The goal of this project was to determine if infection by Candidatus Liberibacter affects the response of Asian citrus psyllid (ACP) to its citrus host plants to understand if the causal pathogen of HLB affects the plant so as to promote its own spread. Initially, we evaluated if uninfected psyllids are attracted more to HLB-infected versus healthy trees. Also, we determined whether this behavior changes when the ACP vector is itself a carrier of the pathogen. We conducted a series of behavioral experiments to investigate whether HLB-infected citrus plants are differentially attractive to ACP as compared with healthy citrus plants. Our results proved that HLB-infected citrus plants released volatile chemicals that were more attractive to ACP adults than healthy plants. Also, more ACP were attracted to HLB-infected plants than to healthy plants in open-air cage experiments. However, subsequent dispersal of ACP adults to healthy plants following their initial choice indicated that final settling preference was for healthy rather than diseased plants. This behavior also occurred under complete darkness, indicating that volatile chemicals were mediating this response more than visual cues such as yellowing of leaves on diseased plants. Initial movement of ACP to infected plants and further dispersal to healthy plants was likely explained by production of deceptive volatile compounds by HLB-infected plants to attract ACP adults so as to facilitate the spread of bacteria. However, we hypothesized that the subsequent movement to new plants could be due to poor nutritional status or a nutritional imbalance in HLB-infected plants. Our next line of research was to identify the specific mechanisms that mediated psyllid response. If we could find the specific chemical cues that mediate greater psyllid attraction to infected versus health plants, we could potentially: 1) make use of such attractants to improve psyllid monitoring, and 2) turns off genes that result in the pathways that lead to production of these chemical psyllid attractants in infected plants. In preliminary experiments in this project, we found evidence of specific psyllid chemical attractants produced by infected plants and potential specific nutritional imbalances that cause psyllids to leave infected plants and finally settle on healthy plants. Elucidating and confirming these specific mechanisms has been the topic of the follow-up project to this one (project 439), which is ongoing.
We have successfully completed our major objective of establishing a continuous tissue culture cell line derived from Diaphorina citri. Several of these cultures have been in continuous cultivation for over two years. We continue to maintain these cells and to attempt to increase the robustness of the cell lines via selective manipulation and transfer of cell patches. As noted in previous updates, suspension cells are more homogeneous than adherent cells and appear to grow faster. A number of more homogeneous looking adherent cell lines appear to be emerging. We have disseminated the cell lines to several laboratories both nationally and internationally. Frozen cell suspensions have also been made for storage of the cell lines.
The objective of this project was to investigate three questions: 1) what is the seasonal pattern of Ca. Liberibacter asiaticus (Las) prevalence in leaf tissue on a grove scale; 2) what are the flushing patterns of citrus and do the flushing patterns affect the prevalence of Las in Diaphorina citri or citrus leaves; and 3) what is the prevalence of Diaphorina citri carrying Las 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 Las 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 ACPs are feeding. This project aims to determine if there is a relationship between the frequency of disease on branches and ACPs. Three sweet orange groves have been sampled from June 2010 for HLB prevalence in branches and ACP. Citrus phenology measurements began in October 2010. 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. Pooling needed to be reduced in the groves with high HLB prevalence as the the season progressed. Simultaneously, ACPs were collected from the same location to compare the seasonal prevalence of Las in citrus branches and ACPs. There were 2 peaks of Las prevalence 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 until March 2011 where a plateau of 0.75 was reached. There was the same general trend observed with ACP although the prevalence levels were 0.15 to 0.20 higher, reaching 0.90 in March 2011, but there was a large decline in ACP prevalence to 0.60 in May 2011. In the low HLB intensity grove, no Las has been detected in since the initiation of sampling in July 2010. However, very low prevalence has been detected in branches. Prevalence has hovered below 0.02 until April 2011 when it reached 0.06. The phenology data was collected at all sites since November 2010. Flush was highly synchronous in 2011 likely because of the extended cold temperatures this winter and the drought conditions. As would be expected with the very dry conditions, there is currently little new flush on the trees. Presentations at Conferences: Parkunan V., Wang N.-Y., Ebert T.A., Rogers M.E. and Dewdney M.M. 2011. Seasonal prevalence of citrus Huanglongbing (Candidatus Liberibacter asiaticus) in a central Florida sweet orange grove. at the 2nd International Research Conference on Huanglongbing January 10-14, Orlando FL. V. Parkunan, T. Ebert, N. Wang, M. Rogers, M. Dewdney. 2011. Seasonal variation of Candidatus Liberibacter asiaticus in citrus branches and in vector, Diaphorina citri, in Central Florida sweet orange groves. Phytopath 101:S138.
Using a food protein-based marking system, we have investigated the movement patterns of the Asian citrus psyllid (ACP)in citrus groves in Florida. We have documented the seasonal activity of movement between abandoned and managed groves; movement between managed groves; and investigated within grove movement. We have also shown that psyllids infected with Candidatus liberibacter asiaticus (Las) move from abandoned groves into managed groves, confirming that abandoned groves can serve as a reservoir of both ACP and citrus greening inoculum. Based on studies conducted from June 2009-Sept. 2010, the seasonal movement of psyllids between abandoned and managed groves can be characterized as primarily local or within grove movement from January-March, dispersal (between groves) from April-September and little or no movement from October-December when ACP populations are at lowest. A study to determine movement of ACP between managed groves and the effects of fallow fields on ACP movement showed that ACP dispersal was not inhibited by fallow fields and ACP dispersed a distance of 300 m in one day crossing an unplanted area to the next grove. A study of long-distance movement determined that ACP are capable of moving at least 2000 m in 12 days. The major factor correlated with ACP movement was the abundance of citrus leaf flush, a resource necessary for ACP for egg laying. Wind direction was not significantly correlated with ACP dispersal, suggesting that ACP may be capable of substantial directed flight. We also confirmed that ACP dispersing from abandoned groves moved the Candidatus liberibacter asiaticus (Las) pathogen into commercial groves. This movement occurred at distances of 100-150 meters. PCR analysis of protein-marked psyllids in this larger study showed that 0.02-0.2 % of captured dispersing psyllids carried the Las pathogen. Infected psyllids were found to move at least 2 km (1.25 miles) from the protein-sprayed area indicating that groves within a minimum of this distance from trees with citrus greening are at risk of infection. We also investigated within grove movement of ACP. A 305 m x 230 m study area was established. The perimeter trees (total 200 trees) of this grove were sprayed with a 20% egg albumin solution and an area in the center of the plot (122 m x 91 m; 192 trees total) was sprayed with a 10% milk solution. Twenty yellow sticky traps (5/side) were placed in the egg-sprayed area, 20 traps in the milk-sprayed area and 20 traps in the unsprayed interior of the plots and removed from the field after 4 days. ACP were removed from the traps and subjected to ELISA analysis to determine if the psyllids had been sprayed with either milk or egg solutions. A total of 48 psyllids were captured on the 60 traps. Of these, only 5 were marked with protein. Three of the 5 were marked with egg protein and captured in the area where egg protein was sprayed. No marked psyllids were captured in the milk-sprayed area. In the unsprayed interior area, 1 psyllid was marked with milk protein and 1 with egg protein. This experiment is currently being repeated. Funding from the CRDF has supported the research of one M.S. degree candidate (expected graduation August 2011) and has resulted in 2 journal publications: Tiwari et al. 2011. Incidence of Candidatus Liberibacter asiaticus infection in abandoned citrus occurring in proximity to commercially managed groves. J. Econ. Entomol. 103:1972-1978. Lewis-Rosenblum et al. 2011. Seasonal short-range movement patterns and long-range dispersal of the Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae) in Florida citrus. In Preparation.
.-aminobutyric acid (BABA) is known to induce resistance against several microbial pathogens, nematodes and insects in several plant species. Several laboratory studies were conducted to determine if a similar effect of BABA occurs against Asian citrus psyllid (ACP) in citrus. Previously, drench applications of BABA to citrus plant roots were found to suppress the growth and development of ACP by reducing the number of eggs, nymphs and adults produced per plant. Head-space volatiles collected from BABA-treated and untreated plants were analyzed using GC-MS. Differences in chemical profiles between BABA-treated and control plants were observed. Identification of these chemicals is underway. To further optimize the use of BABA for ACP management, another set of greenhouse experiments was conducted to investigate the effects of a reduced rate of BABA alone and in combination with a reduced rate of imidacloprid, made as foliar applications. The treatments were evaluated by tracking the growth and development of psyllids. BABA (5mM) alone caused 30.7%, 17.7%, 69.5% and 56.1% reductions in the numbers of eggs, nymphs, and adults per plant, respectively, when compared to control plants. The combination of BABA (2.5 mM) and imidacloprid (312.5 ppm) caused 90.7%, 82.9%, 76.6% and 65% reductions in the numbers of eggs, nymphs, and adults per plant, respectively, when compared to control plants. Likewise, in a follow-up experiment, BABA (5mM) alone caused 15.2%, 45.3%, 59.8% and 56.6% reductions in the numbers of eggs, nymphs, and adults per plant, respectively, when compared to control plants. The combination of BABA (2.5 mM) and imidacloprid (312.5 ppm) resulted in 99.1%, 99.7%, 100.0% and 100.0% reductions in the numbers of eggs, nymphs, and adults per plant, respectively, when compared to control plants. BABA alone or in combination with imidacloprid may be a useful method for managing ACP. In this project, we also investigated the activity of Pyriproxyfen, Buprofezin and Diflubenzuron (Micromite) on ACP eggs, nymphs, and adults to evaluate their potential usefulness as biorational insecticides as part of an integrated pest management (IPM) strategy for ACP control. All three chemicals exhibited strong ovicidal and larvicidal activity against ACP eggs and nymphs, respectively, in age- and concentration-dependent manners. Fewer eggs hatched into nymphs at the higher concentrations tested (80-160 ‘g mL-1). A significantly lower percentage of early instar nymphs (first, second and third) survived and emerged into adults at the higher concentrations tested (80-160 ‘g mL-1) compared with late instar nymphs (fourth and fifth). Furthermore, the chemicals exhibited transovarial activity by significantly reducing the fecundity of females and viability of eggs deposited by females that emerged from treated fifth instar nymphs. Topical application of each chemical to adults also significantly reduced female fecundity and egg viability. Application of each chemical at 160 ‘g mL-1 results in the highest inhibition of egg hatch in younger eggs (0-48 h old) laid before or after treatment and the strongest suppression of adult emergence from early instar nymphs compared with other rates tested. They also markedly reduced female fecundity and egg viability for adults that were exposed either directly or indirectly. The direct (ovicidal and larvicidal) and indirect (transovarial) effects of these insect growth regulators against immature and adult ACP, respectively, suggest that integration of this insecticide as part of an IPM strategy should negatively impact ACP populations over time.
Under Objective 1 and 2 the following was submitted and published in Plant Disease: Soil applications of inducers of systemic acquired resistance (SAR), imidacloprid, thiamethoxam or acibenzolar-S-methyl, at various rates and application frequencies were evaluated for control of citrus canker caused by Xanthomonas citri subsp. citri in a field trial of 3- and 4-year-old ‘Ray Ruby’ grapefruit trees in southeastern Florida. Reduction of foliar incidence of canker produced by one, two or four soil applications of imidacloprid, thiamethoxam, and acibenzolar-S-methyl was compared with 11 foliar sprays of copper hydroxide and streptomycin applied at 21-day intervals. In 2008 and 2009 crop seasons, canker incidence on each set of vegetative flushes was assessed as the percentage of the total leaves with lesions. By the end of the 2008 season, despite above average rainfall and a tropical storm event, all treatments significantly reduced foliar incidence of citrus canker on the combined Spring-Summer-Fall flushes. Sprays of copper hydroxide and streptomycin were effective for reducing canker incidence on shoot flushes produced throughout the season compared to the untreated control, whereas soil applied SAR inducers reduced foliar disease depending on rate, frequency and timing of application. Except for the treatment of four applications of acibenzolar-S-methyl at 0.2 g a.i. per tree or two applications of imidacloprid, SAR inducers were ineffective for reducing foliar disease on the flushes that were present during the tropical storm. In 2009, all treatments significantly reduced the incidence of foliar canker on the combined Spring-Summer-Fall flushes but not all treatments of Spring-Summer flushes with SAR inducers were effective compared to the untreated control. Hence, depending on rate, frequency and timing of application, soil-applied SAR inducers reduced incidence of canker on foliar flushes of young grapefruit trees under epidemic conditions. Under Objective 2 Integration of soil applied SARs with foliar applications of copper sprays for control of canker, we are testing the efficacy of the inducers for control of citrus canker on young fruiting Red grapefruit and Hamlin orange trees. We previously demonstrated that trunk application of imidacloprid is effective for canker control on fruit as 21 day copper sprays. To keep the chemicals out of the soil, we are spraying the SAR inducers on the trunk. These trials involve testing of non-registered acibenzolar-S-methyl and the insecticides imidacloprid, thiamethoxam at elevated rates. Hence, fruit from the treated trees must be dropped and destroyed (called ‘crop destruct’ of nonregistered chemical treatments). Under Objective 3, In 2011 the complementary use of ASM, THIA and IMID soil applications to increase and/or extend canker control in 3-yr-old grapefruit and 2-yr-old Vernia orange trees. The highest incidence of disease trees and/or leaves is in the unreated check in each trial compared with a very low incidence of canker in the integrated SAR treatments. A field trial with soil applied neonicotinoids in Parana, Brazil was evaluated. IMID (Confidor) as a soil drench and IMID (Winner) applied to trunk gave comparable in disease control activity on 2-yr old Valencia orange trees, as well as, the other neonicotinoids tested, THIA and Clothianidin. Clothianidin is now registered for use on non-bearing citrus in Florida, hence all of neonicotinoids registered for non-bearing citrus in Florida have been shown to have SAR-inducing activity against canker as well.
Four CTV-vectored insecticidal peptides have been proposed for evaluation against the Asian Citrus Psyllid (ACP) ACP, Brown Citrus Aphid (BCA), and Citrus Leafminer (CLM). Since our last report, we have successfully cloned all of the sequenced genes from the proposed peptides into CTV vectors. We have recently completed bark flap inoculations of the final two CTV-peptide constructs into Nicotiana benthamiana plants to obtain sufficient material for graft-inoculation into citrus, a process that is expected to take two months. The peptide, indolicidin, which was previously cloned into the CTV vector and amplified in Nicotiana, has been successfully inoculated into five citrus plants. Currently, these plants are being used in bioassays (e.g. host preference, feeding, survival, development, and fecundity) with ACP and BCA. Similar bioassays with CTV-vectored snow drop lectin (GNA) are also ongoing, which we expect to complete in the next three months. In addition, begun evaluating efficacy of CTV-vectored GNA against CLM; however, due to low production of insects from our laboratory colony, we expect that these bioassays will need to be completed next spring/summer when CLM can be obtained from the field. Artificial feeding bioassays continue to be conducted to evaluate the efficacy of synthetic peptides, outlined in this proposal, against ACP. Two artificial feeding methods for exposing ACP to synthetic GNA have been evaluated since the last proposal: leaf uptake and feeding sachets. The first method, wherein ACP were placed on single, detached leaves held in a solution containing GNA, proved to be unsuccessful. Feeding sachets have subsequently been used for artificial feeding assays, with preliminary results suggesting that GNA reduces ACP survival at high concentrations. Development of enzyme-linked immunosorbant (ELISA) assays specific to the peptides under investigation are ongoing. Identifying the concentration of expressed peptides should facilitate comparisons between the efficacy observed in artificial bioassays with synthetic peptides and in planta assays with CTV-expressed peptides. Although we are able to successfully quantify CTV expression with ELISA, we have not been successful in accurately quantifying peptide expression in in CTV-GNA plants with this method.
We have previously shown that dsRNA identitical to two separate Asian citrus psyllid genes (Cathepsin F and Vacuolar ATPase) are toxic when fed to the psyllid in an artificial diet. As a result of this finding, we have worked in collaboration with Dr. Bill Dawson and Dr. Siddarame Gowda to express these dsRNAs in citrus using their infectious Citrus Tristeza Virus (CTV) vector. Such plants have been developed and are now being evaluated for the presence of psyllid gene dsRNA in the phloem tissues. These plants are also being propagated to challenge with psyllids to analyze psyllid preference and performance with respect to mortality and efficacy when either provided with these plants as the sole host plants or provided with a choice of these dsRNA expressing plants and non-dsRNA expressing plants.
This is the second annual report for a three year project on protecting newly planted citrus trees from Asian citrus psyllid (ACP) and huanglongbing (HLB). Key information from the project is summarized here. In an area under high ACP and HLB pressure, regular treatments of insecticides may reduce ACP levels but outbreaks still occurred even under an intensive insecticide program (monthly applications of traditional insecticides). Repetitive (every three weeks) applications of petroleum oil failed to prevent periodic ACP outbreaks. There was little evidence that interplanting orange jasmine with citrus reduced ACP infestations in citrus, regardless of whether the jasmine was treated with pesticides or not – however, problems establishing and maintaining jasmine plants was difficult, thus definite conclusions regarding the value of jasmine as a trap plant could not be made. Regardless of the specific insecticide program evaluated and degree of ACP control achieved, none of the programs entirely circumvented HLB infections. In experiments where infected trees were removed as they were identified, less than 10% trees tested positive for HLB by 12 months after planting. In a test where infected trees were not removed, the incidence of HLB in young citrus generally increased faster with up to 25% trees testing positive within a year. In the experiments where infected trees were removed as they were found and not replaced, 40 to 50% trees had been removed by 33 months after planting and some plots began to look barren. Had the infected trees been replaced with resets, it was likely that resets would have developed the disease at a rate equal to the older young trees and it is doubtful that enough trees would have ever began producing enough fruit to offset planting expenditures. In tests where trees were not removed, at 14 months after planting percentages of trees testing positive for HLB (Ct<32) reached 35 to 50% but then declined to as low as around 15% at 19 months after planting. In experiments where infected trees were not removed, some trees (both healthy and infected) began producing fruit during the second year after planting. During June at 22 months after planting, although infected trees were shorter, they were equivalent to healthy trees with respect to trunk diameter and number of fruit; whether the fruit of infected trees will remain on trees until harvest remains to be determined. In one planting, an average of 5% trees died by 17 months after planting but only about 1% of dead trees were known to be HLB-infected. Among other dead trees, some were killed by freezes and some by Phytophthora. Although HLB may weaken a tree and predispose it to other problems such as Phytophthora, at 22 months after planting there was the same percentage of trees infected by Phytophthora among trees infected by HLB (10.8%) and trees not infected by HLB (10.3%). During the third year of this project, we will continue subjecting young citrus to the various insecticide programs and monitoring the young plantings for HLB. Growth and yield data will be obtained for healthy and HLB-infected trees. Juice quality will be assessed for healthy and diseased trees. The epidemiology of HLB in citrus under different insecticide programs will be investigated. Collectively, the results of these experiments will provide growers insight into the efficacy of different ACP control programs for protecting young citrus from HLB and, if infected trees are not removed, what they may expect from a new young citrus planting with respect to tree growth, survival and fruit production.
A series of research greenhouse research experiments have been completed that show that a number of commercial formulations of plant growth regulators (PGRs) (GA biosynthesis inhibitors) reduce Asian citrus psyllid fitness. Specifically, we found that two of the PGRs tested (prohexadione calcium and mefluidide) reduced ACP fecundity and survivorship while two others, uniconazole and paclobutrazol, reduced fecundity or survivorship, respectively, when psyllids were caged on treated trees 10 days after PGR application. These effects on psyllid biology are independent of vegetative growth, i.e. they occur at PGR rates below which vegetative growth is reduced, and are apparently not a result of PGR toxicity to the psyllids. These results have been summarized in a manuscript currently in review with the Journal of the American Society for Horticultural Science that we anticipate will be published in late 2011. Follow up work has been ongoing to understand the plant biochemical changes occurring in response to PGR treatment that may be correlated with changes in psyllid biology. We have found that plants treated with prohexadione calcium exhibit significant changes in a number of amino acids between 9 and 15 days after PGR application. At least three of these amino acids are considered to be essential dietary nutrients for plant-feeding insects. Analyses of other dietary compounds (carbohydrates, vitamin A and E) are ongoing. We anticipate that these results will be submitted for publicatio in late 2011. One of our proposed objectives is to determine the effect of PGRs on acquisition of Candidatus Liberibacter asiaticus (Las) by the Asian Citrus Psyllid (ACP). Previously, we reported that a population of citrus trees was graft-inoculated with Las-infected budwood for use in experiments. We recently tested for the presence of Las in the grafted plants using quantitative polymerase chain reaction (qPCR) assays and determined that approximately 40% of grafted plants were infected with Las. Subsequently, we have conducted experiments to evaluate the effect the PGR prohexadione calcium on Las acquisition. Healthy and Las-infected plants were treated with prohexadione (sprayed until run-off) and allowed to dry. Twenty adult psyllids of mixed gender were released onto treated plants and enclosed in nylon mesh sleeve cages. As a control, twenty ACP were also released onto healthy and Las-infected plants treated with water + adjuvant. Each treatment was replicated 6 times on two separate dates. Plants and insects were maintained in a temperature-controlled environmental chamber for 28 or 14 days prior to collecting insects. Following each acquisition experiment, qPCR analysis of psyllids revealed that 54% and 51% of psyllids acquired Las from treated plants after 28 and 14 days, respectively, compared with 50% and 40% of psyllids feeding on untreated plants. Although these data suggest that prohexadione does not decrease acquisition, these results are preliminary and may indicate that the residual activity of prohexadione against psyllids does not extend to two weeks. These experiments, which evaluated acquisition after two weeks or one month of feeding, will be replicated two more times. In addition, subsequent experiments will be conducted to determine the rate of acquisition after 24 hours, one week, and during nymph development.
Under Objective 1 (define rates and formulations of copper sprays for more effective control) one trial with Red grapefruit was conducted in Ft. Pierce, FL and one trial with Hamlin orange in Hardee County, each with 14 treatments of formulations, rates and combinations with other chemical treatments of interest. This season, trials had low to moderate disease epidemics as a result of a periodically wet spring, moderately wet summer and an absence of tropical storm events. Evaluations of fruit infection in these trials were made in November 2010 (grapefruit) and January 2011 (Hamlin). Copper formulations containing copper hydroxide or basic copper sulfate (metallic rates of 0.67 to 1.12 kg/ha), varied from low (Hamlin) to moderately effective (grapefruit) for canker control of fruit disease incidence. Copper pentahydrate, at a lower metallic copper rate/ha per application, provided equivalent control to film-forming copper formulations in grapefruit but less control in Hamlin. Greater canker susceptibility of fruit occurred in later season and was likely because of prolonged opening of stomates in cooler weather and enhanced bacterial entry, coincident with more numerous windblown rain events. Although Hamlin fruit disease incidence was higher, copper protection against early season fruit infection was moderately effective for prevention of premature fruit drop. Under Objective 2 (establish the period of fruit susceptibility, residual activity and phytotoxicity of copper). In 2010, we compared the copper fruit residues from different copper-containing products up to 28 days after application to grapefruit. The copper product used and the number of days after application significantly affected the amount of copper residue. These effects were consistent whether the data were analyzed as copper/fruit or copper/surface area. The rate of copper residue decrease was different for the various products tested. Copper products forming films, Kocide 3000 (copper hydroxide), Cuprofix Ultra 40 Disperss (copper sulfate), and Badge X2 (copper hydroxide and oxycloride) decreased at a similar rate, whereas the soluble formulation Magna-Bon CS2005 (copper sulfate pentahydrate) decreased at a higher rate and left about 75% less fruit residue. This was expected as Magna-Bon is applied at approximately 15% of the metallic contained in film-forming coppers. Since Magna-Bon performed as well as film-forming copper products for control of canker on grapefruit the last two seasons, we have hypothesized that the Magna-Bon copper could be locally systemic in fruit rind tissues. This coming season, we will attempt to assay the activity of copper in the rind against Xcc to determine whether the copper is located internally as well as externally. Under Objective 3 ( evaluate the use of streptomycin [Firewall]) As in the past three seasons, applications of Firewall in July and August, were effective for canker control on grapefruit either in combination with a reduced rate of copper or when substituted for copper in the spray program. Based on Firewall’s efficacy and ability to lower the risk of copper phytotoxicity, an application has been submitted by FFVA and FDACS to gain EPA Section 18 emergency registration for use of Firewall against canker on fresh grapefruit. Under Objective 4 (To define risk for development of bacterial resistance to copper and streptomycin in FL citrus groves) a number of factors favorable for the development of copper resistance in Xcc were identified. Findings are being prepared for publication. Under Objective 5 (rapid transfer improved canker management technology to the Florida citrus industry), 2011 canker management recommendations have been submitted for publication in the Florida Citrus Pest Management Guide and to Citrus Industry Magazine. Oral presentations have been delivered at the Florida Citrus Show and at a multi-county extension meeting. Results of the Hamlin trials will be presented at the 2011 FSHS annual meeting.
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). Trees were 8 years old at the initiation of the trial. 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. Five disease ratings have been taken so far with the latest in June 2012 and a slight decline in tree health has been observed, but no significant treatment effects have been detected. The 2011 harvest increased 27% yield in all treatments compared with the 2010 harvest, but no significant treatment effects were observed. Objective 2 is to determine the mechanism of HLB symptom suppression by foliar nutritional application, Rep 1 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 and results are in previous reports. Rep 2 monthly samplings of leaf and root tissue are continuing. Root samples are split for qPCR Las quantification and starch analysis for a quantitative measurement of phloem function throughout the plant. Microscopy will be continued, however the high variability of phloem plugging and collapse even within the same midrib from a symptomatic leaf makes interpretation of results difficult. 72 of the 75 inoculated trees are now positive with disease symptoms developing. No treatment effects have been observed, except that the phosphite alone treatment results in higher than expected infection of roots within 4 months of inoculation which continues to occur at 6 months post inoculation. Now that symptoms have developed and consistently positive tissue has been identified, samples have been collected for microscopy of leaves and roots. The samples have been fixed and sectioning of samples for microscopy is underway. Some trees have begun to show significant signs of decline, but it is too early to tell if decline is more common in a specific treatment. No significant differences in quantity of flush between treatments or Las titer has been identified other than the initial infection of roots mentioned above.
Exp.1 ‘ Test plant from the first and second trials are being observed for the presence of new symptomatic trees and leaf sampling of suspected plants are being PCR tested. We are waiting for laboratory results. Exp. 2- Emerging ACP adults from nymphs reared on inoculum sources plants were used for inoculation in Mar/10. Until now no symptomatic test plant was observed. Exp. 3 ‘ New inoculation of Las in three shoots of each younger trees of each of three sweet orange varieties on screenhouses was done in July/11. The two older class of plants are being prunned to be inoculated in November/11. Some symptomatic trees that appeared before the inoculation were maintained in the screenhouse and the severity progress was assessed during last year project. For Hamlin/Rangpur 99, the annual severity increased from 24.3 to 27.5% (average of 7 trees); for Valencia/Rangpur 99, from 24.5 to 38.5% (average of 5 trees); and for Hamlin/Rangpur 04, from 35.0 to 57.5% (average of 2 trees). As an alternative for this experiment, 30 trees from each five citrus blocks at different scion/rootstock combinations (Hamlin/Swingle 07, Valencia/Volkamer 07, Valencia/Swingle 07, Valencia/Swingle 99, Valencia/Swingle 95), showing very low symptoms severity, were selected in Jun/10 in farms without removal of symptomatic trees but with strong ACP control program. After 400 days, the disease severity increased in average from 6.2 to 9.7% for Val/Sw 95, from 4.6 to 10.3% for Val/Sw 99, from 7.9 to 14.5% for Val/Sw 07, from 8.6 to 23.5% for Val/Volk 07, and from 10.8 to 9.3% for Ham/Sw 07. For all plots the disease severity increased except for Ham/Sw 07. In that plot many selected trees did not show visual symptoms at last assessment (the presence of Las in those asymptomatic trees will be checked). Also it was observed that during drought season, because of water stress and defoliation, the disease severity assement is more complicated and difficult, usually resulting in lower severity. All assessed tree of Val/Sw 95, Ham/Sw 07 and Val/Volk 07 were harvested in July/11 and the average yield were compared with average yield of healthy trees from the same plot. Yield reduction (%) was higher for diseased trees of younger plots Val/Volk 07 (31.6%) and Ham/Sw 07 (35.2%) than older plot Val/Sw 95 (16.5%). In the plot Val/Sw 99, for a mistake the healthy trees were pruned and the selected trees were not. The plot Val/Sw 07 was not harvested yet.
Leprosis infected samples were recently sent to quarantine facilities at the USDA, APHIS, PPQ, CPHST, Beltsville, MD in order to check on the shipment and the viability of the samples for the mite transmission work to begin as soon as the funding is in place. The materials were successfully shipped from Mr. Guillermo Leon of COPIACA, our cooperator in Colombia. Both protein and nucleic acid extractions were done and PCR verified the presence of the virus in the samples. Dr. Jorge Pena, University of Florida, Homestead, FL has begun preparing colonies of endemic Brevipalpus mites for transmission experiments. Mr.Leon in Colombia already has Brevipalpus colonies and will begin transmission experiments there as soon as funding is in place for him to be able to pretest samples for virus. Drs. Schneider and Damsteegt have prepared space for the transmission work at the USDA, ARS, FDWSRU in Ft. Detrick and as soon as we are able to purchase the isolation chamber for the transmission work we will begin the first transmission experiments.
Under Objective 1 (define rates and formulations of copper sprays for more effective control) and Objective 2 (establish the period of fruit susceptibility, residual activity and phytotoxicity of copper) a three year trial with Hamlin orange in Hardee County, with treatments of formulations, rates and number of sprays was presented at the 2011 FSHS annual meeting and submitted for publication in the proceedings. The results are summarized as follows: The objective was to evaluate copper formulations for control of fruit infection and drop in 6- to 8 year-old ‘Hamlin’ trees. Copper sprays were applied at 21-day intervals after fruit reached 0.5 to 1.0 cm dia. The period of susceptibility of fruit to canker infection and fruit drop was established by increasing the number of applications through the fruit growth period. Separate treatments ended at each 21-day interval so that there were four to seven applications per season. In 2008, early season infection occurred during rains before copper sprays commenced in late April. Subsequently, five sprays of copper formulations at rates exceeding 0.5 kg metallic copper/ha significantly reduced incidence of lesions on fruit. Fruit disease and cumulative fruit drop were co-ranked among copper treatments (r = 0.83). Although a tropical storm in early August promoted disease on fruit late in the season, most of the premature drop was due to infection occurring before July. In 2008 and 2009, sprays after the period of early fruit susceptibility did not further reduce canker incidence or fruit drop. In 2009, copper sprays were initiated before significant spring rainfall and the incidence of fruit disease and fruit drop were lower and less well-correlated among copper treatments (r = 0.57) compared to 2008. In 2010, disease on fruit and premature drop were not significantly different from the untreated checks. Overall, there was little difference in efficacy among copper formulations, although there was reduced control using copper sulfate pentahydrate at lower rates of metallic copper. In each season, copper treatments controlled fruit drop by ~50% compared to the untreated check, however as ‘Hamlin’ trees grew from 6 to 8 years of age, canker incidence dropped due to the development of hedgerows which reduced windblown rain penetration into the grove. Hence, fewer copper sprays will be necessary after canopy closure promotes an internal windbreak effect. Under Objective 4 (To define risk for development of bacterial resistance to copper and streptomycin in FL citrus groves) the results were submitted and accepted for publication in European Journal of Plant Pathology. The results are briefly summarized as follows: Streptomycin was tested as an alternative to copper bactericides, which are routinely used for the control of citrus canker where the disease is endemic. A major concern is that excessive use of copper as a bactericide may lead to development of copper-resistant strains of Xcc. In this study, a semi-selective medium was developed to recover copper or streptomycin-resistant strains of Xcc from citrus leaves. The newly developed semi-selective medium was used to monitor the effect of a 21-day-interval copper or streptomycin spray program on Xcc for three consecutive seasons and on citrus epiphytic bacterial populations for two seasons in a commercial grapefruit grove. Although, no copper- or streptomycin-resistant strains of Xcc were isolated after three seasons, a significant increase over time was observed in the frequency of citrus epiphytic bacteria resistant to these chemicals. Under Objective 5 (rapid transfer improved canker management technology to the Florida citrus industry), 2011 canker management recommendations were published in the Florida Citrus Pest Management Guide and Citrus Industry Magazine. Oral presentations have been delivered at the Florida Citrus Show and at a multi-county extension meeting.
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