One of the primary goals of this work is to identify a small molecule treatment that can be used to activate the phage lytic cycle genes encoded by Las prophage, thus bringing about the death of Las bacteria carrying these prophage. All Las bacteria examined to date have been found to carry prophages in their genomes. In periwinkles, but not in citrus, lytic phage particles are formed and can be visualized. We previously reported that relative mRNA expression levels of prophage late genes SC2-gp095 (“peroxidase”), SC2-gp100 (“glutathione peroxidase”) and particularly SC1-gp110 (‘holin’) were much higher in periwinkle than in citrus. We also reported that both the prophage holin (SC1_gp110) and endolysin (SC1_gp035) were functional. We now report that the Las prophage holin not only suppresses growth, but kills E. coli when overexpressed, indicating that strong expression of this gene alone in Las cells would be sufficient to kill the cells, whether or not phage particles were formed. Four differently sized potential promoter-active regions upstream of the Las holin (SC1_gp110) were cloned in front of a promoterless lacZ gene to form reporter constructs. E. coli colonies carrying these plasmids were nearly white on X-gal media, indicating a lack of holin promoter activity to drive lacZ expression. One of these was fused to a promoterless GUS reporter gene, cloned in a wide host range shuttle vector and used to transform L. crescens (Lcr) strain BT-1. This reporter gene exhibited unusually strong GUS activity in Lcr strain BT-1; in fact, this GUS activity was stronger than that observed using the constitutive lacZ promoter in Lcr. Significantly, in both qualitative and quantitative (MUG-based) assays, diluted psyllid extracts strongly and specifically inhibited GUS reporter activity in Lcr expressed by the holin promoter, as compared to the untreated controls. This suppression activity was heat labile (ie., heat treatment of the psyllid extracts destroyed the inactivating activity). In the same quantitative activity assays, psyllid extracts at the same treatment levels failed to inhibit the constitutive lacZ promoter driven GUS activity to any significant level. This work has been accepted for publication in Applied and Environmental Microbiology and is now available. The lack of holin promoter activity in driving the LacZ reporter in E. coli indicates the absence of a transcriptional activator that is present in Lcr and necessary to drive the expression of typically repressed downstream gene. An alternative but less likely explanation is that E. coli carries a repressor that recognizes the holin promoter and silences the expression of the reporter gene. Both hypotheses are being tested in an effort to identify the activator or suppressor. It seems likely that one or more chemical ligands in the psyllid extract is able to penetrate Lcr cells, bind to, and interfere with, phage late gene transcriptional activator(s), (for example, a MarR-like transcription factor) leading to suppression of phage formation in psyllids. Based on the increased transcriptional activity of the holin promoter in periwinkle, (a) similarly acting ligand(s) is/are also likely to occur in citrus. The strong activity of the holin promoter in the absence of psyllid or plant extracts may help explain why liberibacters other than BT-1 have not been cultured to date. Regardless of whether or not phage particles form, expression of the Las holin alone is sufficient to kill Las. Once Las bacteria are separated from any plant or insect host, any host-provided ligand that may suppress transcriptional activity will become increasingly dilute, and prophage late gene expression derepressed. We speculate that factors present in hosts may suppress phage activation and may be necessary, but not alone sufficient, for free living culture of Las bacteria. The assay developed here provides a high-throughput basis to screen for chemicals that may activate Las holins or interfere with holin suppression.
One of the primary goals of this work is to identify a small molecule treatment that can be used to activate the phage lytic cycle genes encoded by Las prophage, thus bringing about the death of Las bacteria carrying these prophage. All Las bacteria examined to date have been found to carry prophages in their genomes. In periwinkles, but not in citrus, lytic phage particles are formed and can be visualized. We previously reported that relative mRNA expression levels of prophage late genes SC2-gp095 (“peroxidase”), SC2-gp100 (“glutathione peroxidase”) and particularly SC1-gp110 (‘holin’) were much higher in periwinkle than in citrus. We also reported that both the prophage holin (SC1_gp110) and endolysin (SC1_gp035) were functional, and that strong expression of the holin gene alone in Las cells would be sufficient to kill the cells, whether or not phage particles were formed. Furthermore, the holin promoter was constitutively “on” in L. crescens (Lcr) strain BT-1, and was strongly suppressed by diluted psyllid extracts and the suppression was heat labile (ie., heat treatment of the psyllid extracts destroyed the inactivating activity). We now report that the psyllid extract suppression activity is also abolished by proteinase K treatment, suggesting that psyllids produce a protein inhibitor of the phage holin. The inhibitor was precipitated with acetone, and size fractionation demonstrated an inhibitor in the size range of 10-50 kDa. This reporter system may be developed into a high-throughput chemical screen for treatments that may interfere with psyllid or plant regulation of the phage lytic system. In addition, two additional phage late genes, SC2-gp095 (“peroxidase”), SC2-gp100 (“glutathione peroxidase”), were functionally characterized. Given that the highly reduced Las genome encodes no known defense against host generated reactive oxygen species (ROS), the putative phage-related ROS scavenging functions annotated as peroxidase (SC2_gp095) and glutathione peroxidase (SC2_gp100) may represent important ‘lysogenic conversion’ genes whose expression may increase bacterial fitness and delay symptom development in the host plant. Both SC2_gp095 and SC2_gp100 were expressed at significantly higher levels in periwinkle than in citrus or insects. SC2_ gp095 alone, and in tandem with SC2_gp100 were separately cloned in a wide-host-range (repW) shuttle vector pUFR071 (under control of the lacZ promoter), and transformed into E. coli and Lcr, a culturable proxy for Las. The transformed Lcr cells showed enhanced in vitro resistance to H2O2, 23% higher enzymatic activity and faster growth rates in culture as compared to Lcr cells transformed with only pUFR071. Moderate enzymatic activity was also evident in transformed Lcr culture supernatants, but not E. coli supernatants, confirming a predicted non-classical secretion potential for SC2_gp095, and suggesting such secretion from Las. Experiments are underway to further characterize the role of SC2_gp095 in planta. We hypothesize that Las peroxidases: 1) mitigate the direct antibacterial effect of reactive oxygen species (ROS) on Las cells, and 2) disrupt systemic cell-to-cell self propagation of ROS (H2O2) -mediated signaling in the host plant. The latter idea may explain the surprisingly long incubation period before symptoms appear. Las peroxidase(s) may be secreted effector(s) that function to suppress host symptoms, a tactic used by most biotrophic plant pathogens.
Surveys conducted throughout three southern California counties (Riverside, Imperial and San Diego) have shown the association of three Eutypella species with citrus branch canker and dieback in this area as well as Neoscytalidium dimidatum, the causal agent of Hendersonula. Morphological and molecular methods have identified the tree Eutypella spp. as E. citricola, E. microteca, and a Eutypella sp. which is closely related to Peroneutypa scoparia. All four fungi have been identified in all counties surveyed with N. dimidatum being the most frequently observed overall, followed by E. citricola, E. microtheca and Eutypella sp. Pathogenicity test on detached shoots show all three species of Eutypella are pathogenic on citrus, however these fungi appear to have a low to moderate virulence. The effect of temperature (25C and 32C) on lesion development was also studied and significant increases (P=0.05) in lesion length were observed for branches inoculated with N. dimidatum and E. microtheca, but not for plants inoculated with E. citricola or Eutypella sp. Greenhouse inoculations were made with the above fungi and symptoms of gumming could be seen after two weeks for plants inoculated with N. dimidatum and E. microtheca. Results from in vitro fungicide screen show that a number of compounds are fairly officious in inhibiting these canker fungi, with strobilurins generally being the most efficacious for all four fungi owing to their effectiveness at low concentrations. Results from a field study assessing azoxystrobin, fenbuconazole, pyraclostrobin, and trifloxystrobin as pruning protestants revealed azoxystrobin to be the most effective fungicide overall in reducing lesion length caused by N. dimidadum and Eutypella spp. Pyraclostrobin and trifloxystrobin significantly reduced lesion length caused by N. dimidatum (P=0.05). Azoxystrobin was the most effective in reducing lesion length caused by E. citricola and E. micropheca, whereas pyraclostrobin was most effective in lesion reduction for the Eutypella sp.
Citrus plants showing symptoms of Huanglongbing (HLB) have revealed extensive phloem plugging because of the increased amounts of callose and phloem proteins, potentially interfering with phloem transport. Transcriptome studies of citrus plants with HLB have shown abundant accumulation of transcripts for callose and phloem protein genes compared to healthy plants. Down-regulation of these over-expressed genes responsible for phloem-plugging by RNA interference (RNAi), potentially would negate the disease severity. Towards this end we have cloned a truncated callose7 and phloem protein genes into the CTV silencing vector individually, and in tandem and generated Citrus macrophylla plants expressing RNA against endogenous callose and pp2 genes. Preliminary studies have indicated no accumulation of callose in these plants similar to healthy plants. However, these plants should elicit less callose in the phloem compared to healthy plants. Presently we are using real-time PCR for quantitatively determine the amounts of RNA for callose and PP2 in Citrus macrophylla RNAi plants. In addition, we graft inoculated sweet orange plants with bark patches from C. macrophylla RNAi plants, to determine HLB symptoms in sweet orange RNAi plants upon challenge inoculation HLB.
For the time period between April 1, 2014 and July 15, 2014, the Southern Gardens Diagnostic Laboratory has run 3996 samples. As has been mentioned previously, the majority of the samples are now research related samples (grower, private companies, State and Federal) as opposed to samples submitted just to diagnose the presence of HLB. Of the 3996 samples, 2966 were samples submitted as “grower” samples and 1030 were submitted as “research” samples and not through the traditional submission process. Some of these research samples were samples from trials submitted to the Southern Gardens psyllid/HLB screening facility that screens plants for resistance to HLB and also does some screening of transgenics and control chemicals submitted by various researchers (private, State and Federal researchers). Also part of the 3996 samples were 376 psyllids. Psyllid assays have been a part of the laboratory testing since 2008 and a database of the percentage of infected psyllids has been maintained. This period of time reflects the “slow” time of year and typically the sample load will begin to increase in the coming quarter and peak October-January.
Citrus huanglongbing (HLB) is associated with three species of Candidatus Liberibacter: Ca. Liberibacter asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus (Laf). The majority of the testing in Florida is focused on detection of Las as this is the only bacterium known to be associated with HLB in Florida to date, while Lam and Las have both been found in Texas. In March 2013, twelve different isolates from citrus and citrus relatives identified as being naturally infected with Ca. Liberibacter species but which would test negative for Las, Lam, and Laf, were inoculated into receptor plants in a greenhouse at Ft. Pierce. From the twelve isolates which were inoculated into receptor plants in the greenhouse, nine isolates have been established. The isolates which were not recovered came from citrus relatives that are not highly graft compatible with citrus. The nine isolates which have been recovered and established have been grafted into plants for the cross protection trial. The challenge inoculation using a Florida isolate of HLB will be done around the first of September. DNA extracts from Liberibacter-like bacteria associated with HLB symptoms are being sequenced using miSEQ and PacBio approaches.
Accumulation of Cu pesticides in soil and development of Cu resistance to citrus canker bacteria demand an alternative that is environmentally-friendly, non-phytotoxic and as effective as Cu compounds. Quaternary ammonium compounds (Quat) are effective antibacterial agent, however they exhibit severe phyto-toxicity to plant species. Therefore, it is not practical to apply quat materials directly to citrus plants. Fixed-Quat is a nanotechnology-enabled formulation that minimizes phytotoxicity of Quat while maintaining its antimicrobial efficacy. Additionally, Fixed-Quat material is designed to exhibit good rainfastness. In this reporting period, optimization of different industrially acceptable synthesis parameters of two previously reported Fixed-Quat nanogel formulations (named hereafter Fixed-Quat NG-A and NG-B) were carried out. Additionally, two new Fixed Quat nanoparticle formulations (named hereafter Fixed-Quat NP-A and NP-B) were synthesized. Fixed-Quat NG formulations are stable thus far (at least 6 months), indicating good shelf-life. Antimicrobial studies of Fixed-Quat NG-A and NG-B were conducted against Xanthomonas alfalfae subsp. citrumelonis (a citrus canker surrogate) using in-vitro microplate Alamar blue assay, bacterial viability (expressed as colony forming units, CFU/mL) and growth curves. Kocide 3000 was used as positive control and silica nanogel was used as negative control. Interestingly, both the optimized Fixed-Quat NG formulations demonstrated complete bactericidal efficacy at concentrations as low as 0.55-1.1 ppm (for Fixed-Quat A) and 3.5-4.4 ppm (for Fixed-Quat B). The MIC values of our previously reported Fixed-Quat NG-A formulation was 2 ppm. Feasibility of producing highly concentrated Fixed-Quat NG formulations has been tested which is industrially attractive for storage and shipping conveniences. We have successfully produced stable Fixed-Quat NG-A and NG-B formulations containing ~ 10000 ppm and ~ 13000 ppm Quat, respectively. Average particle size of Fixed-Quat NG-A and NG-B materials in solution was estimated using Dynamic Light Scattering (DLS) technique. DLS study suggests the formation of sub-micron to micron size hydrophilic NG particles. Fixed-Quat NP-A and NP-B are being characterized. Average particle size of Fixed-Quat NP materials was estimated to be ~450 nm by DLS. Future reports will include research results on Fixed-Quat NP optimized formulations.
Citrus huanglongbing (HLB) is associated with three species of Candidatus Liberibacter: Ca. Liberibacter asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus (Laf). The majority of the testing in Florida is focused on detection of Las as this is the only bacterium known to be associated with HLB in Florida to date, while Lam and Las have both been found in Texas. Nine isolates from citrus and citrus relatives identified as being naturally infected with Ca. Liberibacter species but which would test negative for Las, Lam, and Laf, previously inoculated and established in receptor plants are being challenged by graft inoculation using bark pieces from a lemon plant testing positive for Las. The plants will be monitored for relative titers of the Liberibacter like and Las isolates. In cooperation with ICA in Colombia, we have worked on characterizing a Liberibacter-like bacteria from Colombia using the methods being applied to the isolates in this project and droplet digital PCR. As a result we have announced the finding of a fourth species of Liberibacter, tentatively named ‘Candidatus Liberibacter caribbeanusfrom Colombia. Using the macroarray approach, we are identifying bacterial genome regions which are conserved among the four species of Liberibacter. We are continuing the characterization of the Florida Liberibacter-like isolates using PacBio sequencing, miSEQ and analyses with droplet digital PCR.
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 almost done with 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. All the sequencing data have been uploaded to public database. We analyzed the hormones in the blight diseased trees and healthy trees. Quantitative reverse transcription PCR was used to further compare the gene expression of selected genes of citrus. We sampled for the fourth time and further analysis of those trees are being conducted. Metagenomic analysis of the sequenced samples is being conducted. In addition, the release of 8 citrus genomes including one sour orange, 2 pummelo, 4 mandarin and 1 sweet orange in the database has facilitated our analysis of the metagenomic data. Two manuscripts are being prepared for publication.
We have made progress with the scFv library made with the earlier grant from CRDF. We had previously used the scFv when expressed as part of the M13 phage vector particle in ELISA and dot blot formats. Our efforts in the past quarter have built on that work, and now we are using the scFv alone in tissue print assays of citrus plants to detect ‘Ca. Liberibacter asiaticus’. scFv are expressed and purified from from E. coli cells using a 6X His tag incorporated in the scFv protein. We have produced purified scFv at concentrations in the mg/ml range. Differences are observed among different scFv clones. Results from SDS-PAGE gels are consistent with post translational folding being problematic for some scFv as has been reported in the literature. The tissue print assays continue on nitrocellulose membranes. Color development is observed in the vascular cylinder (phloem) of HLB infected petioles but not in comparable petioles from healthy trees. In some tissue prints, color development is observed in discrete spots outside of the phloem cylinder. Similar results are obtained with all scFv that were selected to bind to proteins expressed on the surface of ‘Ca. Liberibacter asiaticus’. These targets include an ATPase associated with the type IV pilus, a pilus assembly protein, two flagellar proteins, the major outer membrane protein OmpA, and the efflux protein TolC. These results have been attributed to non specific cross reactions of the commercial monoclonal antibody directed at the 6X His TAG. The His tag will not be very useful for tissue printing. In continuing work, we have recently made tissue prints with a modified technique, using ‘Super block’, a commercial product used in Northern and Southern blotting on nitrocellulose membranes and detection with a monoclonal antibody directed at the FLAG epitope on the scFv. This protocol produces remarkably sharper tissue prints with dramatically reduced background, and color tightly focused as a ring in the phloem cylinder of HLB infected, but not healthy petioles. These results vary by manufacturer of the monoclonal anti-FLAG antibody conjugate. We have identified the best supplier. We have also prepared rabbit polyclonal antibodies against the major outer membrane protein (OmpA) and detected them with alkaline phosphatase labeled goat anti rabbit monoclonal antibody. The polyclonal antibodies produce distinct spots of color corresponding to individual phloem cells infected with ‘Ca. Liberibacter asiaticus’. This is a useful assay for ‘Ca. Liberibacter asiaticus’, and we are using it to describe the distribution of ‘Ca. Liberibacter asiaticus’ in infected citrus trees, fruit and seed. We have conducted research with the anti OmpA antibodies and have demonstrated that they are at least as effective as qPCR for the detection of ‘Ca. Liberibacter asiaticus’ in plant samples when used in a tissue print format. We have used these antibodies to characterize the distribution of Ca. Liberibacter asiaticus’ in infected citrus and can unambiguously detect it in all plant and seed parts tested. We have combined this antibody with PCR and have developed a sensitive immunocapture-PCR (iPCR) protocol for the detection of Ca. Liberibacter asiaticus’. Three manuscripts are in draft form related to this work.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. The reimbursable agreement with CRDF was established on September 5, 2012. We continue to study the literature to identify vectors to use for a future scFv library made as part of this project. The goal is to find a suitable vector that is not encumbered by intellectual property and patent issues. I have written twice to a laboratory in Germany which has published results with a suitable vector but have had no reply. We are also optimizing the cloning strategies that will be used to move already selected scFv into transgenic plants. We have obtained the vector, pUSHRL-26, to be used for plant transformation of the scFv constructs from Ed Stover at Fort Pierce and the plasmid has been purified. We have purchased the restriction enzymes and designed primers to be used for PCR to amplify the cloned scFv encoding inserts from vector pKM19. The cloned inserts will be sequenced to confirm that they are correct and then cloned into the transformation vector. The scFv have been modified by the addition of a four amino acid leader sequence (KDEL) and both Sma I and Spe I cloning sites. The KDEL sequence is expected to stabilize the concentration of scFv in phloem cells by facilitating proper folding of the protein in the microtubules and thereby protecting the ScFv from proteolytic digestion. Eleven scFv inserts have been sequenced to be sure that the expected sequences are correct, and five ScFv sequences have been successfully cloned into the recombinant vector pUSHRL-26 for transformation of citrus rootstocks. These inserts include three different scFv that bind to the protein InvA and two that bind to the protein TolC. The protein InvA is produced by CaLas and secreted into the host to prevent the infected host cells from entering into apoptosis, and the protein TolC targeted by the scFv, is in the external membrane and is essential for the removal of antimicrobial substances produced by the plant. The vector is designed to direct expression of the scFv into the phloem cells of citrus, where CaLas grows, and the vector encoding the scFv genes is being introduced into rootstock varieties by Agrobacterium mediated transformation. In the period just ending we have purified 9 scFv genes and cloned them into the plant transformation vector developed by Ed Stover at Fort Pierce. Sequencing confirmed the clones were correct. The Stover laboratory has transformed the constructs into Agrobacterium and the Agrobacterium has been used to transform Carrizo seedlings. Nine transgenic lines have been established with between 150-400 epicotyl explants for each line. These explants include scFv for both TolC and InvA, and are being grown at Fort Pierce for subsequent evaluation.
We have made progress with the scFv library made with the earlier grant from CRDF. We had previously used the scFv when expressed as part of the M13 phage vector particle in ELISA and dot blot formats. Our efforts in the past quarter have built on that work, and now we are using the scFv alone in tissue print assays of citrus plants to detect ‘Ca. Liberibacter asiaticus’. scFv are expressed and purified from from E. coli cells using a 6X His tag incorporated in the scFv protein. We have produced purified scFv at concentrations in the mg/ml range. Differences are observed among different scFv clones. Results from SDS-PAGE gels are consistent with post translational folding being problematic for some scFv as has been reported in the literature. The tissue print assays continue on nitrocellulose membranes. Color development is observed in the vascular cylinder (phloem) of HLB infected petioles but not in comparable petioles from healthy trees. In some tissue prints, color development is observed in discrete spots outside of the phloem cylinder. Similar results are obtained with all scFv that were selected to bind to proteins expressed on the surface of ‘Ca. Liberibacter asiaticus’. These targets include an ATPase associated with the type IV pilus, a pilus assembly protein, two flagellar proteins, the major outer membrane protein OmpA, and the efflux protein TolC. These results have been attributed to non specific cross reactions of the commercial monoclonal antibody directed at the 6X His TAG. The His tag will not be very useful for tissue printing. In continuing work, we have recently made tissue prints with a modified technique, using ‘Super block’, a commercial product used in Northern and Southern blotting on nitrocellulose membranes and detection with a monoclonal antibody directed at the FLAG epitope on the scFv. This protocol produces remarkably sharper tissue prints with dramatically reduced background, and color tightly focused as a ring in the phloem cylinder of HLB infected, but not healthy petioles. These results vary by manufacturer of the monoclonal anti-FLAG antibody conjugate. We have identified the best supplier. We have also prepared rabbit polyclonal antibodies against the major outer membrane protein (OmpA) and detected them with alkaline phosphatase labeled goat anti rabbit monoclonal antibody. These polyclonal rabbit antibodies are very useful in a tissue print detection format. The tissue print assay also preserves the anatomical distribution of CaLas in plant tissues. The sensitivity of the assay is comparable to qPCR but interestingly is more successful for detection of CaLas in asymptomatic plant tissues than is qPCR. This is because although the concentration of CaLas may be locally high in individual phloem cells, the overall concentration of CaLas in tissues can be low in these presymptomatic leaves. The ease and cost of the dot blot assay is also much less than qPCR. We have used the assay to follow the distribution of CaLas in infected trees. The assay shows the distribution of CaLas in roots, stems, leaves, peduncles and seed of citrus samples collected from HLB affected groves in Florida. The results of the tissue print assays have been correlated with results from qPCR.
Sept 18,2014 The objectives of this proposal are 1) to conduct a statewide survey of tangerine and tangerine hybrid groves to determine the proportion of strobilurin resistant Alternaria alternata isolates along with the identification and characterization of resistance-causing mutations; 2) establish the baseline sensitivity of Alternaria alternata to the SDHI class fungicide, boscalid and characterize field or laboratory SDHI resistant mutants to determine the likelihood of SDHI resistance development in Florida tangerine production and 3) Develop an accurate and rapid assay to evaluate sensitivity to DMI fungicides. During this quarter we accomplished: ‘ Boscalid paper (revisions completed and final acceptance in Plant Disease) ‘ Finished isolate screening for DMI baseline project
June 4,2014 The objectives of this proposal are 1) to conduct a statewide survey of tangerine and tangerine hybrid groves to determine the proportion of strobilurin resistant Alternaria alternata isolates along with the identification and characterization of resistance-causing mutations; 2) establish the baseline sensitivity of Alternaria alternata to the SDHI class fungicide, boscalid and characterize field or laboratory SDHI resistant mutants to determine the likelihood of SDHI resistance development in Florida tangerine production and 3) Develop an accurate and rapid assay to evaluate sensitivity to DMI fungicides. During this quarter we accomplished: ‘ QoI fitness paper (accepted in Plant Disease) ‘ Boscalid paper (submitted to and accepted in Plant Disease) ‘ Assay for DMI screening developed and isolates are being screen for baseline assay.
This is a project to continue one of the most fruitful leads that accidentally resulted from our previously funded work. We have found that citrus becomes a source of Huanglongbing (HLB) inoculum for spreading the disease to other plants much earlier than previously thought. The working hypothesis is that the female psyllid finds an area of new flush to lay her eggs. As she is laying eggs, she probes the phloem to feed and transfers Candidatus Liberibacter asiaticus (Las) to the tree. As the eggs develop into nymphs, Las begins to multiply in that localized area of the plant, where the new nymphs then feed and acquire Las. Thus, infection of only a micro area of flush tissue where the nymphs develop is sufficient for the first generation of psyllids to become infected and to be vectors to spread the disease to other trees. Thus, the time-period after a tree becomes infested by infected psyllids until it is a donor for other trees could be as short as 15-30 days or less. The limitation is actually the time for the second generation of psyllids to develop. We are working with a group in the Math Department of UF to develop a model of spread of HLB in new planting of citrus. A manuscript is being prepared reporting these results. We are using this rapid screen to determine whether a peptide can inhibit Las multiplication within 60 days instead of approximately one year. However, this rapid screen measures resistance, but not tolerance. We are still screening using infected psyllids to inoculate plants, but this information allows us to know which plants are inoculated with Las and is greatly improving those assays also. This work is continuing as described above. Our major emphasis is to use this system to screen for the effect of specific RNAi constructs against psyllids. Preliminary results suggest that it is possible that RNAi can reduce the number of psyllids produced and/or reduce the number of psyllids that become infected with Las.
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