We are continuing to solicit sequences that can be used to prepare RNAi molecules that may silence ASP genes resulting in mortality. As these sequences are selected, we are feeding the resulting RNAi molecules to adult ASP using our feeding system. Results comparing mortality of the these RNAi molecules on psyllids will be reported later after the comparisons are complete.
The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. The graduate student, Nan-Yi Wang, whose Ph.D. project this is, continues his studies in Gainesville this term and is making good progress in his classes. He is also conducting research while in Gainesville. We have redesigned the mating gene primers several times but have not found any products with homology to known mating genes. We are evaluating new strategies to approach this program. He has finally had success transforming G. citricarpa with GFP. He found that the fungus is very sensitive to hygromycin, a selectable marker, but was eventually able to transform protoplasts with modifications to his protocol. The isolates are being evaluated to make sure that the growth and other characteristics other than the GFP are similar or the same as wild type The trees have been purchased for the greenhouse experiments but the air conditioner broke down so needed to wait for delivery until the air conditioner in the quarantine house had been repaired.
This is a three-year research project that started in June, 2012. The overall goal of this research is to efficiently deliver antimicrobial molecules into citrus phloem against the HLB bacterium. The quarterly (July~Sep) research focused on designing the W/O (water in oil) and O/W (oil in water) nanoemulsions by evaluating the ideal adjuvant mixtures of solvents (ethanol, acetone, methyl acetate, and ethyl acetate), oils (soy oil, cremorohor, carvacrol, p-cymemen, orchex796) and lipophilic (span80, span85) and hydrophilic surfactants (tween20, tween80). The nanoemulsions were first evaluated for transparency, viscosity, and other physical properties that would make them good candidates for moving molecules into citrus phloem . This evaluation indicated that several O/W nanoemulsion formulations and several W/O nanoemulsion formulations had the desired properties. In addition, seven compounds from the Citrus Research and Education Center, IFAS, UF and one compound, epsilon-L-polyline, from China were tested for their effectiveness against HLB bacterium by our grafted-based chemotherapy method (Zhang et al., 2012). Three effective compounds from the Contest were treated by nano-green (one of the commercial nano-products) and applied to HLB-affected citrus by foliar-spray in the greenhouse. The results will be reported in the future.
The overall objective of this one year project was to rapidly screen and evaluate chemical compounds against citrus HLB using a graft-based screening method (Zhang et al., 2012). A total of 59 chemical compounds were tested based on the ranking of an expert panel and the mode of action of the compounds. HLB-affected lemon scions were soaked in the above solutions overnight in a fume hood and grafted into two-year-old healthy grapefruit rootstocks. First samples for DNA extraction were taken 4 months after inoculation; subsequent samplings were taken at 2 month intervals. Four types of data were collected: Las titer in scions and rootstock based on qPCR Ct values, transmission of the bacteria from the scion to the grapefruit rootstock, survival of the infected/grafted scion and growth of new leaves from the scion. The 59 compounds were placed into four groups based on the above data. (1) Highly effective (Group O): Eight out of 59 compounds were clustered in this group, including Ampicillin, Actidione, Carbenicillin, and Nicotine. All the molecules in this group reduced Las titers to undectable levels (Ct values of more than 38.0) in the inoculated plants (grafted scions and rootstocks). All molecules in this group were antibiotics except Nicotine. (2) Effective (Group I): Twenty-five out of 59 compounds were clustered in this group, including Validoxylamine A, Hygromycin B, Poly-l-arginine, Carvacrol and Gossypol. Some of these compounds, such as Carvacrol, Validoxylamine A and Gossypol were not medical-antibiotics. The effectiveness of group I molecules against HLB bacterium was less than those in Group O, but they could greatly suppress the HLB bacterium with average Ct values of 33.8 and detectable Las in less than 40% of the scions . (3) Partly effective (Group II): Sixteen out of 59 compounds were clustered in this group, including Thujone, Hydroxyurea crystalline, Polymixin B and Neomycin. Compared to the water control, they could partly suppress the HLB bacterium with Ct values of 28.6, and 74.7% scion infection. (4) Non-effective (Group III): Ten out of 59 compounds were classified into this group, including Meso-erythritol, Lincomycin, 2-methyl-4-isothiazolin-3-one and Zlineb. Similar to the water control (CK1 and CK2), the compounds in this group were not effective in suppressing the HLB bacterium with average Ct values of 26.8 and 72.6% scion infection. Three of the 59 compounds were found to be highly phytotoxic to citrus, especially Actidione, Oxytetracycline and M-cresol. The other 56 compounds were not phytotoxic due to more than 80% of scion survival and 40% of scion grown. Based on the this research, two manuscripts will be prepared for publication. The compounds in Group I and Group O may be useful for field control of HLB pending field testing, improved application methods and registration.
This is a three-year research project that started in June 1, 2012. The overall goal of this research is to efficiently deliver antimicrobial molecules into citrus phloem against the HLB bacterium. The objectives focused on will be : 1) Designing nanoemulsions by evaluating the ideal adjuvant mixtures of solvents, oils, lipophilic and hydrophilic surfactants based on the physicochemical characterization of the compounds; 2) Evaluating penetrants to promote uptake, translocation and diffusion of molecules into phloem based on leaf and bark surface characteristics; 3) Developing an efficient transdermal/translaminar delivery process and ideal formulations of compounds for foliar-spray or basal bark applications. Recent studies from Contest (CRDF#400) indicated that some non-medical antibiotics were effective in eliminating or suppressing the HLB bacterium using the graft-based screening method (Zhang et al., 2012). The major limitation to applying these compounds in the field will be to rapidly and efficiently deliver these compounds to the citrus phloem where the HLB bacterium resides. We therefore propose to screen and assess the components of nanoemulsions to improve the efficiency of delivery of the compounds into citrus in the first quarter research of this project. The overall experimental plan has been developed. The physicochemical characteristics of these effective compounds were evaluated and tested, such as solubility. The chemicals were purchased and nanoemulsions were prepared with water in oil (W/O) for lipophilic compounds and oil in water (O/W) for hydrophilic compounds. The HLB affected citrus to be used for this research was identified in the greenhouse and in the field. Some effective compounds combined with one kind of commercial nanoemulsion product (Nano-green) were used to treat HLB-affected citrus by foliar-spray and soil soaking in the greenhouse in June 2012. One OPS student will be hired and will be available in August, 2012.
Having shown that oral uptake of ~300 bp dsRNA fragments matching the coding region to either psyllid Vacuolar ATPase or cathepsin can induce mortality in the Asian citrus psyllid, comparisons were made to determine the optimal dsRNA size. Psyllids were fed either the ~300 bp dsRNAs directly or after processing to siRNAs with the Dicer enzyme. Results showed that the 300 bp dsRNAs induced greater mortality and that observed with processed siRNAs. Furthermore, non-linear dose dependent toxicity of the ~300 bp dsRNAs suggesting complex interactions that have not yet been characterized with respect to dsRNA induced toxicity in insects.
The project was initiated during this quarter. Initiation involved Hiring of personnel and adaptation of facilities to support project operations including increasing plant production to support psyllid colonies and increasing psyllid rearing. Experimental protocols were also optimized for large scale screening to be conducted during this time. Meetings were held with Innocentive review panel to chose the dsRNAs that will be used in the screening process. Initial testing of dsRNAs were performed and are now being replicated with 3 dsRNA targets.
Florida growers have reported that supplementary foliar nutrient applications maintain productivity of HLB-infected trees. However, efficacy and sustainability of the nutritional approach for HLB disease management has not been validated. The main cause of visible HLB symptoms, yield reduction, and tree decline appears to be disruption of phloem tissue, which blocks the flow of photosynthate and nutrients from source to sink tissue. If supplemental nutrition is a sustainable approach, it is expected that foliar nutrients will reduce or eliminate damage and plugging in citrus phloem tissue caused by the bacterium and possibly reduce spread or replication of the bacterium in infected trees. A greenhouse study is underway to evaluate a mixture of foliar nutrients representative of that used by Florida growers for HLB management. Infected and non-infected Hamlin trees under different combinations of nutritional treatments are being monitored for bacterial titer in phloem tissue and development of disease symptoms, including phloem cell morphological changes: plugging, necrosis, and starch accumulation. Initial results show no difference in the infection rate or bacterial populations in leaf midribs. Multiple microscopy techniques including TEM, light, and fluorescence microscopy with callose-specific dyes are being used to monitor phloem plugging and necrosis as the infection progresses. A complimentary field trial is evaluating bacterial titer, yield, and tree health in a south Florida Hamlin grove with a mixture of healthy, asymptomatic (PCR+), and HLB symptomatic trees.
The objective is to evaluate soil-applied neo-nicotinoids and other SAR inducers on HLB disease progress in newly planted citrus trees subjected to psyllid-mediated infection or graft-inoculation. One yr-old Hamlin trees were planted in May 2009 and treated as follows: 1) untreated check (UTC), 2) foliar insecticide to control psyllids, 3) soil-applied imidacloprid/thiamethoxam (IMID/THIA) to induce SAR, 4) soil-applied IMID/THIA plus foliar insecticides, 5) graft-inoculated UTC, 6) graft-inoculated with IMID/THIA. There were 50 trees per treatment (5 blocks of 10 trees). In 2009, the effect of SAR inducers on HLB infection progress was inconclusive perhaps attributable to the interaction of IMID/THIA with psyllid control which may have an uncontrolled effect on psyllid transmission. In 2010, the SAR inducer acibenzolar-S-methyl (ASM, Actigard 50WP) which does not control psyllids was substituted in treatments 3, 4 and 6. At 24 months after treatments began, 105 trees were PCR+ (35%) in the trial. Higher number of PCR+ trees occurred in the UTC (20), the UTC with graft inoculation (22), and the IMID/THIA/ASM with graft-inoculation (28). A lower number of PCR+ trees occurred in the treatments with SAR inducers (11), foliar insecticides (12), and foliar insecticide plus SAR inducers (12). Two years after treatments were initiated, the effect of SAR on HLB disease progress has been minimal, which indicates a lack of promise for SAR inducers in HLB management.
The objective is to evaluate soil-applied neo-nicotinoids and other SAR inducers on HLB disease progress in newly planted citrus trees subjected to psyllid-mediated infection or graft-inoculation. One yr-old Hamlin trees were planted in May 2009 and treated as follows: 1) non-treated check (UTC), 2) foliar insecticide to control psyllids, 3) soil-applied imidacloprid/thiamethoxam (IMID/THIA) to induce SAR, 4) soil-applied IMID/THIA plus foliar insecticides, 5) graft-inoculated UTC, 6) graft-inoculated with IMID/THIA. There were 50 trees per treatment (5 blocks of 10 trees). In 2009, the effect of SAR inducers on HLB infection progress was inconclusive perhaps attributable to the interaction of IMID/THIA with psyllid control which may have an uncontrolled effect on psyllid transmission. In 2010, the SAR inducer acibenzolar-S-methyl (ASM, Actigard 50WP) which does not control psyllids was substituted in treatments 3, 4 and 6. At 17 months after treatments began, 65 trees were PCR+ (22%) in the trial. Higher number of PCR+ occurred in the UTC (14), the UTC with graft inoculation (13), and the IMID/THIA/ASM with graft-inoculation (18). Lower number of PCR+ trees occurred without graft inoculation in treatments with SAR inducers (6), foliar insecticides (8), and foliar insecticide plus SAR inducers (6). At this time, the effect of SAR on HLB disease progress is minimal, which indicates a lack of promise for use of SAR inducers in HLB management.
Objective 1: On Young fruiting Hamlin trees, elevation of imidacloprid (IMID) to 2X the rate per season to compensate for the higher tree volume produced canker control effects on leaves and fruit that matched those of 6 applications of copper sprays (the recommended program for canker control on young fruiting trees). This control is attributed to SAR-mediated reduction in canker incidence on the foliage not to control of the citrus leafminer interaction. Trunk application of IMID produced canker control on leaves and fruit that was equivalent to that of the IMID soil application. The advantage of trunk application is that IMID is not reaching the soil which alleviates the risk of leaching in the soil profile. Objective 2 Integration of soil applied IMID with foliar applications of copper sprays for control of canker. IMID applied once at the beginning of the season followed by 11 CH sprays gave the best control in the 2010 trials. This confirms that SAR and copper could be integrated for augmentation of canker control for young fruiting trees. Objective 3 is to evaluate of the complementary use of acibenzolar S- methyl (ASM), thiamethoxam (THIA )and IMID soil applications to increase and/or extend canker control in 2-yr-old grapefruit trees. In 2010, ASM at different frequency of soil application with THIA and CH sprays at 21 day interval in 2 yr-old Ray Ruby grapefruit and 1-yr-old Vernia sweet orange was highly effective for suppression of foliar canker compared to the non-treated check in each trial.
for April 2011 The objective of this project was to investigate three questions: 1) what is the seasonal pattern of Ca. Liberibacter asiaticus prevalence in leaf tissue on a grove scale; 2) what are the flushing patterns of citrus and whether these flushing patterns affect the prevalence of Ca. Liberibacter asiaticus in Diaphorina citri or citrus leaves; and 3) what is the prevalence of Diaphorina citri carrying Ca. Liberibacter asiaticus on a grove scale and how does it compare the results from the citrus trees in the same grove. In 2008 and 2009 Ebert and Rogers demonstrated that the prevalence of Ca. Liberibacter asiaticus in the Asian citrus psyllid (ACP) varied seasonally but the pattern between seasons was not consistent. It was suggested that perhaps the reason for the differences between the years related to the flushing patterns of citrus and the prevalence of the bacterium in the leaves where psyllids are feeding. This project aims to determine if there is a relationship between the frequency of disease on branches and psyllids. The postdoc working on this project is still processing the backlog of samples but preliminary results from 2 groves is available. Two groves are in Polk County near Lake Alfred (high HLB levels) and Lake Wales (low HLB levels) and the third is in Lake County (moderate HLB levels). The Polk County groves are Hamlin sweet orange and the 3rd is Early gold. We are restricted in site selection by the presence of significant populations of ACP. In each grove, two hundred trees were selected from 10 consecutive rows. One leaf/tree was randomly picked every fortnight. For qPCR detection of Las, the midribs of five random leaves were pooled to obtain 40 samples/date. An estimated Las prevalence in the branches was generated from the pools with PooledInfRate v3. Simultaneously, ACPs were collected from the same location to compare the seasonal prevalence of Las in citrus branches and ACPs. Las prevalence peaked twice in the moderately infected grove at approx. 0.50 mid-October 2010 and April 2011, dipping to 0.20 in February 2011. Las prevalence in ACP had a similar pattern but the main peak reached 0.75 in December. In the highly infected grove, there has been a steady increase of Las prevalence in branches since July 2010 to Jan 2011 an appears to continue. There was the same general trend observed with ACP although the prevalence levels were 0.15 to 0.20 higher. The prevalence rates in the low HLB incidence grove are not available yet. The phenology data continues to be collected at all sites every 2 weeks. To date, flush has been synchronous between trees.
Report for September 2011 The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. This project was initiated in August. The graduate student, Nan-Yi Wang, whose Ph.D. project this is, started his studies in Gainesville at the end of August and progressing well in classes. He is also conducting research while in Gainesville. No substantial progress has been achieved thus far.
Objective 1: To evaluate topical applications to the trunk of bactericides and Pentra-bark, a penetrant proven effective for trunk application of systemic insecticides. In the greenhouse, 0.5-1.0 cm dia trunks of Hamlin orange trees (1 yr old) were painted with a Magna-Bon (Copper sulfate pentahydrate), Cop-R-Quik (copper nitrate), a copper phosphite (CP), an experimental copper (EXP) or oxytetracycline (OTC) applied with 0.1% Pentra-Bark or left non-treated. After for 2-3 weeks, the leaves were observed for phytotoxicity. No phytotoxicity was observed, potential bactericidal activity in the plants is being assayed with detached immature leaves inoculated with Xanthomonas citri subsp. citri (Xcc) as a Gram negative bacterial surrogate for non-culturable Candidatus Liberibacter asiaticus (Las). In July, the bactericide treatments were scaled-up in a field trial by painting 1-2 yr-old nursery trees exposed to highly infected psyllid populations at USDA-ARS Picos farm in Ft. Pierce.
Objective 1 is to conduct a field evaluation of nutritional sprays for control of HLB and HLB symptom expression and yield. The field study was set up May 2010 in Southern Grove, Hendry Co., FL. Six treatments were located in 4 plots of 150 trees per treatment (interior 10 trees in each block were identified for PCR, leaf nutrition sampling, tree health and yield evaluation). Treatments were 1) non-treated check; 2) Nutri-Phite sprayed 4 times bimonthly; 3) N-Sure sprayed bimonthly; 4) Agra Sol Mn/Zn/Fe plus Nutri-Phite plus triazone urea sprayed bimonthly; 5) Keyplex 1400 DP plus Nutriphite plus triazone urea sprayed bimonthly; 6) Wettable powder nutrients (Diamond R #2) plus Nutri-Phite P+K sprayed bimonthly. The materials were applied to both sides of the tree in 125 gallons per acre with an airblast sprayer driven at 2 mph to obtain thorough coverage. Three disease ratings have been taken so far and a slight decline in tree health has been observed, but no significant treatment effects have been observed. There were no significant treatment differences in yield at the first harvest, after the initiation of treatments the previous April. Objective 2 is to determine the mechanism of HLB symptom suppression by foliar nutritional application, Rep 1 from a prior 1 year grant using Hamlin sweet orange trees inoculated with HLB and treated bimonthly with the nutritional sprays treatments 1, 2, 3, and 5 from objective 1 has finished. Monthly monitoring of Infection rate and disease development did not show obvious treatment differences except a possible increased rate of decline in treatment 2 compared to all other treatments. Trees that were only PCR+ in root tissue showed an unexpectedly fast decline across treatments. After pruning trees at 6 MPI for canopy management, sampling at 7 MPI showed a slight reduction in titer in the new flush of all treatments except treatment 1, where no Las was detected until 8 MPI. This suggests that treatments 2,3, and 5 may potentiate movement of Las to new flush where psyllids are most likely to feed and acquire Las. Sectioned midrib samples were observed by light microscopy at 6 and 8 MPI and 9 MPI. At 6 MPI reduced phloem plugging and necrosis was observed in treatments 3 and 5, however these treatments had some symptomatic leaves without detectable Las. These leaves had abnormal starch buildup preferentially in phloem tissue instead of mesophyll cells. At 9 MPI there was significant variation in plugging between midribs within a treatment even with highly similar symptoms and Las titer. All treatments had a full range of phloem damage observable in midribs from similarly symptomatic leaves ranging from severe plugging and collapse to apparently healthy phloem. Rep 2 plants began bimonthly nutritional treatments in June and were inoculated at the end of August and monthly samplings of leaf and root tissue are underway. Root samples are split for qPCR Las quantification and starch analysis for a quantitive measure of phloem function throughout the plant. Month 1 post inoculation samples have just been harvested and are being processed for qPCR.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
