For the time period between July 15, 2014 and October 15, 2014, the Southern Gardens Diagnostic Laboratory has run 5732 samples for a total of 9728 samples since April 1, 2014. Of the 5732 samples, 2861 were samples submitted as “grower” samples, 2411 were submitted as “research” samples and 460 were psyllid samples. Although submitted as grower samples, a significant portion of the “grower” samples were also research samples based on the submission data included with the samples. Thus, the trend in recent years towards a shift in the type of sample continues with the significant emphasis of the lab samples being geared towards private and publicly funded research.
Prolonged use of Cu compounds in citrus industry has significantly raised concerns pertaining to Cu accumulation in soil and development of Cu resistance to target organisms. To protect citrus crops from devastating citrus canker disease a suitable alternative to Cu compounds is highly desirable. “Fixed-Quat” is designed to be film-forming, locally systemic, non-phytotoxic. All the chemical ingredients of “Fixed-Quat” material are environmentally-friendly and USEPA approved. Superior anti-bacterial efficacy of Quat compounds must be maintained to maximize the benefit of Fixed-Quat material as an alternative to Cu biocides. In our earlier reports we have discussed synthesis, optimization and characterization of two Fixed Quat NG formulations and results on their antimicrobial efficacy against Xanthomonas alfalfae subsp. citrumelonis (a citrus canker surrogate) and Escherichia coli. In this reporting period we focused on developing two Fixed-Quat nanoparticle (NP) formulations (named hereafter Fixed-Quat NP-A and NP-B). Traditional Stober sol-gel synthesis method was used with certain modifications to load Quat compounds in Fixed-Quat materials. Fixed-Quat NPs form thin film on plant surface once spray applied. The material formed fairly smooth film when deposited over a silicon wafer by spin-coating technique. Particle size in vacuum was characterized by the Scanning Electron Microscopy (SEM) technique. Particle size in solution was estimated by using Dynamic Light Scattering (DLS) Technique. Both the SEM and DLS size measurements are in agreement and the average particle size was estimated to be in the sub-micron range (400-600nm). Antimicrobial studies of optimized Fixed-Quat NPs have been conducted against Escherichia coli and Xanthamonas alfalfae subsp. citrumelonis using a number of assays including in-vitro microplate alamar blue assay, bacterial viability expressed as colony forming units (CFU/mL) and the determination of the Minimum Inhibitory Concentration (MIC). Antimicrobial efficacy of the Fixed-Quat NPs were compared against Kocide 3000 (as a Cu control). The MIC of Fixed-Quat NP-A was determined to be as low as 4.4 and 1.1 ppm against E. coli and X. alfalfae respectively, whereas, the MIC values of Fixed-Quat NP-B were 8.8 and 4.4 ppm against E. coli and X. alfalfae, respectively. The difference in the MIC values has been attributed to the types of the Quat compounds used. Industrially-viable concentrated formulations of Fixed-Quat NP-A and NP-B were prepared at 17740 ppm and 22250 ppm with respect to the respective Quat content.
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. To date we have purified 19 scFv genes, and cloned them into the plant transformation vector pUSHRL-26 developed by Ed Stover at Fort Pierce. This includes 9 antibodies directed at InvA and 10 directed at the external opening of the TolC protein. 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. Nineteen transgenic lines have been established or are in development 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 by inoculation with psyllids infected with CaLas.
Sept 2014 The objectives of this project are to optimize Guignardia citricarpa ascospore extraction procedures and qPCR with automated extraction system, determine if prototype passive ascospore traps will capture a sufficient number of Guignardia citricarpa ascospores to be an effective monitoring tool and monitor for G. citricarpa ascospores in six locations around state. We have designed and built prototype spore traps. The first one was placed at CREC near our Burkhard trap so that spore counts could be compared. Once we were satisfied with the design and that we had made them so they would not break, further traps were constructed. Six traps were deployed at different heights (0.5, 1.0, and 1.5 m) near our Burkhard traps in the Immokalee area. We were only able to use 2 sites because one of our Burkhard traps was struck by a mower and is in many little pieces. We are counting the slides for the traps but it is slow because we are not able to use a dye to better visualize the slides. We moved from Vaseline to a Silicone grease for the slides because we found spores stuck better. Work on the PCR has been initiated.
Objective1: Reduce sampling and analysis time from 10 minutes (currently) to < 1 minute. We have reduced the sampling time to 3 minutes with an additional 3 minutes for analysis. Objective 2: Develop a VOC sampling method to collect VOCs from a significant portion of the tree. The sampler is completed and has undergone initial field testing. Changes were made to improve the performance. Additional field tests are underway now. The sampler is hand-portable and will operate on battery power for up to 8 hours. It uses a demountable odor trap that retains the odor signature after the sample was capped for 5 days. Objective 3: Develop an algorithm for identification of HLB. This task will be completed when we build the new library for detecting HLB-infected citrus trees. Testing is underway now in South Texas. Objective 4: Develop software to implement the disease detection algorithms (example output is a red/yellow/green light). A user interface has been developed to easily browse samples and immediately identify incorrectly gathered samples. This will be implemented on the program deliverable. Objective 5: Improve the durability of the instrument. The instrument modifications are on track for delivery at the end of the program. Objective 6: Deliver two hand-portable engineering prototypes. The first unit has been delivered and is being used at the UC Davis confinement unit. The second will be delivered at the end of the program. Objective 7: Demonstrate viability in field tests of engineering prototypes in Florida and California. This testing is underway in South Texas. The initial trial was completed in August, 2014 with additional testing scheduled in September. The biggest hurdle is optimizing the sampling technique. We attempted to use a bag to better confine the odors but this may also disturb the tree sufficiently to significantly affect the odor signatures. Sampling optimization continues.
Recent finds of huanglongbing (HLB) in commercial groves and residential areas in South Texas have necessitated intensification of surveillance efforts to ensure a cost-effective detection of ‘Candidatus’ Liberibacter asiaticus (CLas) as a precursor to implementing management measures. Since the probability of HLB detection over a given area is directly proportional to the number of tree samples tested in epidemiological studies, the sensitivity of qPCR for detection of CLas in composite (pooled) plant DNA samples was evaluated. One unit DNA from a range of known CLas-positive samples with Ct values 25, 30 and 32 were spiked separately into 4-fold increments of CLas- samples (up to 99 units) units of DNA from known negative samples. Resulting composite DNA were used as template in multiplex qPCR assays using probe-primer sets specific to CLas, CLam, and a plant cytochrome oxidase-based internal control gene (Li et al., 2006; J. Microbiol. Methods 66:104-115). The optimal composite ratio of one unit CLas-positive to four units CLas-negative individuals was validated using 31 composite DNA samples derived from 155 field survey samples and the results compared with tests performed on each individual in two independent machine runs. CLas was consistently detectable when one unit of DNA from a positive tree is pooled with four units of DNA from a healthy tree. A comparative analysis of the 155 field-collected tissue samples indicate that qPCR analysis of composite DNA samples could result in 77.5% true positive & negative, 3.2% false positive and 19.4% false negative HLB detections. The use of composite DNA samples could enable a cost-effective routine quick screen of large number of survey samples in disease surveys considering that the number of required analyses and associated reagents is reduced by pooling DNA samples from five individuals into one and analyzing the pooled sample. As a consequence, larger numbers of survey samples can be handled, including samples from asymptomatic trees. Cohorts of DNA extracts from single trees included in the CLas-positive composite DNA sample could then be tested individually to identify the culprit tree(s). The net amount of time required to perform the composite and individual qPCR assays was comparably equal since time expended pooling cohorts of individuals into composite samples are balanced by time saved setting up qPCR reactions for a condensed number of DNA templates. Based on current estimations, the ability to pool together five DNA samples into one composite sample could result in a saving of up to 40% of the total cost of testing individual samples. This is because the cost of DNA extraction will remain constant since DNA will be extracted from each individual while only one-fifth of the cost of qPCR analysis of the isolated DNA is needed for the composite DNA qPCR assay. However, it is worth noting that the composite DNA assay resulted in a significant amount (19.4%) of false negative samples. This should be taken into consideration especially when the assay is being considered for diagnosing very sensitive samples such as nursery trees.
This project provides the infrastructure to support eight CRB-funded projects investigating detection of the putative causal agent of huanglongbing (HLB), Candidatus Liberibacter asiaticus (CLas) in citrus prior to symptom development. The eight projects are all using the same cohort of plants, so that results of the various methods are comparable. The citrus cultivars used include Washington Navel, Tango Mandarin, and Lisbon Lemon grafted onto Carrizo rootstock. All of the plant material used for experiments is from the UC-Riverside Citrus Clonal Protection Program and is tested periodically to determine disease-status. The second cohort of experimental plants was grafted with scion buds on May 5, 2014. It is anticipated that these plants will be ready for CLas graft infection early in the fall. The rootstock for the third cohort of experiment plants was planted (from seed) on May 22, 2014. The seed has germinated and the seedlings are growing. The CLas (California Hacienda Heights strain) is maintained in Washington navel, Tango mandarin, Lisbon lemon, Mexican lime, and Volkameriana plants to have material to infect experimental plants and to maintain the CLas-positive Asian citrus psyllid (ACP) colony. All plants and psyllids are tested regularly with qPCR to determine the presence of CLas. The CT values for the CLas source plants varied from 24.0 ‘ 30.17 for positive plants, and the CT values for the control (not infected) plants were greater than 40. The CLas-positive colony of ACP has an infection rate of between 30-90%, depending upon how long the ACP adults have been exposed to CLas-positive plants. In general, the CT values for adults from the CLas-positive colony vary between 19.13 and 24.04 when tested with qPCR. This colony will be used to infect citrus plants in experiments in the future. A CLas-negative colony of ACP is also being maintained and CT values for that colony are greater than 40. Experiments have begun on determining the most efficient method of exposing experimental plants to CLas-positive ACP. Because of space limitations in the quarantine, mass releases of CLas-positive ACP into a greenhouse cannot be done. Studies are being conducted to determine optimum sizes of sleeve cages to use and length of exposure. This project provides addition support by providing the conference call line, and compiling minutes of the research group monthly meeting. The minutes and other pertinent materials for the group are archived on a secure website. Researchers in the projects have access to a website where all of the data generated can be compiled. This project maintains and updates the site.
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 small area of flush tissue where the nymphs develop is sufficient for the next 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. When the psyllid progeny move to the next tree for infection and reproduction, the three that they are leaving has only about the top 4-6 inches of flush that is infected. The rest of the tree is still noninfected. The virus then spreads downward from the point and eventually causes disease symptoms in the tree, long after the disease has spread to other trees. 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 information learned from these experiments to greatly speed up our screen of antimicrobial peptides and transgenic plants. A major new project in collaboration with other labs in Florida and California 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.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, June 2014. The lab has been in operation for 7.5 years, and as of August 2014, we have processed more than 68,700 samples. Additionally, more samples have been received for research for the entire period of diagnostic service supported by grant funding of individual researchers. The quarter from April through June 2014 saw a 50% increase in total number of samples processed when compared to the same quarter in 2013. There were 325 growers samples received during that time frame, which is well over double the number seen during the second quarter of the previous year. For the calendar year to date, the HLB Lab has seen a 50% increase in number of growers samples received compared to 2013 numbers. The first two quarters of 2014 saw a 10% increase in the total number of samples processed over January-June 2013. While overall numbers still reflect a decline when compared to peak sample processing of 2009-2010, there is a slight rebounding trend seen with increasing grower submissions over both 2012 and 2013 levels. Of interesting note is the fact that the number of individual submissions of growers samples in the first 7.5 months of 2014 has already surpassed the total number of submissions from either of the previous two calendar years. This correlates to the smallest average submission size (# of samples received at a time) seen since the all-time low during the lab’s first year of operation. These increases in sampling may be attributed to replantings and analysis of new young trees. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant.
This project aims in developing a method for efficient inoculation of plants with HLB that could be an alternative to currently used grafting and psyllid-mediated inoculation. We chose to use a Pulse Micro Dose Injection System (PMDIS) for injecting a suspension prepared from tissues collected from citrus trees infected with HLB into leaves and stems of receptor citrus seedlings with a purpose to transmit the bacterium and initiate infection in the latter plants. Since the HLB bacterium is distributed sporadically throughout the infected trees and is present in low quantities, we tested different types of tissue (stems, leaves, seed coats of infected citrus plants as well as tissues of HLB-infected psyllids) as resources of the HLB bacteria for preparation of the inoculum along with testing different extraction buffers for preparation of the bacterial suspension. While doing PMDIS-mediated inoculations, we tested different parameters of injection. We included plants of many species into our experiments: citrus (sweet orange, grapefruit, Citrus macrophylla, Mexican lime, and others) as well as tobacco, periwinkle, papaya. The outcomes of inoculations were evaluated by observation of inoculated plants for symptoms development along with PCR tests with HLB bacterium-specific primers. We have done a large number of inoculations in which we tested different conditions. However, at this point, this new approach was not very successful. Although some infections of citrus plants using PMDIS were achieved, infection rates were far less than those seen upon graft-inoculation of plants with HLB-containing tissue. In order to improve the inoculation technique, we contacted Dr. Carlos F. Gonzalez, Professor at the Center for Phage Technology, Faculty of Genetics, Department of Plant Pathology and Microbiology,Texas A&M University who uses a similar injection system for injection of bacteriophage into grape vines as a part of phage therapy for control of Pierce’s disease. A scientist from my lab who has worked on this project visited Gonzalez’s lab and participated in injection system-based inoculations in order to learn the protocol that Dr. Gonzalez’s lab uses. He also brought back some new nozzles that Dr. Gonzalez uses with his injection device. We have tested those nozzles to see if they would increase the efficiency of our inoculation procedure. Although the funding for this project already ended, we will continue our work. We have a few sets of plants from latests inoculation tests that we maintain in our greenhouse. We will monitor these plants in order to see if any of the changes that we have recently done to improve the procedure have worked. We believe that the key to success in this type of inoculations is a concentrated inoculum containing viable bacteria. Once Ca. L. asiaticus culture becomes available, we will use it with our injection device. The bacterium culture will provide much better inoculum to be tested using this approach. In the meantime, we are testing our injection system with Liberibacter crescens culture.
The goal of this project is to develop a method for rapid and efficient inoculation of plants with HLB using a Pulse Micro Dose Injection System. We tested many sets of plants that we have injected with the HLB bacterium-containing extracts using different conditions. Using this supplemental funding, we attempted to improve the efficiency of our inoculation protocol. We made a contact with Dr. Carlos F. Gonzalez, Professor at the Center for Phage Technology, Faculty of Genetics, Department of Plant Pathology and Microbiology,Texas A&M University who uses a similar injection system for injection of bacteriophage into grape vines as a part of phage therapy for control of Pierce’s disease. A scientist from my lab who was involved in this project, visited Gonzalez’s lab and participated in injection system-based inoculations in order to learn the protocol that Dr. Gonzalez’s lab uses. He brought back some new nozzles that Dr. Gonzalez uses with his injection device. We have tested those nozzles to see if they would increase the efficiency of our inoculation procedure. We also are testing our injection system with Liberibacter crescens culture. We obtained a USDA-APHIS permit for working with this bacterium. Liberibacter crescens culture was received from Dr. Triplett’s lab and has been to inject several different plant hosts. We also have been assisting Dr. Dean Gabriel with injections of his L. crescens culture into citrus plants using our device. Although the funding for this project already ended, we will continue our work. We have a few sets of plants from latests inoculation tests that we maintain in our greenhouse. We will monitor these plants in order to see if any of the changes that we have recently done to improve the procedure have worked. We believe that the key to success in this type of inoculations is a concentrated inoculum containing viable bacteria. Once Ca. L. asiaticus culture becomes available, we will use it with our injection device. The bacterium culture will provide much better inoculum to be tested using this approach.
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. The majority of Candidatus Liberibacter asiaticus (Las) strains described carry bacteriophage similar to SC1 and SC2 of Las UF506. 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. This could be part of the explanation as to why Las has to date not been cultured (Fleites et al. 2014). Furthermore, the holin promoter (a “late” phage gene promoter that is switched on only when the lytic cycle is activated) was constitutively “on” in L. crescens (Lcr) strain BT-1, and was strongly suppressed by diluted psyllid extracts and the suppression was heat labile and protease sensitive, suggesting that psyllids produce a protein inhibitor of the phage holin. Activity of the promoter in Lcr was suppressed by aqueous extracts from psyllids applied outside of the Lcr cells, indicating cell penetration, which is unusual. The suppressor activity was sensitive to heat and proteinase treatment, indicating direct repression by a protein, and size fractionation demonstrated the the size was 10-50 kDa. To further explore promoter activation in Lcr and suppression by psyllid extracts, electrophoretic mobility shift assays were performed. The mobility of the holin promoter fragment was decreased in the presence of both the psyllid and Lcr extracts, and was outcompeted by analogous unlabeled promoter DNA, indicating sequence-specific DNA binding. Using small, overlapping holin promoter fragments as competitor DNA, the binding sites of the proteins were further delineated. The two DNA-binding proteins were purified by DNA affinity capture. Identification of these repressor and activator proteins and target sequences is underway using MALDI-TOF. In addition to analysis of the late gene promoter, we have identified an early gene promoter of the phage that may be regulated by a chromosomally encoded bacteriophage repressor in Las. Japanese Las strain B430 appears to be nonpathogenic in citrus but parasitic, with a reduced titer relative to other Las strains. B430 appears to be missing all 6 prophage genes examined, including the functional peroxidase. The nucleotide sequences of B430 chromosomal genes flanking the expected prophage insertion site are identical to Las strain Psy62. A B430 ortholog of a Psy62 chromosomally encoded bacteriophage repressor, was found to exist as a mixture of mutated gene variants in the same plant. The mutated repressors appeared in 9 / 9 UF506 strains transmitted from citrus to periwinkle, and UF506 has only a wild type repressor, suggesting selection for loss of the repressor in periwinkle. This provides a second potential target for activating the Las lytic phage in psyllids and in citrus.
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.
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