The primary goal of this project is to develop cryotherapy as a practical, reliable method of eliminating graft transmissible pathogens from Citrus and citrus relatives without inducing juvenility. Permits have been obtained to ship pathogen infected citrus material from Riverside, CA and from the Exotic Disease Quarantine Laboratory, Beltsville, MD to Ft. Collins, CO, and materials from Ft. Collins, CO can be sent under permit to Riverside for lab testing. In the first 18 months of the project, we have developed a cryotherapy protocol which works well with any type of citrus or near citrus relative. Two technicians from Riverside and one technician from Beltsville have been trained in Dr. Volk’s lab on the technique. Initially we concentrated on therapy from citrus viroids and Citrus tatterleaf virus, and were able to eliminate these pathogens, the most difficult to remove by traditional shoot tip grafting (STG) using the cryotherapy protocol. Presently we are testing the protocol for elimination of huanglongbing, Citrus mosaic virus, and citrus variegated chlorosis (in Beltsville), citrus stubborn, Citrus psorosis virus, Citrus leaf blotch virus, Dweet mottle virus, citrus concave gum disease, and Citrus vein enation virus. Efficiency of therapy with cryotherapy is being compared with results obtained by shoot tip grafting with most pathogens. For Citrus tatterleaf virus, we have recovered 32 plants which are clean from 32 recovered plants; viroids 11 clean from 14 recovered plants; Citrus leaf blotch virus 6 clean of 6 recovered plants; Citrus psorosis virus 25 clean of 25 recovered; and Citrus Stubborn 10 clean of 10 recovered. Because we wait at least 12 weeks to conduct lab testing of recovered plants, we have a constant backlog of plants to be tested. In Riverside, we are testing recovered plants after they have been grafted from the test tube to a rootstock, thus representing plants that would be continued through the indexing required for a variety release. The research is continuing.
The primary objective of this research is to identify diverse germplasm which has a tolerance to huanglongbing (HLB) and its psyllid vector, Diaphorina citri. An earlier germplasm screening block containing 87 different accessions of citrus and citrus relatives and planted in the field at Ft. Pierce for four years provided evidence of different types of tolerance to HLB. As a result of the first trial, this is a second trial which includes accessions of Poncirus trifoliata, hybrids with P. trifoliata, sour orange types, and kumquats. For this present trial, 97 different accessions were selected based on the population structure which suggested that the accessions contained genetic material from P. trifoliata, kumquat, or sour orange. Seed was collected from the University of California Citrus Variety Collection; heat and fungicide treated using standard protocols, and shipped to Ft. Pierce where the seedlings were grown under greenhouse conditions. The trees in Riverside used for seed collection were tested for Citrus leaf blotch virus, Citrus psorosis virus, HLB, and Xylella fastidiosa prior to shipment of the seed. Seven seedlings of each accession were inoculated with HLB while in the greenhouse in March 2013, seven seedlings remained un-inoculated. The seedlings were planted in the field in late spring 2013 and the first visual assessment was made in November 2013. Sampling for HLB will continue biannually and incidence of Asian citrus psyllid will be made this summer season.
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. Application of crytherapy to recover plants free of HLB from budwood approaches 95 percent efficiency. 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. The inoculum bud survival is very high, greater than 98 percent. The entire indexing procedure can be done with the plants in the Jiffy pots. A trial was performed to compare the results obtained by using the mini-plant biological indexing compared with the traditional standard indexing protocol, using indicator plants 10-14 months old, for 15 accessions. The results from the mini-plant index revealed the presence of Citrus vein enation virus in one accession which had been missed using the traditional standard indexing protocol. The advantage of using the mini-plants for biological indexing is that 35 indicator plants may be contained in the space normally occupied by 3 indicator plants; quicker turnover of the small plants for indexing means more accessions may be tested per year; biological indexing for pathogens expressing only under cool temperatures may be indexed for year-round as the smaller plants may be placed close to the cool pads in the greenhouse, and because they occupy less space, the whole trial can be done utilizing the cooler temperatures.
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 Aurantioideae with genera sexually compatible with citrus like Microcitrus, Eremocitrus and Poncirus. Research conducted this quarter (from Jan, 2014 to March, 2014) consisted of: a). Second batch of pollinations conducted in Riverside (Citrus Variety Collection) using HLB tolerant and susceptible accessions. This year we have utilized certain mandarin cultivars known to be more tolerant to HLB than others based on field trials in Fort Pierce by collaborators. The crosses between these ‘tolerant’ mandarins and ‘resistant’ citrus relatives (as determined by our previous trial) is likely to yield useful resistance/tolerance to HLB. We have now performed over 1100 crosses using mandarin and pummelo as seed parents. Based on previous year’s results, we were able to select seed parents that are likely to set fruit when crossed with the pollen from HLB resistant citrus relatives. b). Seeds collected from previous year’s pollinations were germinated in the greenhouses in Riverside for confirmation of genotypes. These experiments are in progress. A part of the seeds will be sent to collaborators in Fort Pierce in the next few weeks for field evaluation of resistance. c). One batch of selected HLB tolerant and susceptible seedlings that were psyllid challenged in Fort Pierce (Hall lab) were used for making RNA extractions in Fort Pierce. The samples are now being processed and analyzed. The experiment continues in Fort Pierce and sampling will be done periodically according to our original plan. We are on track as per our proposed milestones.
The goal of this study is to understand the role of biofilm formation and quorum sensing (QS) in X. citri ssp. citri infection of citrus fruit and to prevent its infection by interfering with biofilm formation and QS. Three compounds exhibited a significant reduction in biofilm formation both on polystyrene surface and in glass tubes compared to the untreated control, where the level of biofilm formation were reduced to 50% and 60% of control, respectively. Plant test in greenhouse showed that treatment with the three compounds prior to infection could reduce biofilm formation of Xac on leaf surface, reduce the formation of canker lesions on spray-inoculated grapefruit leaves with the wild-type strain. Effects of the three compounds on Xac on detached immature citrus fruit were also tested using spray inoculation. Preliminary results showed that these small molecules affected Xac 306 infection of unwounded and wounded citrus fruits at sub-inhibitory concentrations. We have completed testing the effect of those compounds in different combinations with copper based bactericides in controlling Xac infection of grapefruit plants in the greenhouse. The sensitivity of biofilm and planktonic cells of Xac 306 to copper (copper sulfate) were evaluated by measuring the MICs. Biofilms are less susceptible to copper than planktonic cells. Effect of the selected compounds on sensitivity of Xac planktonic cells and biofilm cells to copper sulfate was also investigated. In the NB medium, planktonic cells exhibited a MIC of 0.50 mM CuSO4 without biofilm inhibitor. In the presence biofilm inhibitors at sub-MIC concentrations , the MICs of CuSO4 against Xac 306 planktonic cells were decreased to 0.25 mM. In a in vitro biofilm system test, the combined use of copper sulfate and the compounds individual or both resulted in significantly increased killing compared to killing by copper sulfate alone. The results have been published by Phytopathology in a manuscript entitled: Foliar application of biofilm formation-inhibiting compounds enhances control of citrus canker caused by Xanthomonas citri subsp. citri. One patent is filed based on the results. We also identified multiple new biofilm inhibitors. The effect of those biofilm inhibitors to control citrus canker is being investigated. We tested the survival of both biofilm deficient and QS mutants on fruit surface. Effects of biofilm formation inhibitors on Xac infection on detached immature citrus fruit were tested using spray inoculation. The inhibitors affected the infection of Xac on both unwounded and wounded citrus fruits. We are testing more potential biofilm inhibitors. We continue characterizing how quorum sensing and biofilm formation contribute to Xac infection of citrus fruit. Multiple virulence genes involved in quorum sensing and biofilm formation are being investigated. The field trial is ongoing to test the effect of the identified biofilm inhibitors to control citrus canker. One new compound is able to inhibit QS at a concentration of 100 .M based on observation of bacterial phenotype of aggregate formation. Plant test in greenhouse showed that the QS inhibitor (100 .M) treatment could reduce the formation of canker lesions and bacterial population on spray-inoculated grapefruit leaves simultaneously with the canker bacterium Xcc 306. Repeated experiment in greenhouse is underway.
The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. In the enhencement project, we are expanding the field test to include two more field trials in two more locations, larger scale of field test, test more beneficial microbes, and test different approaches to enhance the survival of the beneficial microbes in the soil. The field trial site were identified. For one site, six applications were conducted. For another site, the first application was conducted in February, 2014. We conducted the background survey regarding HLB disease incidence, severity, Las titers, Phytophthora, nematodes, root (root density and health status), and microbial diversity right now. The following up study is ongoing.
The goal is to develop short term approaches to control citrus Huanglongbing (HLB) using antibacterial-producing bacteria. Recent studies indicate that HLB severely damaged citrus roots. The destructive effect of HLB on roots partly results from Candidatus Liberibacter asiaticus (Las) infection of the roots. In this study, we will conduct the following objectives: Screen bacteria that can produce antimicrobial compounds against Liberibacter crescens and related Rhizobiaceae bacteria; purify and characterize antimicrobial compounds produced by the screened bacteria; illustrate the regulation of antimicrobial production by producing bacteria at different environmental conditions and with different inducers; investigate the growth of the producing bacteria in different conditions; and control HLB by delivering antimicrobial compounds using the producing bacteria in the soil. We have isolated 84 strains of Streptomyces spp., Bacillus spp., Paenibacillus spp., and Pseudomonas spp. producing antibacterials from Florida citrus groves. We have also acquired 28 different species of Streptomyces spp., Bacillus spp., Paenibacillus spp., and Pseudomonas spp. strain producing different antibacterials. Field trials are being conducted.
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. 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. Greenhouse assays suggested that a consortium of three Bacillus and relative isolates (AY16, PT6 and PT26A) may delay the development of both HLB symptoms and pathogen population on citrus leaves after root inoculation. The potential of the consortium to recover the tree decline from HLB infection is being evaluated in greenhouse. The growth conditions of the three strains were optimized using a small fermenter. Three antifoam agents, A204, PPG200 or M-Oil did not affect the growth of the three bacterial strains. The initial neutral to alkaline pH values (7.0 ~ 8.0) favor growth of the three bacteria in LB, while acidic pH (5.0 ~ 6.0) suppress bacterial growth. The optimal cultural temperature was determined to be around 30C with average bacterial population of 109-1010 cfu/ml after 20-hour incubation, although the bacteria may grow slowly under room temperature (~ 23C). The shelf life of three different formulations of the bacterial culture is being evaluated under room temperature. In a six-month time course, the bacterial populations in LB broth, OPB broth and tape water are comparatively stable with initial and final both at ~ 108cfu/ml. Four field trials are being conducted including more beneficial bacteria. For one of the field trial, six applications have been performed. We are evaluating the survival of the beneficial bacteria in the soil. The application method has been changed during application to improve the survival of microbes in the soil. For two trials, four applications were conducted. For another field trial, the application started in February, 2014. We have surveyed the HLB disease incidence, and Las population. We are investigating the survival of the applied microbes in the soil.
Management of phloem-limited bacterial diseases is very challenging. These bacteria employ unusual and sometimes unique strategies by which to optimize their niche occupation and obtain their nourishment from the host plant. Their location within the living (sieve tubes) plant cells, rather than in the intercellular spaces, offers different challenges and opportunities for them to avoid the host plant’s defense system. Phloem is also difficult for any bactericides to reach to control the pathogen population. Among the phloem-limited bacterial diseases, citrus Huanglongbing (HLB, greening) is one of the most devastating diseases. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las). The goal of the proposed study is to develop HLB management strategies which boost plant defense to protect citrus from HLB by exploiting the interaction between Las and citrus and understanding how Las manipulates plant defense. Recently, we compared the gene expression of PR1, PR2 and PR5 in healthy trees and Las infected citrus plants. The expression of PR1, PR2 and PR5 was significantly reduced in HLB diseased grapefruit as compared to healthy grapefruit after inoculation with Xac AW. We also tested whether infection by Las can make citrus more susceptible to infection by Xanthomonas citri subsp. citri. We also sprayed four times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB in one grove. Multiple compounds showed control effect. To further test those compounds, we have selected two more groves to expand the field test. The disease index of the two groves have been investigated and treatments already started. The follow up investigations are ongoing, including monitoring the HLB symptoms, disease incidence and Las titer in leaves. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We also compared the SA levels in HLB infected and healthy Valencia citrus. We are continuing to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove. We have applied different compounds at three separate field trials. Four compounds were shown to have positive effect on controlling HLB based on two year field test results. We are also testing the mechanism of those compounds showing positive effect on HLB control. For example, the analysis of gene expression of pathogenicity related genes and callose synthase CalS1 in treated plants and nontreated plants is underway. We have investigated the effect of those compounds on disease severity, yield, juice content and quality. We will repeat those treatments for one more year. Currently, the treatments are being conducted.
The goal of the research is to control citrus HLB using small molecules which target essential proteins of Candidatus Liberibacter asiaticus (Las). In our previous study, structure-based virtual screening has been used successfully to identify five lead antimicrobial compounds against Las by targeting SecA. SecA is one essential component of the Sec machinery. Those compounds showed promising antimicrobial activity. However, further work is needed to apply the compounds. We will evaluate the important characteristics of our antimicrobial compounds including solvents and adjuvants, phytotoxicity, antimicrobial activities against multiple Rhizobia, antimicrobial activity against Las, application approaches, and control of HLB. Those information are critical to for the practical application of those antimicrobial compounds in controlling HLB. We also propose to further optimize the five lead compounds. In addition, we propose to develop antimicrobial compounds against lipid A of Las. The lipid A substructure of the lipopolysaccharides (LPS) of Sinorhizobium meliloti, which is closely related to Las, suppresses the plant defense response. Las contains the complete genetic pathway for synthesis of lipid A. We hypothesized that Las uses lipid A to suppress plant defense. Thus, targeting lipid A could activate plant defense response. Lipid A is also an ideal target and has been targeted for screening antimicrobial compounds for multiple pathogenic bacteria. We are optimizing the compounds in collaboration with IBM. Two compounds with slightly higher binding affinity than C16 were identified. Currently, we are evaluating the best range of composition ratio among each component (%weight) of AIs, solvents and surfactants. The following characteristics are being evaluated: 1) emulsion stability and ease of emulsion; 2) stability of diluted concentrate; 3) freeze-thaw stability; and 4) phytotoxicity to citrus species. We have successfully identified one formulation suitable for all five compounds without phytotoxicity. Using the formulation, we have tested all five compounds against eight different bacterial species including Liberibacter crescens. We are repeating the results for one more time. Field test is being conducted. We have sprayed once. Greenhouse trial is being conducted.
At the request of the CRB, 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 (FRB) 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 2014-15, loads found to be infested with viable egg masses may be denied entry into that country. Morse and Dr. Andy Cline of CDFA conducted 6 training sessions for county agricultural inspectors in different areas of California during Nov. 2013 — at the prompting of APHIS, county inspectors have agreed that all loads of citrus shipped to Korea would be sampled to determine the percent of fruit infested with unhatched FRB egg masses. Field FRB control trials were run at Lindcove by Grafton-Cardwell (15 treatments evaluated) and in Pauma Valley by Morse (10 treatments evaluated). None of the treatments results in 100% control but it appears that 2 treatments are relatively effective — either 2 ground sprays of bifenthrin, 2 trunk sprays of bifenthrin, or 2 foliar sprays of combinations of carbaryl, cryolite, and/or thiamethoxam. A method of analyzing bifenthrin residues on trunks was developed in collaboration with Dr. Jay Gan (UCR Dept. of Environmental Sciences). Studies were conducted on 3 dates to determine bifenthrin levels on trunks necessary to cause beetle paralysis after they walk over the trunk spray (“LC”50 of 0.04874 ug/cm2). The persistence of 5 formulations of bifenthrin trunk sprays were evaluated in Riverside by taking residue samples 6, 12, and 18 weeks after trunk application. 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. To better study Delegate resistance, we started a greenhouse colony from an area reporting poor control and have confirmed that the population is resistant to Delegate (very flat dose-response line). So that we can study the mechanism and genetics of resistance, we will select for higher levels of resistance. We are examining contaminants of export citrus using 10 randomly selected cartons per load from a variety of citrus packing houses. To date, we have processed 29 such loads with a diversity of commercial varieties examined. Genetic identification of insects appears fairly routine but we will have to continue to work on methods for mite identification — initial extractions have been problematic.
At the request of the CRB, 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 (FRB) 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 2014-15, loads found to be infested with viable egg masses may be denied entry into that country. Morse and Dr. Andy Cline of CDFA conducted 6 training sessions for county agricultural inspectors in different areas of California during Nov. 2013 — at the prompting of APHIS, county inspectors have agreed that all loads of citrus shipped to Korea would be sampled to determine the percent of fruit infested with unhatched FRB egg masses. Field FRB control trials were run at Lindcove by Grafton-Cardwell (15 treatments evaluated) and in Pauma Valley by Morse (10 treatments evaluated). None of the treatments results in 100% control but it appears that 2 treatments are relatively effective — either 2 ground sprays of bifenthrin, 2 trunk sprays of bifenthrin, or 2 foliar sprays of combinations of carbaryl, cryolite, and/or thiamethoxam. A method of analyzing bifenthrin residues on trunks was developed in collaboration with Dr. Jay Gan (UCR Dept. of Environmental Sciences). Studies were conducted on 3 dates to determine bifenthrin levels on trunks necessary to cause beetle paralysis after they walk over the trunk spray (“LC”50 of 0.04874 ug/cm2). The persistence of 5 formulations of bifenthrin trunk sprays were evaluated in Riverside by taking residue samples 6, 12, and 18 weeks after trunk application. 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. To better study Delegate resistance, we started a greenhouse colony from an area reporting poor control and have confirmed that the population is resistant to Delegate (very flat dose-response line). So that we can study the mechanism and genetics of resistance, we will select for higher levels of resistance. We are examining contaminants of export citrus using 10 randomly selected cartons per load from a variety of citrus packing houses. To date, we have processed 29 such loads with a diversity of commercial varieties examined. Genetic identification of insects appears fairly routine but we will have to continue to work on methods for mite identification — initial extractions have been problematic.
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.
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