Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care costs tend to be higher than commercial groves due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. During the recently completed quarter (October 1 to December 31, 2013), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ Plant Improvement Team o Scion for HLB tolerance under evaluation o Evaluation of Valencia clones on new rootstocks established o Sugarbelle harvested ‘ Dr. Singh continued to evaluate herbicide tolerance of selected USDA rootstocks to various residual herbicides continues ‘ 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 continues ‘ Dr. Futch evaluations: o Continued evaluations of trifoliate rootstocks for HLB tolerance ‘ Applications of the ‘Boyd Program’, Keyplex and Ben Hill Griffin programs continued in the ‘commercial scale’ nutritional trial. ‘ Conducting late summer/early fall fertilizer and pest management programs for the groves o Herbicide program on schedule o Dormant psyllid management continued o Initiated mechanical weed/middle management for evaluation ‘ Applications of seven nutritional treatments continue in MFCF replicated nutritional programs evaluation and plant growth data taken in July ‘ MFCF evaluating topping treatments evaluated ‘ Commercial Trials: o Eurofins evaluations on disease and insect management continue o Evaluations of Agri Quest Citrus Root Health Improvement Project continue o Keyplex nutritional trial evaluations continue o DuPont demonstration for row middles management with Matrix Herbicide established o Syntech trial for GLP evaluation ‘ 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
We have continued to focus on the treatments and analysis of naturally infected trees in the field to evaluate the efficacy of our treatments. Twenty four trees were chosen and first sprayed with our 8 spray treatments that included 6 therapeutic treatments and 2 control treatments. The plot was first sprayed on June 25, 2013 and then again in October 30 2013 with 3 trees per treatment. The therapeutic treatments included: 2, L- Arginine, 2, gibberellin in combination with 6-benzyl adenine (BA), and 2, atrazine in combination with sucrose. The remaining two treatments were control treatments that accounted for the surfactant Silwet, K-phite and fertilizer LDKP3XTRA that were added to all treatments. After the spray treatments the trees were sampled at day 0, 3 and 6 collecting 6 leaves from around each of the trees. The leaves from each tree was pooled and extracted for DNA, RNA and protein. The first spray yielded 72 samples that have been processed. The analysis of the DNA revealed that 40% of the plants were infected and that infected trees were unfortunately not always randomly distributed among each of the three replicates for each treatment. To overcome this in October 30, 2013 we sprayed an additional 6 infected trees per treatment. We have analyzed 10 biomarkers at both 3 and 6 days after treatment: 5 related to immune responses pathways (EDS1, PR1, WRKY70, WRKY48, WRKY54), 2 involved in gibberellin pathways and 3 related to sucrose and starch metabolism, a pathway highly linked with HLB syndrome. As we expected, Gibberellin-2-oxydase was clearly induced at Time 6 days in response to GA + BA treatment. Same trend was observed for one arginine and one atrazine + sucrose treatment. Relating to genes involved in sucrose and starch metabolism, we interestingly observed that at least at one concentration, the six treatments were able to reverse the trend of expression of invertase and sucrose synthase induced by HLB infections. GPT2 is strongly induced by HLB in leaf tissues and it is believed to be strongly associated with the starch accumulation in infected leaves. GPT2 is a transporter present in the chloroplast and responsible of the glucose-6-phosphate transport leading to the HLB-induced starch biosynthesis. Interestingly, we observed a strong down regulation of this gene in all six treatments only at three days after treatments. Data showed that some treatments may allow boosting Citrus immune responses. Indeed, WRKY70, a well-known transcription factor involved in biotic stress response was clearly enhanced by BA + GA spray and one Arginine treatment at both Time 3 and 6 days. Arginine and atrazine treatments induced expression of PR1, a SAR-related protein at Time 6 days. Conversely, WRKY48 was not significantly regulated by any six spray conditions. Another key player in biotic stress responses, WRKY54, was higher in abundance in both arginine and atrazine treatments. One concentration of GA + BA showed similar effect. The transcript abundance of EDS1 was unchanged in comparison to both controls. Taken together, these data look promising and indicated that, at least at one concentration, the three therapeutic strategies were able to significantly affect expression of HLB-regulated key biomarkers. However this effect is temporary and will need to be validated by the next gene expression data performed at different season time. Based on this finding we have planned another experiment integrating the eight conditions in two treatments (Arginine + GA + BA or Atrazine + GA + BA) and two control (K-phite and LDKP3XTRA fertilizer that were included also in the other two treatments). These 4 treatments were sprayed on March 11, 2014. We have performed a preliminary proteomic analysis for few treatment samples that allowed identifying around 250 proteins, mostly belonging to Citrus host response. Few pathogen proteins were identified and might be considered possible target to improve early detection. Different protein extraction protocols were tested and we are focusing on one that was the most effective allowing ~1300 proteins to be detected. These data will be functionally analyzed with data mining tools, integrated with previous transcriptomic findings to depict an overall view of the HLB-related syndrome in infected leaves.
Our earlier report showed (Dec-2014) the induction of new flush and flowering in HLB-infected trees following the first spray (Oct-2004) application of 10 ‘M strigolactone (SL). The second spray in January-2015 further induced the vegetative and reproductive growth in greenhouse grown HLB-infected ‘Valencia’ trees. We observed SL induced spring flush in fall and summer flush in spring. The number of vegetative branches were more numerous in SL treated HLB-infected trees in comparison to all other treatments (Healthy, Healthy + SL, HLB alone). SL can be used to regulate canopy architecture in citrus. Early induction of vegetative growth during dormant period of psyllid activity would prevent transmission of Clas through young foliage. Due to early induction of reproductive growth the fruit diameter is larger in SL treated HLB-infected trees in comparison to other treatments. In addition number of fruit and number of flower were also higher in SL treated HLB-infected trees in comparison to untreated HLB trees. Following 2 spray applications of SL over a period of 7 months the number of fruits remained higher in SL treated HLB-infected trees. Field evaluation of SL activity on fruit drop was also conducted on ‘Valencia’ tress at CREC and Weirsdale, Marion County. The fruit drop was 50% in HLB-infected tress. However, only 30% fruit drop was observed in SL treated HLB trees. At anatomical level, SL induced the formation of novel phloem in stem of ‘Valencia’ tress in greenhouse conditions as well as in field grown sweet orange ‘Pineapple’ trees. New phloem tissue in SL treated HLB trees is approximately five times larger in diameter than non-treated HLB trees. Phloem tissue remained functional without any plugging under SL treatment. Both cambium and phloem tissues were poorly visible in HLB-infected trees. SL also induced the starch mobilization at ultrastructural level. Lower accumulation of starch was observed in stems of SL treated sweet orange ‘Pineapple’ trees in comparison to untreated HLB trees. Starch mobilization provides indirect evidences for the availability of inorganic phosphate in cells. Third spray application of SL was done in Apr-2015 on greenhouse grown ‘Valencia’ trees and data will be collected in July-2015 for fibrous root growth, development, and anatomy.
HLB is characterized by impaired phloem performance, hormonal imbalance, poor root function and architecture, limited nutrient supply resulting in immature fruit drop and ill tree health/death. Our goal is to restore the proper functioning of the phloem tissue and consequently restore tree health by using the newest plant hormone ‘Strigolactones’ (SL). As opposed to other growth regulators, SL are carotenoids derived hormones demonstrated to induce cambial activity, fruit growth and development and symbiotic arbuscular mycorrhizal (AM) fungi association in many crops. SL applications have resulted in substantial increases in vascular tissue including phloem. Besides SL regulates root and shoot architecture by increased formation of primary roots, lateral roots, and elongation of root hairs. Application of SL either foliar and/or drench to citrus trees in nano- to micro- mole quantities, should to induce new phloem, roots in and regulate shoot architecture of HLB trees resulting in restore tree health. month intervals. Strigolactone (SL) application in green house trial resulted in early induction of new flush and flowering in HLB-infected trees in comparison to control tress. Similar results were observed in HLB-infected trees in field experiments. Second, SL spray application was done on green house plants after an interval of three months to observe the effect of SL on fruit retention and development. In addition, SL was also applied on individual branches of Sweet orange ‘Pineapple’ to further confirm the vegetative growth promontory role of SL. Control trees were also sprayed with SL in Wiersdale, Marion County. We are also developing a model system which included one leaf-one stem-one root system of citrus to observe the effect of SL under greenhouse conditions. For all field and greenhouse experiments, measurements of fruit size and petiole diameter have been recorded. In addition, tissue samples were collected for further anatomical studies.
HLB is characterized by impaired phloem performance, hormonal imbalance, poor root function and architecture, limited nutrient supply resulting in immature fruit drop and ill tree health/death. Our goal is to restore the proper functioning of the phloem tissue and consequently restore tree health by using the newest plant hormone ‘Strigolactones’ (SL). As opposed to other growth regulators, SL are carotenoids derived hormones demonstrated to induce cambial activity, fruit growth and development and symbiotic arbuscular mycorrhizal (AM) fungi association in many crops. SL applications have resulted in substantial increases in vascular tissue including phloem. Besides SL regulates root and shoot architecture by increased formation of primary roots, lateral roots, and elongation of root hairs. Application of SL either foliar and/or drench to citrus trees in nano- to micro- mole quantities, should to induce new phloem, roots in and regulate shoot architecture of HLB trees resulting in restore tree health. month intervals. To carry out this project, a Postdoctoral associate was hired. The individual is already established in the lab and commenced with the preparations for all types of experiments. Experimentally, both parts of the project have been started. Greenhouse experiments: For the greenhouse trial,s 50 Valencia on Carrizo trees were purchased. Trees were planted on 3.5 gal pots and placed in a greenhouse. An automatic irrigation system was established and trees will be allowed to acclimate for 30 days prior to the start of treatments. Field experiments: Field grown Hamlin HLB-affected trees have been selected in a block at CREC. Trees have been flagged, area under the tree has been cleaned, fruit per tree counted and experimental fruit flagged. We have purchased the growth regulator strigolactone and are poised to begin the first sprays during the first week in September.
The goal of the project is to identify a Bacillus thuringiensis (Bt) toxin with toxicity against Asian Citrus Psyllid (ACP) and to improve its toxicity by genetic modification with a psyllid gut binding peptide. The peptide will be identified by screening a phage display library for peptides that bind to the gut of the ACP. Addition of the selected peptide to the identified Bt toxin will result in significant enhancement of toxicity of the modified toxin relative to the wild type toxin toward the ACP. Durign the current reporting period, the focus was on trypsin activation of the partially purified Bt toxins from selected Bt strains provided by USDA ARS, MD. Trypsin activation of Bt toxins was carried out at Iowa State University. Briefly, Bt toxins were solubilized using sodium carbonate pH 10.5 + 10mM DTT for three hours at 37’C and dialyzed against 50 mM Tris-Cl pH 8.5. Bt toxins were then incubated with bovine trypsin at a final concentration of 10% of the toxin concentration at 37 ‘C for 1 h. Removal of trypsin was carried out using benzamidine sepharose. The samples were boiled in denaturing SDS sample buffer for 5 min, separated on 10% (wt/vol) SDS/PAGE and stained with Coomassie blue. Different SDS-PAGE profiles of trypsin-treated Bt toxins were apparent with multiple molecular weight protein bands ranging from ~18 kDa to ~150 kDa. Based on the similarity of the molecular mass of the toxin profiles, eleven Bt strains are classified into the following six groups. These groups are distinct from the five groups reported in the previous update. Group one: Five Bt strains, each with three activated toxin bands of ~60, ~65 and ~70 kDa. Group two: Two Bt strains, each with two activated toxin bands of ~60 kDa. Group three: One Bt strain with one activated toxin band of ~60 and ~65 kDa. Group four: One Bt strain with one activated toxin band of ~70 and~73 kDa. Group five: One Bt strain with activated toxin bands of ~70, ~90, and ~150 kDa. Group six: One Bt strain with activated toxin bands of ~18, ~20, ~22, ~28,~30, ~35, ~49, ~60, and ~70 kDa. Two milligrams of each of four trypsin-activated Bt toxin samples were sent to Dr. David G. Hall, USDA-ARS for ACP membrane feeding assays for toxicity analysis.
ACPS -grapefruit research at IRREC: The overarching goal of this component is to develop ACPS and high-density plantings for commercial grapefruit on the east-coast. To date, the progress of this project is as follows: Irrigation installation was completed in October 2013. We are currently testing and evaluating the irrigation systems to fine-tune their performance and eliminate inefficiencies and any errors made during installation. The first experimental block of citrus was planted in November 2013. ‘Ray Ruby’ grapefruit trees on ‘Sour orange’ and ‘US-897’ rootstocks were planted on 8 acres on the citrus research grove at the UF-IFAS-IRREC station in Ft. Pierce. ‘Ray Ruby’ / ‘Sour orange’ trees were planted at a density of 152 trees/acre with microsprinklers and will be fertilized with granular, dry fertilizer: these plantings will serve as the ‘grower standard’ control treatment. ‘Ray Ruby’ trees on both ‘Sour orange’ and ‘US-897’ rootstocks were planted in staggered-set, tramline configurations at a high-density of 421 trees/acre: half of these plots will be fertigated with microsprinklers and half will be fertigated with in-line drip tubing. Funds from this project were also used to partially support the construction of a 5-acre high-density block of ‘Ray Ruby’ / ‘Kuharske’ grapefruit at the IRREC. In this block, all trees are being irrigated with microsprinklers but are planted at 3 different densities: 126, 189, and 421 trees/acre. Funds from other sources were used to cover the costs to complete this research block. These trees were also planted in November 2013. Data collection on tree growth, psyllid abundance, and CLas presence will begin in late December 2013. We plan to hold a field day for stakeholders in late winter/early spring of 2014. This field day will exhibit the two trials listed above. A Ph.D. graduate student was recruited and hired through the UF-Horticultural Sciences Department to assist in executing this objective. Currently, this student is in Gainesville completing her required coursework. The student will be available to monitor the progress of the research blocks full-time starting in the summer of 2014. A full-time OPS worker was hired and began work in August 2013. The balance of this worker’s wages came from other funding sources. This employee has been extremely instrumental in completing the irrigation installation and maintenance of the research block. This employee will continue to help maintain these research blocks and assist the graduate student (described above) with data collection.
Experiments to determine the efficacy of different nano-particle systems to deliver nutrients and antimicrobial molecules to citrus leaves: The objective of this study is to deliver water-soluble antimicrobial compounds into citrus plants affected by greening disease. The delivery of the antimicrobial compounds will be accomplished using nanoparticulate carrier systems (e.g. liposomes and polymer nanoparticles). In the first quarter of this project, dye-doped lipid and polymer nanoparticles that have encapsulated water-soluble dyes as surrogate for antibiotics have been prepared. Liposomes have been prepared using e.g. 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane, and cholesterol through the thin lipid film hydration method. Initial studies have been made to encapsulate water-soluble dyes such as sulforhodamine B and fluorescein. In order to minimize the leaching of the dyes, cationic lipids are incorporated in the lipid composition. Primarily, the particle size is being restricted to less than 100nm by the use of appropriate filtration membrane and the choice of hydrating solvent. Characterization of the developed dye-doped lipid structures as well as optimization to minimize leaching is under progress. In addition, dye-doped polymeric nanoparticles comprised of environmentally safe materials such as poly (lactic-co-glycolic acid) have also been prepared. Initial studies have been performed to encapsulate hydrophilic dyes in the polymer matrix. Using the double emulsion method, polymer nanoparticles that have successfully encapsulated fluorescent dyes have been synthesized. The size of the dye-doped particles lies between 200-300 nm. The engineered particles are being characterized for their particles size, distribution, absorbance, and fluorescence properties. Future studies involve the encapsulation of select dyes that have low interference from the auto-fluorescence of citrus plants and to study their uptake.
Experiments to determine the efficacy of different nano-particle systems to deliver nutrients and antimicrobial molecules to citrus leaves: The objective of this study is to deliver water-soluble antimicrobial compounds into citrus plants affected by greening disease. The delivery of the antimicrobial compounds will be accomplished using nanoparticulate carrier systems (e.g. liposomes and polymer nanoparticles). In the first quarter of this project, dye-doped lipid and polymer nanoparticles that have encapsulated water-soluble dyes as surrogate for antibiotics have been prepared. Liposomes have been prepared using e.g. 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane, and cholesterol through the thin lipid film hydration method. Initial studies have been made to encapsulate water-soluble dyes such as sulforhodamine B and fluorescein. In order to minimize the leaching of the dyes, cationic lipids are incorporated in the lipid composition. Primarily, the particle size is being restricted to less than 100nm by the use of appropriate filtration membrane and the choice of hydrating solvent. Characterization of the developed dye-doped lipid structures as well as optimization to minimize leaching is under progress. In addition, dye-doped polymeric nanoparticles comprised of environmentally safe materials such as poly (lactic-co-glycolic acid) have also been prepared. Initial studies have been performed to encapsulate hydrophilic dyes in the polymer matrix. Using the double emulsion method, polymer nanoparticles that have successfully encapsulated fluorescent dyes have been synthesized. The size of the dye-doped particles lies between 200-300 nm. The engineered particles are being characterized for their particles size, distribution, absorbance, and fluorescence properties. Future studies involve the encapsulation of select dyes that have low interference from the auto-fluorescence of citrus plants and to study their uptake.
A new high-capacity indirect hot air portable heater was purchased for further study on the effect of heat treatment of HLB inoculum as well as tree health. The heater works by convection, heating the air as it passes over a flame while a fan moves the heated air to the heat treatment tent as opposed to the electric heaters used in the previous treatments which use radiant heat transfer. The goal of using the dry hot air heater was to provide supplementary heat to increase the temperature more rapidly and test the ability of heat treatment late in fall and winter as well as to resolve the issues that were created by electric heaters used in the first part of this study. In November, five trees were tested at temperatures ranging from 50-55’C (122-131’F). A new portable tent was built to accommodate the new hot air heater and the larger trees subjected to the test. In all five tests, it was observed that the temperature within the tent rose much more rapidly than when the electric heaters were used. However, at this point, it is too early to assess the differences in leaf damage due to heat stress. It seems that hot air can increase the temperature more uniformly and rapidly than electric heaters, but it also increases the amount of leaf drop in the canopy after treatment. The original 36 trees that were heat treated in the summer are still undergoing physiological tests to evaluate the overall health of the trees. Six month post-treatment tests are currently underway. The goal of these tests is to quantify the change in health of the tree due to the heat treatment. This data will be used to support evidence of fruit yield and fruit quality improvement. A mathematical model was developed using the finite difference method to simulate heat transfer in the citrus canopy. This model will help to predict the amount of heat needed for different sizes of trees. Two types of heat transfer, conduction and convection, were applied within the model. The conduction heat transfer model will simulate heat transfer through the trunk and branches, while the convection heat transfer will simulate heat transfer around the canopy under the enclosure. The input parameters included the trunk diameter, thermal conductivity of the trunk, heat transfer coefficient of air, and air velocity from the fan. The output parameters were temperature and heating duration, in which a mapping of temperature with heating duration was produced. Regressions and model fitting was done based on the plots generated by the data. Model calibration and validation were done by comparing the data from the model with experimental data obtained from the preliminary results.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. Although we previously reported that LasAI and LasAII target host mitochondria, we had to co-inoculate the gene silencing suppressor P19 to have a detectable expression of LasAI and LasAII (PLoS ONE8:e68921, 2013). After optimization for a variety of parameters that are critical for efficient gene expression in plants, high expression level of LasAI/LasAII were detected without co-inoculation of P19. Transgenic Arabidopsis plants expressing lasAI or lasAII showed a different degree of impaired growth. In particular, the LasAI contains domains responsible for abnormal growth of the root and/or meristem. Transient expression of LasAI and three different LasAI domains, LasAI-N-terminal, LasAI-repeat, LasAI-C-terminal allowed us to visualize the sub-cellular localizations of different domains. Because of high level expression of these effector proteins, we developed a novel in vitro screening system that can evaluate small molecules against these Las effectors. We are currently screening potential chemicals that could interfere LasAI localization in the mitochondria. The library consists of more than 30 million compounds obtained from the small molecule libraries of the TPIMS (Torrey Pines Institute for Molecular Studies). Interestingly, a few groups of compounds showed interference activity against the mitochondrial localization of LasAI, indicating that some potential inhibitors against these Las effectors may be screened out in the following screening process. Meanwhile, to study the function of LasAI in citrus, transgenic citrus were generated to express LasAI, LasAI N-terminal, LasAI C-terminal, LasAI repeat region, LasAII and GFP control, respectively. Transgenic citrus expressing different domains of LasAI are under evaluation. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein was able to detect this antigen both in the transgenic plants and in the Las-infected plants. Meanwhile, the Western blot results revealed unique formation of this protein in E. coli and plants. Citrus plants with high level expression of this gene displayed HLB-like symptoms, yellow shoot and impaired growth. Further characterization of this effector revealed its unique sub-cellular localizations. Understanding the molecular mechanism of how the effector induces HLB-like symptom is underway. Polyclonal antibodies against LasAI and LasAII were generated and tested fro their sensitivity and specificity. The detection of LasAI protein transiently expressed in Nicotiana benthamiana using Western blot was well established. However, the detection of LasAI from infected citrus and psyllid hosts remains to be optimized.
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. During this past quarter, additional selections of mandarin and sweet orange materials have been forwarded to Ft. Collins for therapy using cryotherapy and shoot tip grafting. Recovered plants are allowed to grow for 12-14 weeks following therapy before testing for the presence of HLB. With pre-treatment and cryotherapy, results indicated the procedure is very effective at eliminating Citrus tatterleaf virus and citrus viroids; pathogens most difficult to eliminate by thermaltherapy and by shoot tip grafting. Additional selections of several varieties of citrus infected with huanglongbing have been forwarded to Ft. Collins for therapy, and will be tested in another 6-8 weeks. In Riverside, a system has been developed using Jiffy pots for the seedling used as indicator plants, allowing for a growout of about 75 days from the seed planting until the young plants can be inoculated and used as indicators. Bud survival is very high, greater than 98 percent. The entire indexing procedure can be done with the plants in the Jiffy pots. Another trial has been initiated whereby the results from using the mini-plant biological indexing will be compared with the traditional standard indexing protocol, using indicator plants 10-14 months old, for 15 accessions.
The project entitled ‘Further characterization of HLB resistant clones of selected citrus varieties’ (project no. 758) is aimed at conducting experiments to understand the basis of HLB tolerance in genera closely related to Citrus like Microcitrus, Eremocitrus and Poncirus. This quarterly research (from Oct 2013 to Dec 2013) consisted of: a) Conclusion of pollination experiments conducted during the spring of 2013: Out of 1000 pollinations conducted, we obtained about 50 usable fruits of pummelos, mandarins and Microcitrus (crosses were made between different HLB tolerant/resistant varieties with susceptible cultivars). As expected, some of the intergeneric hybrids did not set fruit. We are now collecting the seeds of the pollinated fruits and will be ready to test for the parental genotypes using a qPCR assay developed in our laboratory. b) Progress in greenhouse experiments for further testing and evaluation: Batch one plants consisting of HLB tolerant and susceptible citrus and relative accessions that were raised in October 2013 are now ready for psyllid challenge under greenhouse conditions in Fort Pierce. We have made arrangements for sample collection at various time points and subsequent RNA seq analysis as proposed. Since germination of certain genotypes was poor in the previous batch, fresh seed was collected and germinated in Fort Pierce greenhouses. We have good germination from the current batch and would include these in the HLB challenge assays as soon as they attain an acceptable size. c) Metagenomic analysis of several HLB resistant field plants has been conducted. The results of 16S rRNA sequencing from resistant and susceptible plants is now being examined to understand the role of the microbiome in the HLB tolerance phenomenon.
The research program objectives are to develop an effective and sustainable phage-based biocontrol system for Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker. During the third phase of year 1, we have expanded the phage bank with activity against Xac to 13 groups based on host range studies and type IV pilus dependency for infection. Host range diversity is indicative of multiple receptor sites being targeted by phages. Such diversity is a necessary component for the development of functional cocktails and for the prevention of phage resistant derivatives in the field when phage therapy is applied. Although type IV pili have not been implicated as virulence factors in Xac, their role in the pathogenic process is well documented in plant, human and animal bacterial pathogens. Electron microscopy studies of 16 purified phages from 10 groups indicate that we have isolated and purified 11 podophages (7 groups) with short non-contractile tails that exhibit capsids ranging from 52-65 nm in diameter, three siphophages (2 groups) with long non-contractile tails (122-130 nm) that exhibit capsids of 56-57 nm in diameter, and two myophages (2 groups) with long contractile tails (141-149 nm) and capsids of 96 nm in diameter. We had previously reported that phages CCP501-CCP505 had genomes that ranged from 40-44.5 kb, as determined by restriction enzyme digest analysis (REDA), and that phage CCP501 exhibited phage phiKMV-like architecture with a single subunit RNA polymerase indicative of a virulent lifestyle. We now report that phages CCP504 and CCP507 also have similar architecture and are therefore virulent. Preliminary genomic analysis using multiple restriction enzymes indicates at least two to three REDA types in host range groups 1-5, and four unique REDA types in groups 6-10. Ongoing sequencing, annotation, and abortive lysogeny experiments will confirm the virulence of phages in the expanding phage bank. The availability of a large bank of virulent phages with a diversity of attachment sites is a necessary first step in developing a sustainable phage based control strategy. We have recently obtained a USDA-APHIS permit to conduct detached leaf assays with citrus in the laboratory. We will conduct population dynamics studies to monitor the effect of phages on Xac populations, which have been introduced to detached leaves. In the next period, we will conduct first round efficacy greenhouse protection and therapeutic evaluation (in cooperation with Dr. Nian Wang, University of Florida) and UV sensitivity and protectant studies.
‘Candidatus Liberibacter asiaticus’ (Las), the causal agent of citrus greening, has not been successfully cultured. However, one species of the genus, Liberibacter crescens, has recently been cultured under laboratory conditions. The focus of our project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins) using L. crescens strain BT-1. It is hypothesized that once Las is successfully cultured, the protocols developed for L. crescens will be adaptable to Las. During the second phase of the project we have developed a protocol to obtain reliable and uniform bacterial lawns in overlays; a necessary first step for the detection, purification and propagation of phage. Standardization of the assay now allows us to move forward with the evaluation of environmental extracts (plant, water, soil or insect) for phage, testing of a large archive of on-hand phage stocks, and screening for tailocins and/ or temperate phages that may have activity against L. crescens. Using the overlay assay, we have initially tested 272 individual phage lysates from diverse hosts and seven broad host tailocins with no activity against L. crescens detected. Utilizing the same system, 37 plant (weeds, citrus, alfalfa, rice, fresh papaya pulp and seed) and 21 citrus psyllid extracts, as well as 27 water samples and supernatants of four UV-induced Rhizobium species were evaluated for phage that replicate and lyse L. crescens; no phage plaques were detected. Classical bacteriocins and tailocins production assays using diverse bacterial isolates from our large collection have also been initiated. We have identified two bacterial isolates that exhibit antimicrobial activity against L. crescens. The nature of the activity is currently under investigation. Exploiting the developed detection system, we will expand our screening for phages, tailocins and microbial activity against L. crescens.
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