We have completed the analysis greenhouse treated trees and concluded that each of the therapeutic spray applications could be validated by corresponding changes in gene expression of pathways that would be impacted by the sprays. We analyzed the transcription of 52 individual genes using Real Time PCR (qRT-PCR), 30 gave us valuable information on how each of these treatments was working. Our focus is on using these spray applications on field trees. On June 25, 2013 we treated a young orchard of Valencia on Swingle rootstocks in the Indian River Region of Central Florida. We treated 3 trees per treatment for a total of 24 trees for the 8 treatments. Six of these were 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 were control treatments that accounted for the surfactant Silwet, K-phite and LDKP3XTRA that were added to all treatments. We have sampled leaves at day 0, 3 and 6 collecting 72 samples of leaves that were flash frozen in liquid nitrogen and used to extract DNA for CaLas titer determinations and RNA for the real time PCR analysis for gene expression and pathway analysis. The analysis of the DNA revealed that greater than a third of the plants were infected. Average CLas titer in field treated trees prior to spraying was ca. 1×107 cells per g fresh weight leaf tissue. The analysis of the 72 RNA samples is underway with 9 of the 30 genes measured earlier. We are conducting phenotypic analysis of all 24 trees. Trees were rated for the presence of HLB symptoms, stem diameters and canopy height and diameter were developed. These measurements will continue throughout the duration of the experiment and represent a phenotypic analysis of the trees during this experiment. We are currently doing another sampling of DNA and RNA from the 24 trees, a 90 day time point from the first spray in June 2013. We plan to examine the expression of the 9 genes to compare the expression levels 3 months post spray treatment. We are also planning a second spray of the same 24 trees but will also spray an additional 48 trees in the same location, 6 each for each of the treatments. We will do a CaLas titer estimation of all 72 trees but isolate RNA from 48 trees 6 tree replicates for each of the 8 sprays. We plan the 2nd spray for early October 2013 and a third spray in Feb 2014. The sprays coincide with the flush observed in this location to have a maximum impact of the spray treatments.
At the request of the CRB, as of 10-1-11, Grafton-Cardwell and Morse merged their core entomology research efforts under a single project, 5500-501 (Morse’s portion of the project is 5500-501b). We have always coordinated our research efforts but this arrangement formalizes the situation. This report summarizes the Morse lab’s recent research under this coordinated project (all arthropod research except ACP which is a separate project, i.e. 5500-189). Our major effort this last year has focused on helping the industry to deal with Fuller rose beetle in relation to citrus exports to Korea. For the last several years, Korea has put increasing pressure on the CA industry to reduce egg mass levels on export fruit and there is a concern that in 2013-14, loads found to be infested with viable egg masses may be denied entry into that country. Morse presented a summary of recent research on Fuller rose beetle at the Korea Export meeting held in Tulare the morning of 9-4-12 and two talks, one on bean thrips and a second on mite contaminants of export citrus, during the 9-4-12 afternoon Australia / New Zealand Export meeting. An update on the FRB situation was given to the CCM Marketing committee 2-20-13, to growers and PCAs in Exeter 2-21-13, and to the CCQC Board in Pomona 3-26-13. A meeting with packing house personnel, CCM, and CCQC was held 4-12-13 in Exeter, a FRB field day at the Lindcove REC on 4-22-13, and an FRB presentation at a CCQC meeting in Visalia 6-17-13. We contributed to a 24(c) application requesting that the present 2ee label allowing use of 0.5 lb ai/a/yr bifenthrin as a trunk spray be expanded to allow a total of 1.0 lb ai/acre. To support the 24(c) request, three field studies were conducted to determine fruit residues after either 4 applications of 0.25 lb ai/a spaced out every 6 weeks (4 week PHI) or 2 applications of 0.5 lb ai/a spaced 12 weeks apart (9 week PHI). Studies were run in a commercial grove north of Bakersfield (Morse lab), at Lindcove (Grafton-Cardwell), and in Riverside (Morse). A method of analyzing bifenthrin residues on trunks was developed in collaboration with Dr. Jay Gan (UCR Dept. of Environmental Sciences) and is being used to analyze levels of residues on trunks at various dates post application with different application rates. Data from a FRB adult bioassay run at Lindcove concurrent with trunk residue analysis allows us to determine when trunk sprays are no longer effective and an additional study was recently completed at Lindcove evaluating the impact of sprinkler irrigation impacting the trunk. We have also set up a large FRB experiment at a site in the Pauma Valley that has very high levels of FRB. Citrus thrips resistance to Delegate has been confirmed in the San Joaquin Valley. Two products nearing registration on citrus that will be useful in control of citrus thrips (as well as other pests) are Bexar and Closer. Fall fruit scarring evaluations were taken at a spring 2012 mandarin field trial comparing the efficacy of abamectin, Delegate, Bexar, and Closer but citrus thrips levels were relatively low at location in 2012 (despite extreme levels in 2011), making it difficult to draw strong conclusions. We are examining contaminants of export citrus using 10 randomly selected cartons per load from a variety of citrus packing houses. To date, data have been collected from 11 navel 1 Valencia, 1 pummelo, 5 grapefruit, and 4 mandarin loads (22 total).
A field trial was established during the period on Header Canal Road (St. Lucie county) where a planting of Corymbia torelliana (Myrtaceae), a relative of Eucalyptus, was established in fields formerly occupied by citrus and infested by Diaprepes root weevil. Males and females, mating pairs and egg masses of Diaprepes were observed throughout the plantation. In addition to documenting the susceptibility of this fast-growing tree to Diaprepes, the infestation presents an excellent opportunity to study the distribution and movement of Diaprepes in the field in response to the deployment of potential attractant blends. The simple and open architecture of the tree (compared with citrus trees) is favorable for comprehensive sampling of Diaprepes. We initiated a survey of 20 acres of C. trorelliana to describe the distribution of males and females and determine the degree of aggregation prior to deployment of male pheromone and kairomone blends. Post-deployment surveys will determine the extent to which odor sources influenced distribution. If movement in response to deployment of volatile blends can be demonstrated in the field, design of appropriate traps will be pursued. Collections of volatile compounds from the headspace over field-collected and lab-reared female adult Diaprepes were initiated during the period. Extracts will be concentrated and analyzed by GC-MS and GC-coupled electroantennography to determine the presence of a female-produced pheromone. Synthesis of the male-produced pheromone methyl (E)-3-(2-hydroxyethyl)-4-methyl-2-pentenoate was completed in-house. This will be used in olfactometer trials to identify an optimal blend of pheromone and kairomones, and in the field as described above.
CREC-ACPS project at Auburndale A Citrus Research and Education Center Field Day was held on December 13, 2012, at the Auburndale ACPS experiment and jointly at the St. Helena Road rootstock experiment of Jude Grosser and Fred Gmitter. About 200 growers attended the morning’s presentations and demonstrations in the citrus experiments. See http://128.227.177.113/ACPS/Documents/Fielddayhandout2012mergedV1.pdf for a full discussion of the day’s proceedings. Highlights of the ACPS field research for year 4: The tour consisted of four stops, shown in the tour plan. Selected trees had been harvested early and fruit yield and vital statistics were displayed on posters at each harvested tree in each location. ‘ Stop 1 is to view and discuss the grower standard treatment ‘ Stop 2 is a walk-through the high density (363 trees/acre) ‘Hamlin’ trees with moderate to strong size control caused by high fruit set and C-35 rootstock. ‘ Stop 3 is to discuss the fertigation strategy used in year 4, and to view the prototype custom hedger on a 4 foot-wide tractor, to be used for hedging and topping in high density groves. ‘ Stop 4 is a walk-through to view the comparison between the microsprinkler-OH, Swingle rootstock and the drip-OH high density, C-35 rootstock performance. The field day was a great success despite the inclement weather. The high, early fruit yield on HLB-asymptomatic trees was self-evident. Also striking was the very high yield achieved per acre on relatively small trees (maximum height 7 feet). The weak performance of HLB-symptomatic trees, especially the high rate of pre-harvest fruit fall, was an issue that generated much discussion among growers, researchers and consultants. The actual plot harvests which were picked a week later will be analyzed and presented in the next quarterly report (March 2013). CREC-ACPS project at block 22, CREC The new 4-acre ‘Valencia’ orange ACPS experiment which was planted in August, has established well during the mild fall weather, and the growth is already above average due to the intensive daily fertigation schedule. Regular plot measurements of tree growth, soil properties and foliar nutrition will begin in the spring of 2013.
IRREC-ACPS project (Barrett Gruber, co-PI) The second objective of this project is to establish an ACPS solid-set trial at the UF-Indian River Research and Education Center (IRREC, Fort Pierce). The goal of this objective is to evaluate the performance of ACPS for citrus fresh-market citrus varieties and regional disease problems such as canker. After receiving funds in July 2012, RTK-DGPS mapping was used to stake the end-of-tree-rows on 8 acres of research property at IRREC and was done to obtain an estimate of total tree numbers. This mapping was used to plan for two different planting densities: 8′ . 25′ (218 trees / acre, ‘conventional modern planting’); and, 9′.1/4(3’+19’+3’+25′) (387 trees / acre, ‘higher density ACPS planting’). After estimates were obtained, a total of 3,098 new grapefruit trees (1,305 Ray Ruby/US897, 1,305 Ray Ruby/Kuharske, and 488 Ray Ruby/Swingle) were ordered on contract from a certified, commercial nursery. The nursery growing these trees provided a revised estimate of delivery. As of June, 2013, the revised delivery estimate for these trees is late July and August of 2013. The irrigation systems for the ACPS grapefruit trial at IRREC have been designed and we are in the process of ordering the necessary poly-tubing, emitters, valves, pumps, tanks, and controllers. 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 would be able to start contributing work towards this objective in August, 2013, and the summer of 2014. A part-time OPS technician will also be hired in fall, 2013, to begin contributing work towards executing this objective. An OPS technician has not yet been hired because we are awaiting delivery of trees from the nursery (see above). CREC-ACPS project at Auburndale A comprehensive survey of HLB incidence in this 14-acre ‘Hamlin’ experiment was completed in May 2013. The total percentage of HLB-symptomatic trees now stands at 44%, and has more than doubled during the past year. Clearly HLB disease is the most serious threat to the long-term survival and production of trees in this experiment. However this spring we documented that the most seriously declining trees were also impacted by Diaprepes root weevil larvae, sting nematodes, and Phytophthora – all having negative effects on root health. The appearance symptoms on HLB-affected trees was most severe during the dry spring season, despite efforts to optimize both nutrition and water relations with daily computerized fertigation. When the summer rains began in June, there was an immediate improvement in the appearance of the trees, seen as a reduction in HLB symptom severity. Fruit set on all 7 ACPS treatments in the experiment is very good on asymptomatic trees and about average on symptomatic trees. We anticipate picking fruit yields of 800-900 boxes/acre from asymptomatic trees of the best treatment in year 5 – a measure of the potential for ACPS to produce early, high yields. The highest average yield in year 4 was 622 boxes/acre.
CREC-ACPS project at Auburndale The ‘Hamlin’ orange harvest from this ACPS experiment at 4 years demonstrated the continued synergistic interaction between hydroponics fertigation, high density planting, and superior rootstock performance (C35 better than Swingle). The average yield of 622 boxes/ac for the best treatment combination of drip OHS fertigation, high density planting of 363 trees/acre with C35 rootstock was produced with only 186 lb N/ac and about 7 acre-inches of irrigation water. Conventionally grown large citrus trees typically use more nitrogen and water to produce this amount of fruit, after more years. Every year saved in the citrus production cycle represents a year of production costs and resources (water, fertilizer, pesticides etc) saved. Moreover, every year saved due to accelerated production helps to offset losses caused by encroaching diseases, and, potentially early return on the investment. The cumulative 4-year fruit yield from the best ACPS treatment was more than double the cumulative yield from the conventionally grown citrus treatment (218 trees/acre, Swingle rootstock, dry granular fertilizer, standard microsprinkler irrigation with scheduling by grower). The fruit/juice quality of the 4th year harvest was only significantly different for Brix, Acid, and Ratio (see table below) and overall quality was good. We emphasise that all the fruit yield and quality data presented represent the asymptomatic trees in the experiment; fruit yield of HLB-symptomatic trees was reduced by up to 90%, mainly due to pre-harvest fruit drop. Treatment Brix Acid (%) Ratio Solids (lb/box) Fruit weight (g) Juice (%) STD-Sw (218′) 10.41 a 0.643 a 16.2 b 5.33 164.3 56.9 MS-OH-Sw (218) 9.95 b 0.623 a 16.0 b 5.15 173.6 57.5 Drip-OH-Sw (218) 9.90 b 0.567 b 17.5 a 4.60 170.3 57.8 Drip-OH-Sw (303) 10.00 b 0.588 b 17.0 ab 5.19 161.5 57.7 Drip-OH-C35 (303) 10.16 ab 0.588 b 17.29 a 5.26 167.1 57.6 Drip-OH-C35 (363) 10.51 a 0.593 b 17.8 a 5.22 159.7 55.1 Significance (F-pr) 0.011 ** <0.001 *** 0.007 ** 0.519 NS 0.116 NS 0.096 NS LSD (0.05) 0.380 0.0263 0.970 1.22 11.12 2.43 ' Trees per acre CREC-ACPS project at block 22, CREC The mild winter season of 2012/13 allowed most of these young 'Valencia' trees to produce leaf flush throughout the normally dormant months of December to February. The first soil samples collected in the spring showed early signs of rhizosphere acidification in the ammonium nitrate - based hydroponics fertigation treatment (calcium-free) versus the non-acidifying calcium nitrate - based fertigation treatment (calcium-containing). The soil samples were collected at a radius of 4 inches from the drip emitters, to a depth of 6 inches. The average soil pH differences of about 0.5 units were significant and quite dramatic, given the short duration of the trial at that time. Ammonium nitrate is an acidifying nitrogen source, primarily from the oxidation of ammonium to nitrate ions in the soil (nitrification). Calcium nitrate on the other hand does not oxidise and remains neautral or slightly basic in reaction with the rhizosphere.
Experiments to determine the efficacy of different nano-particle systems to deliver nutrients to citrus leaves were started by optimizing conditions for experimental treatments. In general, the experimental design consists of delivering of Mg to citrus leaves by means of three different nano-particle systems. Given their chemical and physical properties, we have selected PAMAM-dendrimers (~ 5 nm), kaolin particles (~1-5 um) and liposomes (~70 nm). To maximize efficiency, conditions of Mg deficiency were created by irrigating trees with a nutrient solution without Mg. Trees developed Mg deficiency symptoms within a few months and are now ready for experimental use (Phase 1). In phase 2, kinetic studies on the binding or carrying capacity of the different nano-particles to Mg were conducted. Concentration dependent kinetic experiments indicated that each PAMAM-dendrimer nano-particle is capable of binding a specific amount of Mg ions. Binding of Mg was demonstrated to be reversible depending on the pH of the environment. Concurrently, kaolin particles were treated similarly and showed a stronger Mg binding capacity than PAMAM-dendrimers. Chemical analysis showed that, although kaolin particles already contain Mg, they have sufficient exchangeable binding sites to bind additional cations. Liposomes, by their hydrophobic nature, will trap solutions of any concentration as they form spherical vesicles from lipid sheets. Greenhouse treatments have commenced with PAMAM dendrimers and the corresponding control of aqueous Mg solution at similar concentration. Treatments with other nano-particles will follow.
Experiments to determine the efficacy of different nano-particle systems to deliver nutrients to citrus leaves were started by optimizing conditions for experimental treatments. In general, the experimental design consists of delivering of Mg to citrus leaves by means of three different nano-particle systems. Given their chemical and physical properties, we have selected PAMAM-dendrimers (~ 5 nm), kaolin particles (~1-5 um) and liposomes (~70 nm). To maximize efficiency, conditions of Mg deficiency were created by irrigating trees with a nutrient solution without Mg. Trees developed Mg deficiency symptoms within a few months and are now ready for experimental use (Phase 1). In phase 2, kinetic studies on the binding or carrying capacity of the different nano-particles to Mg were conducted. Concentration dependent kinetic experiments indicated that each PAMAM-dendrimer nano-particle is capable of binding a specific amount of Mg ions. Binding of Mg was demonstrated to be reversible depending on the pH of the environment. Concurrently, kaolin particles were treated similarly and showed a stronger Mg binding capacity than PAMAM-dendrimers. Chemical analysis showed that, although kaolin particles already contain Mg, they have sufficient exchangeable binding sites to bind additional cations. Liposomes, by their hydrophobic nature, will trap solutions of any concentration as they form spherical vesicles from lipid sheets. Greenhouse treatments have commenced with PAMAM dendrimers and the corresponding control of aqueous Mg solution at similar concentration. Treatments with other nano-particles will follow.
The overall goal of this project is to identify a Bacillus thuringiensis (Bt) crystal toxin with toxicity to Asian Citrus Psyllid (ACP) and to further enhance toxicity by genetic modification. The long term goal of the proposed research is an effective management strategy for the psyllid and associated HLB disease that is more sustainable, less costly and more environmentally benign than the repeated application of broad spectrum insecticides. Our focus for the current reporting period has been on isolating Bt toxins from the selected Bt strains. Nine Bt strains with known toxicity against Dipteran and Lepidopteran insects were selected. These strains recovered from storage, checked for stability and amplified for 4 days on T3 plates (Travers et al. 1987) at 30.C until spores and crystals have formed. The strains then harvested in sterile water and shipped to Iowa State University at room temperature. This work was carried out at collaborative USDA-ARS laboratory in Beltsville, MD Purification of the toxins from Bt strain suspension was carried out Iowa State University. Briefly, Bt strain suspension centrifuged at 14,000 rpm for 15 min at 4.C. Pellet gently sonicated on ice and washed with cold 0.5 M NaCl. Final pellet resuspended in 50 mM Na2Co3, pH 10.5 containing 10 mM DTT for the Bt toxin solubilization in the incubator shaker at 37.C for 3 hr. Solubilized Bt toxins harvested by centrifugation. Small aliquot of Bt toxins analyzed by SDS-PAGE while remaining Bt toxin preparation snap frozen in liquid nitrogen and stored at -80.C until further use. SDS-PAGE profile of isolated Bt toxins exhibited multiple high intensity protein bands ranging from molecular weights of ~22 kDa to ~150 kDa (Figure 1). Three out of nine Bt strains showed presence of a unique protein band running above 250 kDa molecular weight marker protein. Toxin profiles of nine Bt strains are categorized in the following five groups based on the similarity of toxin profiles. Group one: Three Bt strains with similar toxin profiles Groups two and three: Two Bt strains each with similar toxin profiles Groups four and five: One Bt strain each with unique toxin profile. Isolated Bt toxins will be activated in vitro using bovine trypsin, analyzed on SDS-PAGE, quantified and will be sent to Dr. David G. Hall, USDA-ARD, Fort Pierce, FL for ACP membrane feeding assays for toxicity analysis. References: 1. Travers, RS, Martin, PAW and Reichelderfer, CF (1987) Selective Process for Efficient Isolation of Soil Bacillus Spp. Appl Environ Microbiol 53: 1263-1266.
Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus canker. The pathogen can cause extensive damage to twigs, leaves, and fruit of susceptible citrus varieties. The overall objective of this research program is to develop an effective and sustainable phage based bio-control system for citrus canker. Therefore, only virulent and non-transducing phages will be used for the development of the phage based bio-control system for citrus canker. During the first phase of the project, we have focused on the isolation and clonal purification of phages that are active against isolates of Xac. A total of 45 environmental (plant, soil and water) samples were directly assayed for the presence phage forming plaques on three individual Xac hosts. The same samples were also used for the enrichment of phages specific to the three individual Xac hosts. Therefore, a 45 X 2 X 3 matrix was used to enrich for viable phages forming plaques on Xac. Using this combined approach, we have observed plaque formation by direct plating of six samples that showed the presence of as high as 2 X10^2 plaque forming units (PFU)/gram of tissue and an average of 5 X10^1 PFU/ml in several water samples. Enrichment of samples with individual hosts yielded 19 high titer lysates that formed plaques on one or more hosts, indicating a potential diversity in receptors. The next phase will concentrate on purification, morphological characterization, preliminary receptor identification and life style determination. We will continue to process samples in order to isolate additional phages through the duration of the project.
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, selections of tangelo, grapefruit, mandarin and pummelo material have been forwarded to Ft. Collins for therapy using cryotherapy and shoot tip grafting. Recovered plants are tested for presence of HLB 12-14 weeks post treatment. A total of 98 plants recovered from cryotherapy have been tested for HLB using qPCR. The COX internal control cot values ranged from 18.4 to 20.6; all but three recovered plants tested negative for HLB with cot values of 40 or higher. Three plants had cot values of 38.9, 37.6, and 38.7, respectively, and may test positive later. HLB positive controls had cot values ranging from 17.4 to 20.0. All recovered plants will be retested to verify the freedom of HLB on a continuing basis. More HLB infected material has been sent to Ft. Collins for therapy. In Riverside, we have demonstrated that very young plants (60-90 days post planting) can be effectively used for biological indexing to verify the presence of Citrus psorosis virus, citrus concave gum disease, Citrus leaf blotch, and Citrus vein enation virus during the late spring-early summer in the greenhouse. Cooler temperatures needed for the biological indexing was achieved by placing the plants near the evaporative cooling pad.
The goal of this 2-year research project (no. 758) is to understand the HLB tolerance phenomenon observed in certain citrus relatives in a previous 4-year field trial. We identified certain cultivars of Microcitrus, Poncirus and Eremocitrus to have tolerance/resistance to ‘Candidatus Liberibacter asiaticus’. This quarterly (from May 2013 to July 2013) research consisted of: a) Conducting breeding trials and b) starting greenhouse experiments for further testing and evaluation. Breeding experiments: We used HLB tolerant Eremocitrus, Microcitrus and Poncirus to conduct pollinations with HLB susceptible mandarins and pummelos to generate tolerant/susceptible F1 population. Selected plants were used as both seed and pollen parents in the crosses. We conducted 960 pollinations in various combinations and we are waiting for the set fruit to mature. We are also developing fast qPCR methods to test the progeny for hybrid genotype. We have started greenhouse experiments in Fort Pierce (Stover’s green house) with seed from HLB tolerant and susceptible accessions identified from the previous trial. These seedlings will be challenged via psyllid feeding and tested under green house conditions for HLB tolerance phenotype. The future work will consist of evaluation of these plants for HLB tolerance, collection of tissue at various time points and analysis of HLB tolerance mechanisms.
Our efforts are directed toward understanding the biology of different strains of the Liberibacter pathogen, the D. citri insect vector, and bacterial endosymbionts of D. citri through bioinformatic analyses of genome sequences. Liberibacter sequences and select endosymbiont sequences are analyzed and incorporated into a publically available online genome viewer for use by the larger research community. Recently released genome sequences for Ca. Liberibacter asiaticus strain gxpsy and Ca. Liberibacter americanus PW_SP have been added to the GBrowse genome viewer at the CG-HLB Genome Resources Website (http://citrusgreening.org), joining Ca. Liberibacter asiaticus psy62, Ca. Liberibacter solanacearum, and Liberibacter crescens. In addition to linking genes to resources in other online databases, the genome viewer also displays prediction of subcellular locations, informative for identification of gene products interacting with the host, and predicted functional domains. Comparative analyses include mapping regions of difference between strains to identify genetic sources of differences in host range, vector preference, and other differences in the biology of individual strains. For different Liberibacter species such as CLas and CLso, the regions of difference are significant in length and are represented in the viewer as large sequence blocks. In contrast, strains CLas psy62 and CLas gxpsy are highly similar. Identification of regions of variation among these closely related strains is ongoing and is expected to provide insight into genes that may be evolving faster owing to host detection or a role in virulence. Analysis of variation in 10 isolates of Clas sequenced by Bob Shatters (ARS, Fort Pierce) will contribute further data on CLas evolution and epidemiology. CLam PW_SP was not sequenced to completion so the individual contigs making up the draft genome sequence have been joined into a pseudomolecule for easier access within the genome viewer. Although pathogenic on citrus, the CLam sequence differs significantly from those of the CLas strains, shedding light on gene products necessary for virulence on citrus hosts. In addition, we are continuing collaboration with Wayne Hunter (ARS, Fort Pierce), Justin Reese (Genformatic), and Shannon Johnson (Los Alamos) on an improved assembly of the D. citri genome sequence, using the previously generated Illumina reads together with data generated PacBio technology. We have recently screened the improved assembly for the presence of genes similar to immunity related genes in Drosophila and pea aphid, the latter being the closest sequenced insect relative of D. citri, to determine how the D. citri innate immune system compares with that of these other insects, and to identify potential immune vulnerabilities that can be exploited for improved insect control. The new D. citri genome data is also being used to re-analyze and improve sequences of its bacterial endosymbionts, previously characterized by our group (Saha et al, 2012. PLoS ONE 7(11):e50067). Of these, Wolbachia is of greatest interest owing its demonstrated role in immunity in other insects as well as success in its use for control of other insect pests.
Development of alternative or complementary approaches for effective management of citrus greening is highly desirable and will greatly help the citrus industry due to the difficulty to control the HLB disease. Considering the highly destructive nature of HLB disease and the lack of control measures, there is a huge potential to develop antimicrobial small molecules against the causal agent thus to suppress the population of Ca. L. asiaticus in planta and to reduce the innoculum for psyllid transmission. The most common targets for antimicrobial agents development include receptors, proteins and enzymes, DNA, RNA and ribosomal targets. Among them, proteins have become the major target due to their druggable characteristics. In this study, we presented our research on screening small molecule inhibitors against SecA. SecA is one essential component of the Sec machinery which provides a major pathway of protein translocation from the cytosol across or into the cytoplasmic membrane. The Sec pathway was also shown to be required for virulence of Ca. L. asiaticus in our study. SecA is the protein translocase ATPase subunit, which is involved in pre-protein translocation across and integration into the cellular membrane in bacteria. In our study, we expanded our previous study in identifying lead antimicrobial compounds with higher activities by targeting SecA using various computational techniques like homology modeling, virtual screening, molecular docking & minimization. Due to the uncultivable nature of Ca. L. asiaticus, we tested the potential inhibitory effect of the selected compounds against Agrobacterium tumefaciens, which is phylogenetically related to Ca. L. asiaticus. Twenty compounds were selected for biological activity study against SecA of Ca. L. asiaticus and A. tumefaciens. Five compounds were found to inhibit the ATPase activity of SecA of Ca. L. asiaticus in nano molar concentrations and showed antimicrobial activities against A. tumefaciens with MBC ranging from 128 ug/ml to 256 ug/ml. These compounds appear to be suitable as lead compounds for further development of antimicrobial compounds against Ca. L. asiaticus. Those findings were published in the article entitled: Identification of small molecule inhibitors against SecA of Candidatus Liberibacter asiaticus by structure based design. on Eur J Med Chem (http://dx.doi.org/10.1016/j.ejmech.2012.05.035). To test the application potential of those compounds on plants, the phytotoxicity studies were performed on the five compounds against citrus. At higher concentrations (0.25mg/ml), all five compounds showed phytotoxicity. When the concentrations were diluted to 0.025mg/ml, the toxicity went down to mild to low. Currently, we are testing the inhibitory effects of the five compounds again Ca. L. asiaticus in planta. We are evaluating different solvents and adjuvants for the five compounds. Their antimicrobial activities against Liberibacter crescens, which is closely related Ca. L. asiaticus and could be cultured, have been tested. Currently, we are optimizing these five antimicrobial compounds to identify compounds higher antimicrobial activity. Fourteen new small molecule inhibitors were identified. Further studies are being conducted on those 14 compounds. We are also in collaboration with IBM to further expand this study.
The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. Currently, we are setting up the experiments to test different Microbe Products in management of HLB. We have developed a culture collection of approximately 400 bacteria initially isolated from the root and rhizosphere of citrus. These bacterial isolates have been screened for various beneficial traits . We are also evaluating the antagonistic activity of these bacterial strains against some well-known plant pathogenic fungi. Especially we have screened a bacterial isolate designated as 43A which possess multiple plant growth promoting activity and is also able to antagonize different fungi. We are also testing the plant growth promoting activity of 24 isolates using seed germination pouch in greenhouse. We have also selected several Bacillus spp. possessing multiple beneficial traits to develop bacterial consortium which can be further developed as carrier based bioformulation. Assay for compatibility between isolates using antagonistic survival tests showed that all the selected beneficial bacteria are compatible with each other. Plant growth promoting activity of six selected isolates was evaluated using the model plant Arabidopsis grown in vitro. The results suggested that three isolates could promote plant growth. The plant growth promoting activity of these six isolates was tested using citrus (grapefruit) seedlings in greenhouse. Three isolates could promote growth of grapefruit seedlings. Those isolates are being used to evaluate their potential to prevent citrus seedlings from Las infection vectored by psyllids in a greenhouse. The three strains seem to delay the development of both HLB symptoms and pathogen population on citrus leaves after root application. Several isolates also exhibit inhibitory activity again the citrus canker pathogen Xanthomonas citri ssp. citri. Currently, we are testing the effect of the selected isolates to control HLB in greenhouse using seedlings and in citrus groves. We have selected two groves and conducted a survey of HLB disease index. We are also evaluating the titers of Candidatus Liberibacter asiaticus in the selected trees. We tested the resistance of our isolates against different antibiotics and only two strains showed minor tolerance to spectinomycin. All the test strains showed strong resistance to copper indicating the copper application in the grove will not affect their survival in the soil. The shelf life of the LB culture and OPB culture of the three strains have been tested. We applied beneficial bacteria in two separate field tests for the first time. Following up study is being conducted.
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