bacteriol. 192:834-840, 2010, 4) PLoS ONE 7(9): e46447. doi:10.1371/journal.pone.0046447, 2012; 5) PLoS ONE 7(5): e37340. doi:10.1371/journal.pone.0037340,2012, and 6) BMC Microbiol. 13:112, 2013.
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. We have surveyed 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 selected 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 for the first batch of samples and are currently analyzing the data. The publication of Sweet orange genome significantly helps our analysis. Now we are aligning the reads from DNA samples to sweet orange genome and C. clementina genome (V1.0), about 30%-40% reads could not mapped on these three citrus genomes. Those unmapped reads which are not citrus sequences are being used for metagenomic analysis. We also analyzed the RNA-seq data. Totally 2383 citrus genes were down-regulated while 2017 genes were up-regulated by citrus blight. Meanwhile, two methods were used to analyze these differentially expressed genes: GSEA (Gene set enrichment analysis) which is Gene ontology based method and Mapman-Mapman pathway based method. Root samples were collected again from 12 trees in the selected citrus grove at St. Cloud in March 2014. Interestingly, further test in April indicated that two previous healthy trees became citrus blight positive. Further analysis of those trees are being conducted.
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 (January 1 to March 31, 2013), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ Plant Improvement Team o Planted additional trees evaluating scion rootstock interaction of Dr. Grosser’s new rootstock selections for HLB tolerance ‘ Dr. Singh initiated a trial evaluating herbicide tolerance of selected USDA rootstocks to various residual herbicides ‘ Evaluation of NpHuric to decrease pH and bicarbonates demonstration initiated ‘ UAS of America evaluation on supplemental materials applied to the soil and/or foliage to increase tolerance to the affects of HLB and citrus canker initiated ‘ Dr. Futch evaluations: o Continued evaluations of trifoliate rootstocks for HLB tolerance o Established an herbicide trial for Dow Agroscience ‘ Applications of the ‘Boyd Program’, Keyplex and Ben Hill Griffin programs continued in the ‘commercial scale’ nutritional trial. ‘ Harvest of Valencia occurred in early June with significantly diminished yields due to extreme fruit drop ‘ Conducting spring fertilizer and pest management programs for the groves o Summer Oil application near completion o Herbicide program on schedule o Beginning evaluation of ‘chemical mowing’ as a cost savings vs. mowing in a portion of the grove ‘ Applications of seven nutritional treatments continue in MFCF replicated nutritional programs evaluation ‘ Commercial Trials: o Eurofins evaluations continue o Evaluations of Agri Quest Citrus Root Health Improvement Project continue o Keyplex nutritional trial evaluations continue o Bayer demonstration of Optiva programs for citrus canker management continue ‘ 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
The analysis of the green house trees used for each of the therapeutic spray applications and controls has been completed. The six therapeutic treatments and the two controls have been previously described. We analyzed the transcription of 52 individual genes using Real Time PCR (RT-PCR), 48 of these gave us interpretable data of these about 30 gave us valuable information on how each of these treatments was working which was very encouraging data. The focus of our analysis was to analyze target gene responses to elucidate treatment effects on two set of genes: 1) the metabolic syndrome (starch and sucrose metabolism) induced by the source-sink disruption, 2) and the immune dysfunction induced by the disease (including hormone biosynthesis and metabolism, signaling, small molecule crosstalk, defense responses). Our spray applications appear to improve the HLB induced metabolic syndrome with respect to starch and sucrose metabolism, we observed the up-regulation photosystem II reaction center, sugar signaling, Sucrose phosphate synthase, alpha-amylase, sucrose synthase and the down regulation of invertase and ARF GAP domain proteins. Our spray applications also appear to improve the innate immune responses and this was observed by the upregulation of WRKY transcription factors 48, 54, 59 and 33, Pathogenesis related protein1, EDS1, MYC2, TGA5, Gibberellin-2-oxygenase, ERF1, NNLTP, SSN1, Zinc ion binding TF, RGA1 with the down regulation of Jasmonic acid biosynthesis. Taken together, these findings encouraged us to treat trees in a young orchard naturally infected with HLB and this study was initiated in a grower orchard near Fort Pierce. On June 25, 2013 we applied our six therapeutic and two control treatments to an infected young orchard of Valencia orange on Swingle rootstocks in the Indian River Region of Central Florida. We chose 24 trees of medium height 5-8 ft, 12 trees in each row. Three trees were sprayed for each of the 8 treatments and each of the treatments was randomly distributed in the two rows. The six therapeutic treatments included: 2, L- Arginine, 2, gibberellin in combination with 6-benzyl adenine (BA), and 2, atrazine in combination with sucrose. The surfactant Silwet, LK-phite and LDKP3XTRA were added to all treatments. We have sampled leaves at day 0 and 3 and will do two additional sampling of leaves at day 6 and 12. We plan to extract DNA and RNA from each of the time points to estimate the bacterial titer and the level expression of the genes describe above to see if we can replicate our observations in the green house with trees naturally infected in the field.
*** Selection of nano-particles (Edgardo Etxeberria, co-PI) *** The previously reported results described using urea as a non-ionic nutrient with nanoparticles. In this report, we describe tests of nanoparticles with an ionic plant nutrient, chloride. I. Chlorine-36 Nanoparticle preparation Cl- was used as a model nutrient anion. A defined concentration of 0.1 mM dendrimer solution was mixed with increasing concentrations of NaCl-36 (Fig. 1) and incubated overnight in constant agitation. After 24 hr, the solution was filtered to exclude unbound Cl36 and Cl36- bound to nanoparticles determined by scintillation spectroscopy. II. Cl36-Nanoparticle application to leaf tissue. Samples of Cl36 bound dendrimers were applied to a leaf surface at 2 concentrations (100 and 50 .M). An aqueous Cl solution of 170 nmoles was used as control (Fig. 2). Leaves were incubated in a humid chamber for 24 hr. Leaves were rinsed with distilled/deionized water and allowed to dry. The amount of Cl36 remaining in individual leaf fragments was determined by scintillation spectroscopy. III. Results Cl36 applied water washed attached to leaf surface % attached 36.6 nmoles (dendrimer bound) 20.67 nmoles 15.93 nmol 43.5% 170 nmoles 152 nmoles 18 nmoles 0.6 % IV. Analysis Nanoparticles are capable of delivering four times the amount of Cl to the leaf when compared to soluble Cl. Nano-particles physically attach to the leaf surface and are capable of delivering additional Cl with time. *** Electrostatically charged spraying and conventional spraying efficacy with or without surfactants *** 200 citrus rootstock trees were planted in the greenhouse in pots and allowed to establish. A reduced fertilization program was implemented to develop nutrient deficiencies in the trees. Nutrient-deficient trees are required for reliable detection of nutrient uptake applied as foliar fertilizer sprays with the computer-controlled sprayer platform. Visible deficiency symptoms of the major nutrients started to develop after 3 months but micronutrient deficiencies were just beginning to show at 6 months and the treatment testing will therefore commence in the 3rd and 4th quarters.
*** Electrostatically charged spraying and conventional spraying efficacy with or without surfactants *** Results of the foliar spray experiment with field-grown young citrus using a strontium (Sr) tracer to mimic the uptake of Ca were measured at 48 hours and 7 days after the treatments were sprayed on the trees. The ANOVA for leaf Sr measurements after 48 h showed that there were highly significant differences in Sr concentration in the leaves in response to 1) Sr application, 2) Sr rate, 3) adjuvant application, and 4) adjuvant B was more effective at the higher (full; 100%) Sr application rate than at the 50% rate. Results, in mg/kg Sr (leaf DM basis) are as follows: Rate: None (0%) 50% 100% Adjuvants: None A B None A B Control: 102.1 +Sr: 161.6 279.7 293.9 181.5 402.4 333.9 It was noteworthy that both surfactants nearly doubled foliar Sr concentrations at the low Sr fertilization rate (50%) and that these foliar element concentrations were higher than those achieved at the 100% Sr rate WITHOUT surfactants. Highly significant treatment differences and a similar Sr concentration trend in the foliage was still detectable after 1 week: mg/kg Sr (leaf DM basis) Rate: None (0%) 50% 100% Adjuvants: None A B None A B Control: 91.3 +Sr: 146.1 302.3 259.5 165.5 380.5 296.7 The efficiency of Sr absorption and retention when applied as a standard foliar nutrient spray on these field-grown trees was significantly improved by the addition of two surfactants, one (A) an experimental product, and the other (B) a well known commercial siloxane-based product (Induce, Helena Chem).
*** Electrostatically charged spraying and conventional spraying efficacy with or without surfactants *** Since the potted citrus trees took longer than expected to develop comprehensive nutrient deficiencies that are necessary to reliably detect the uptake of added foliar nutrients as concentration increments, we decided to use a tracer method for quicker results, while allowing additional time for the greenhouse trees to become nutrient-depleted for testing later. Strontium (Sr) has been used as a calcium (Ca) nutrient analog to evaluate Ca transport processes and distribution in plants. Since natural Sr concentrations in soils and crop plants are present at extremely low background levels only, any artificial addition of Sr to the crop system as a foliar or root application can be easily detected with standard ICP instruments used in agricultural laboratories. The objective of this study was to determine the efficacy of two spray surfactants (1 experimental, and 1 commercial) on the foliar uptake Sr nutrient tracer. We obtained strontium nitrate for mimicking the foliar spray of calcium nitrate to citrus (formula weight: 211.63 g/mol) and applied the following foliar spray treatments, replicated four times, to 1-yr old HLB-asymptomatic citrus: Treatments were factorial, consisting of 2 Sr rates and 2 surfactants, plus an independent untreated control in a completely randomized design: 1) 100%, 7.4 g/L strontium nitrate 2) 50%, 3.7 g/L strontium nitrate 3) 100% + Expt surfactant A 4) 50% + Expt surfactant A 5) 100% + surfactant B (Induce) 6) 50% + surfactant B (Induce) 7) Control – 0%, no surfactant Rainfall of 7.7 mm was measured in the afternoon, about 6 hours after spray treatments were applied. Leaves were sampled from the trees 48 h and 7 d after treatment, and were processed and analyzed for Sr, using the normal methods recommended for nutritional analysis (acid+detergent wash, dry at 70C, grind, mix and submit to Waters Agricultural Labs). Analysis of variance was used to determine statistical significance of the treatment factors and the least significant differences were calculated to separate treatment means. Results are presented in the following quarterly report.
In olfactometer trials, we confirmed that Diaprepes were attracted to the frass of the opposite sex. We continue to document the attraction of males to female odor by field-collected Diaprepes males. We 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. We continue to investigate the synergistic interaction between pheromones (male and female) and plant odors (kairomones) previously identified to be slightly attractive to males. We will continue these studies to further develop an attractant for Diaprepes abbreviatus. Synthesis of the pheromone in our laboratory is proceeding and we will have sufficient quantities to continue olfactometer assays and field tests. All tests to date of the synergy between male pheromone and kairomones have been done with a natural pheromone source (male frass) but can now be confirmed quantitatively with synthetic pheromone.
The bacterial endosymbiont, Wolbachia, is of interest to the citrus greening research community as a potential route for manipulating behavior of its host insect, Diaphorina citri, vector of citrus greening disease. Our recently published study, ‘Survey of endosymbionts in the Diaphorina citri metagenome and assembly of a Wolbachia wDi draft genome’ (Saha et al PLoS ONE), emphasizes draft genome assembly and annotation of candidate host interaction factors of the Wolbachia strain in a Florida isolate of D. citri. This sequence should prove a useful reference for comparison among other D. citri Wolbachia strains as they are evaluated for potential vector control. The Wolbachia wDi draft genome sequence together with genome features such as the 54 predicted ankyrin proteins, implicated in host interaction in other Wolbachia systems, can be viewed using the genome browser at http://citrusgreening.org/. Also new to the CG-HLB genome resources website is the genome sequence and annotation of L. crescens (Leonard et al, Standards in Gen. Sci). This strain holds great potential for use in experimental characterization of L. asiaticus gene product function, and through genome comparison with pathogenic strains, can shed light on the gene products potentially determinative for pathogenicity and in vitro culture. Within the genome viewer, functional insights can be accessed using the hyperlinks from individual genes to lists of similar proteins at NCBI. Links to metabolic pathway predictions have also been implemented and predictions of protein locations (of particular interest given the disproportionate role of surface located proteins to be involved in host interaction), are being generated using SignalP. Genome regions divergent from Ca. L. asiaticus psy62 and Ca. L. solanacearum are highlighted. A summary of the research and tools available at the CG-HLB genome resources website was presented at the 3rd international Research Conference on HLB in February 2013. A genome sequence for Ca. L. asiaticus strain gxpsy has been recently deposited at NCBI and incorporation into the genome viewer is ongoing. We are currently using our expertise with datasets generated by a variety of next generation sequencing technologies to assist Bob Shatters in validation of genome assemblies generated for 10 isolates of L. asiaticus from sequence reads generated using Ion Torrent. Validation and comparison of sequence differences observed among isolates from Florida and other citrus growing regions worldwide has the potential to enhance understanding of the epidemiology and biological variation of this pathogen. A manuscript describing the D. citri transcriptome, including the repeat characterization conducted by us, has been accepted for publication. Our assistance with the D. citri genome project is ongoing.
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. Emphasis is being placed on cryotherapy with conventional shoot tip grafting being used for comparison purposes. The project also includes determining the effectiveness of using young indicator plants for biological indexing to verify elimination of graft transmissible pathogens. In the past quarter, four shipments of HLB infected material from Florida, including a tangelo type, a grapefruit type, a mandarin type, and a pummelo, have been shipped to Ft. Collins, CO where they have been treated by cryotherapy. Plants recovered by cryotherapy and/or shoot tip grafting are held for 12 weeks before being initially tested by laboratory diagnostics for the target pathogen(s); this material will be tested initially for Las in late April. Plants testing negative for Las will be held and retested in 8-10 weeks. From Riverside, mandarin tissue infected with Citrus tatterleaf virus, several viroid groups, citrus stubborn (Spiroplasma citri), and Citrus psorosis virus have been sent to Ft. Collins, and treated with cryotherapy. In Riverside, a trial has been established where the results of biological indexing for Citrus psorosis virus, Citrus leaf blotch virus, Citrus concave gum, Citrus vein enation virus, and citrus viroids on young plants are being compared to the results from the conventional protocols for biological indexing for eleven accessions.
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 four times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB in one grove. Multiple compounds showed control effect. To further test those compounds, we have selected two more groves to expand the field test. The disease index of the two groves have been investigated. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We also compared the SA levels in HLB infected and healthy Valencia citrus. 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 using yeast two hybrid system.
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. All the 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. We are repeating this assay to eliminate any false positives. 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 studying 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 total RNA of grapefruit infected with wild-type Xac and mutant were extracted and microarray analysis is being conducted. Quantitative reverse transcription PCR is being used to study expression of selected plant genes. Anatomical analysis of the inoculated tissues were conducted using transmission electron microscopy.
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