June 2014 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: All preliminary experiments with the DMIs are completed and tests are underway. Our main limitation is suitable incubator space at the moment. A student has been found to work with the SDHI compounds. Objective 2: Data from spray trial have been taken and are still being analyzed. Sites have been selected for then next trial and planning has begun for the 2014-2015 season. Objective 3: Suitable sites to conduct the spatial studies have been located. Blocks were scouted and initial disease incidence and severity were collected. Objective 4: Asymptomatic and symptomatic fruit from a known symptomatic were harvested and a set of experiments were initiated to look at the effect of postharvest fungicides and heat treatments. In vitro heat treatments were also conducted and heat did have an effect on G. citricarpa growth on petri dishes. Some experiments were also started on fruit with heat and carvacol. Experiments are on-going and results are expected soon.
Sept 18, 2014 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. Guignardia mangiferae sequencing is complete and the assembly is continuing. To get better resolution with the G. citricarpa genome, the organism was resequenced, giving far better resolution from the genome data. Analysis of the gene structure is being continued. A manuscript on mating type genes is in preparation with all of the data for figures collected. Materials and methods were drafted and are under revision. We decided to run another set of confirmatory experiments after the results of the last experiments were somewhat inconclusive. Sample collection for the greenhouse inoculation experiment continues and the samples are being fixed and embedded. We moved from weekly sampling to biweekly sampling. Some fungal penetration structures have been observed in the samples. Irradiated and autoclaved citrus twigs were inoculated with a replacement series of G. citricarpa and D. citri inoculum. In initial results of the first trial of the experiment, there was greater initial colonization by D. citri than G. citricarpa.
June 4, 2014 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. Guignardia mangiferae sequencing is complete and the assembly is continuing. To get better resolution with the G. citricarpa genome, the organism was resequenced, giving far better resolution from the genome data. A manuscript on mating type genes is in preparation with all of the data for figures collected. Materials and methods are being written. We continuing the confirmation of some of the results in one section. An inoculation experiment with conidia on leaves was preformed and samples continue to be collected at regular intervals for microscopic observation for when infection structures are formed and when the fungus starts to spread through the leaf. The paraffin embedding technique was fine tuned. Irradiated and autoclaved citrus twigs were inoculated with a replacement series of G. citricarpa and D. citri inoculum. The fungal structures and qPCR are being counted bi-weekly
Reduction/suppression of inoculum and fruit infection in grapefruit using trunk applications of non-insecticidal SAR inducers:SAR induced by soil-applied insecticides provides substantial benefits for canker disease management on young citrus trees that may be augmented with ASM. Currently, use of neo-nicotinoids insecticides for control of the psyllids and leafminer is limited to trees less than 2.75 m tall. Soil drench or trunk applications of IMID and THIA and ASM are bing compared to untreated controls and 9 or 10 standard 21-day interval copper sprays for protection of foliage and fruit on 5 to 7-yr old ‘Ray Ruby’ grapefruit trees. Actigard is commercial SAR inducer which is non-insecticidal and does not have a tree size restriction for use. Collaboration with Syngenta in an EPA-approved Experimental Use Program (EUP) in two east coast grapefruit groves is being repeated again in 2014 to support the labeling of soil applied Actigard integrated with copper sprays for control of canker on bearing grapefruit. Reduction/suppression of fruit infection in citrus trees using foliar applications of soluble copper novel bactericidal formulations in comparison with film-forming copper formulations: In 2014 trials, fixed copper formulations (e.g. copper hydroxide and copper oxide) sprayed at 2 to 4 lb of product per acre (0.75 to 1.4 lb per acre of metallic copper) are being compared with commercial and experimental soluble copper formulations at much lower metallic rates in grapefruit and Hamlin blocks. Improving performance of copper formulations: Reducing wind speed below 18 mph greatly increases the efficacy of the copper film for protection of the fruit surface from infection through natural openings and rind wounds. 6 and 7 yr-old red grapefruit blocks surrounded by a 20-30 ft tall Corymbia torelliana windbreaks are under study. In each block, weather stations are deployed to measure wind gusts in proximity to the windbreak. Cu bioavailability and residual activity of copper formulations: Copper residues of fixed and soluble copper formulations continue to be evaluated at different periods of season as rainfall and fruit expansion rates vary.
Objective 1: Assays of non-bearing trees soil drenched with the chelated copper formulations Magna-Bon (MB) and Copper Quik (CQ) confirm that copper concentration in leaves of non-sprayed field trees is well above nutritional concentrations. HLB preventative treatments of MB and CQ were applied at 3 month intervals during two growing seasons at 1,2,4,5 or 10 oz/tree in 8 oz of water as soil drenches to visually asymptomatic 1 and 2 year old Valencia trees on Volkamer lemon and Swingle citrumelo, respectively. The solid plantings, located in Sebring and Avon Park, were surrounded by adult trees with 100% incidence of HLB symptoms. After two seasons, leaf samples for determining PCR status of trees in each trial were collected in December 2013 and analysis completed in early 2014. In the 4 year old block of Valencia on Swingle, PCR positive trees across all treatments ranged from approximately 40 to 80% incidence and in the 3 year old block of Valencia on Volkamer lemon ranged from 8-15% incidence. Incidence of PCR positive trees drenched with MB or CQ drenches was not significantly different from untreated trees. Soil copper treatments will be continued in the 3 year old block of Valencia on Swingle for one more season. Second year PCR results from a trial at USDA Picos farm in Ft. Pierce treated with MB, CQ and copper or zinc phosphite drench treatments of Hamlin on Swingle trees treated since planting still has a low incidence of HLB positive trees. Drench treaments at the Picos and Sebring sites will be continued for another season.
Objective 1: Assays of non-bearing trees soil drenched with the chelated copper formulations Magna-Bon (MB) and Copper Quik (CQ) confirm that copper concentration in leaves of non-sprayed field trees is well above nutritional concentrations. HLB preventative treatments of MB and CQ were applied at 3 month intervals during two growing seasons at 1,2,4,5 or 10 oz/tree in 8 oz of water as soil drenches to visually asymptomatic 1 and 2 year old Valencia trees on Volkamer lemon and Swingle citrumelo, respectively. The solid plantings, located in Sebring and Avon Park, were surrounded by adult trees with 100% incidence of HLB symptoms. After two seasons, leaf samples for determining PCR status of trees in each trial were collected in December 2013 and analysis completed in early 2014. In the 4 year old block of Valencia on Swingle, PCR positive trees across all treatments ranged from approximately 40 to 80% incidence and in the 3 year old block of Valencia on Volkamer lemon ranged from 8-15% incidence. Incidence of PCR positive trees drenched with MB or CQ drenches was not significantly different from untreated trees. Soil copper treatments will be continued in the 3 year old block of Valencia on Swingle for one more season. Second year PCR results from a trial at USDA Picos farm in Ft. Pierce treated with MB, CQ and copper or zinc phosphite drench treatments of Hamlin on Swingle trees treated since planting still has a low incidence of HLB positive trees. Drench treaments at the Picos and Sebring sites will be continued for another season.
This research seeks to determine whether young trees infected with CLas and displaying typical HLB symptoms can be brought to maturity and produce an economically viable yield. This will be achieved by managing a 58 acre grove of 3-year-old ‘Valencia’ / Kuharske Carrizo trees using a combination of three different foliar and three different ground applied nutritional programs. Factorial AxB treatments consist of A) ground-applied: 1) Liquid/dry+Ca (BHG standard), 2) Liquid+Ca, 3) Liquid/dry-Ca B) foliar-applied: 1) BHG standard-Ca, 2) BHG standard+Ca, 3) “Prescription”(+Ca). The prescription treatment was designed to be dynamic, customized for optimization, with feedback based on frequent leaf tissue analyses, visual symptoms, and the growth of the tree canopies and yield. There are six replications of treatments, with two being pure replications. The grove still continues to look better, despite the nearly 100% HLB incidence. Foliation of canopies is dense, and leaf color going into Spring season was a healthy green. Fruit yields per plot were measured on 19 March 2014, and analyzed together with the fruit quality data determined in the packing house. Selected data is summarized below: FOLIAR FERT Fruit yield (boxes/ac) STD-Ca STD+Ca Prescrip SOIL FERT Liq/Dry 171.8* 130.2 134.9 Liq 150.6 121.3 134.5 Liq/Dry-Ca 146.4 133.9 113.0# Only the yield of the treatment marked with an * was significantly higher than the worst yield (#); all others were non-significant and can be considered the same. The best treatment yielded 1.13 boxes per tree at age 5; considerably less than healthy trees of the same age class would yield, but nevertheless showing the increasing potential of HLB-impacted young trees to recover and gradually increase their fruit yielding capacity. As previously mentioned, HLB-impacted young blocks should be planted at higher densities to offset the per tree yield losses caused by the disease. Theoretically the best treatment in this experiment could yield 410 boxes/acre at age 5 if the planting density was 363 trees/acre (8×15′) instead of the 151 trees/acre (12×24′) to which it is currently set. Statistically speaking, all the different fertilizer strategies chosen for testing in this block appear to be working equally well. However an economic analysis of the treatments will undoubtedly show that the less expensive standard fertilizer program for both soil and foliar nutrition contributes to the profitability in this grove. In fact the best treatment (*) consists of standard BHG liquid/dry (with calcium nitrate) soil fertilization and standard foliar nutrition (no Ca). Interestingly, it appears that Ca is best applied to the soil and not the foliage. The reader should understand that mitigation of HLB with plant nutrition is a strictly site-specific practice and the results obtained in this experiment are not necessarily reproducible in another grove elsewhere in Florida.
A new class of inhibitors were discovered based on discovery of enzymes present in salivary sheaths. Two of the six tested inhibitors blocked sheath formation. An additional six different proteins belonging to the same functional class of proteins were identified based on their potential to bind to salivary sheath subunits. These proteins were tested for sheath inhibition, but none caused inhibition of sheath biosynthesis or alterations in sheath appearance. The ultimate goal of this screening approach is to identify a diverse set of sheath inhibitors that can be used in combination to function as effective topically applied blockers of psyllid feeding on citrus. If feeding is inhibited at the initiation of feeding and prevents the psyllid from successfully reaching the phloem, this method of psyllid control will prevent the psyllid form acquiring or transmitting the bacterium that causes citrus greening.
As a result of our compositional structural and enzymatic analysis of psyllid sheath structure and biosynthesis, we identified a class of natural products (Generally Regarded as Safe ‘GRAS’ compounds) that could inhibit sheath biosynthesis. These compounds were selected based on the probability that their structure would be competitive with the substrates used in the sheath polymerizing process. Twelve such compounds were selected and tested for sheath inhibition. Of the twelve, the presence of 10 in the media in which these insects fed resulted in aberrant sheath formation. These are now being tested in dose studies and in combination. The doses and combinations showing the best response will be used in greenhouse application studies. The discovery of GRAS compounds that have affect sheath formation offers the possibility of a rapid commercialization process if they are shown to be effective in whole plant application trials. Some of the tested compounds are available at relatively low costs that would be compatible with commercialization if efficacy as a topically applied compound can be verified.
THis project was an extension of a previously funded CRDF grant. Funding for both overlapped during this year with just minor funds available in the base 330 project. Therefor results presented in the March 2014 report are the same for both projects and are repeated in the quarterly reports: As a result of our compositional structural and enzymatic analysis of psyllid sheath structure and biosynthesis, we identified a class of natural products (Generally Regarded as Safe ‘GRAS’ compounds) that could inhibit sheath biosynthesis. These compounds were selected based on the probability that their structure would be competitive with the substrates used in the sheath polymerizing process. Twelve such compounds were selected and tested for sheath inhibition. Of the twelve, the presence of 10 in the media in which these insects fed resulted in aberrant sheath formation. These are now being tested in dose studies and in combination. The doses and combinations showing the best response will be used in greenhouse application studies. The discovery of GRAS compounds that have affect on sheath formation offers the possibility of a rapid commercialization process if they are shown to be effective in whole plant application trials. Some of the tested compounds are available at relatively low costs that would be compatible with commercialization if efficacy as a topically applied compound can be verified.
Continued proteome analysis of isolated psyllid salivary sheaths was conducted using a more complete psyllid protein database. This analysis was performed through collaboration with Dr. Michelle Cilia, Research Molecular Biologist USDA-ARS, BioIPM Research Unit and Boyce Thompson Institute for Plant Research that has allowed the identification of a candidate enzyme that may be involved in the sheath polymerization process. The identified gene encoding the protein was then identified in the psyllid genome showing that it is indeed of psyllid origin. Further analysis of sheath formation showed that inhibitors of this enzyme blocked sheath formation. This research has allowed us to understand the nature of the sheath polymerization process and this knowledge has opened the door for natural product screening to identify natural product inhibitors to sheath formation.
In earlier phases of this study, a “Bugphone” microcontroller device that detects Asian citrus psyllid male vibrational calls and plays back female replies was tested for its ability to attract males to a sticky cone attached to a small tree. In these studies, 20-30% of tested males have responded to the Bugphone, but for various reasons, only a small percentage of the males that approached the cones were captured. In the enhancement phase of this study, considering other ways of interfering with mating that did not necessarily involve trapping, we purchased outdoor speakers that can be operated with small amplifier systems to produce signals interfering with male and female ACP duetting calls in field environments. Initial studies of the capability of the systems to disrupt mating have begun in a laboratory setting to determine the best sound pressure levels and signal timings. We have joined forces with Dr. William Eisenstadt and students at the University of Florida Department of Computer and Electrical Engineering to consider options that use low power because initial versions could operate for only 1-2 days before replacement batteries were needed.
This proposal aims to continue improvement to a novel psyllid trap and to use the trap to gather new information on the behavior, biology, population dynamics and biological control of ACP/Candidatus Liberibacter asiaticus. Lab and field testing was and continues to be conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps and behaviorally active components. 570 – Grant Obj. 1: We continue to conduct field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulations of visual cues as well as trap physical components and component orientation. This within Florida research component is continuing and we now added several new locations in Puerto Rico to exploit the significantly higher ACP populations there that make the research easier. Doing this quarter we have successfully tested >20 trap prototype configurations in Puerto Rico in cooperation with Dr. David Jenkins and have now eliminated a number of configurations that are inferior as well as identified the most successful trap under field conditions. We have had no success with trap additions related to odor cues reported to be potential attractants in the literature. We are continuing this research with new ideas but now have a trap identified a “standard trap” that can be used to capture and preserve the DNA of the psyllids and their associates in situ. Obj. 2: We have initiated the areawide psyllid sampling objective to detect and develop new biological controls for use against ACP. We are continuing the sampling in the northern most citrus populations in Alachua County and around Ft. Pierce with the in Florida and Puerto Rico. The standard trap is being used for this work and performs well enough to complete this effort, i.e., where ACP occur the trap captures and preserves them in proportion to their populations. So far we have not identified any new pathogens. We have obtained cooperation with other USDA-ARS research personnel with expertise in the identification and rearing of entomopathogenic fungi and they have evaluated samples toward this end from Florida and Puerto Rico. This effort will be continued.
This proposal aims to continue improvement to a novel psyllid trap and to use the trap to gather new information on the behavior, biology, population dynamics and biological control of ACP/Candidatus Liberibacter asiaticus. Lab and field testing continues to be conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps and behaviorally active components. This enhancement grant is to further use the trap for detection and identification of biological control agents as well as to develop novel methods to manipulate psyllid behavior to improve the deployment and efficacy of biological control agents. This research was begun this quarter and is ongoing in Florida and Puerto Rico. We have have successfully solicited some producers with conventional and organic groves where we are conducting our experiments. No results to report so far but we are actively collecting field data toward the objectives that will be forthcoming in the next report.
We are evaluating and attempting to develop optimal RNA interference constructs to target Diaphorina citri, the psyllid vector of Ca. Liberibacter asiaticus, the causal agent of citrus greening. We are using two psyllid species for our work, D. citri and Bactericera cockerelli, the latter being the potato psyllid. Using B. cockerelli offers the opportunity to use herbaceous plants and make more rapid progress that can then be applied to citrus and D. citri. So far, 7 aMIRNAs targeting the BC-ATPase gene (pAMIRA1, pAMIRA1c, pAMIRA2, pAMIRA2c, pAMIRA3, pAMIRA3c, and pAMIRA2PE) and 4 pAMIRNAs targeting the GFP gene (pAMIRG1, pAMIRG1c, pAMIRG2, and pAMIRG2c, as controls) have been generated. We cloned some of these into three expression vector systems: a TMV-based viral vector, a geminivirus-based viral vector and a binary plasmid. We are evaluating these in plants now. Both the TMV and binary plasmids appear to be functioning as expected although different amiRNA product patterns are observed after northern blot hybridization analysis. The geminivirus-based system did not work originally, we have re-generated the constructs and they are under evaluation now. We believe that the geminivirus system offers the best potential for generating high amounts of specific amiRNAs in plants. We are now preparing small RNA cDNA libraries from plants for each system and will sequence the small RNA products to assess the quantity and quality of specific miRNAs. We are also using RT-qPCR to assess target mRNA knockdown in psyllids after feeding on plants infiltrated with the above constructs. We are using psyllids per sample with replicated samples for each treatment. Simultaneously, we are testing B. cockerelli on specific transgenic plants that are engineered to generate interfering RNAs to different B. cockerelli mRNA targets. We are using a leaf disc assay and RT-qPCR to assess target mRNA knockdown, and using whole plants to assess mortality effects on adults and psyllid nymphs.
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