Improved method of Salivary sheath isolation was developed and used to show that the primary component of the sheaths produced from adult psyllids feeding on an artificial diet is carbohydrate. A postdoctoral research associate was recruited and identified during this time as was technical staff to support maintenance of Asian citrus psyllids. Greenhouse facilities were remodeled to provide room for increased psyllid production needed for this research and plant material was obtained to be used as host for psyllid colonies. Meeting with Torrey Pines Institute for molecular studies were held and experimental procedures were developed for peptide library screening.
Protection of ‘Hamlin’ orange fruit from infection by Xanthomonas citri subsp. citri, the cause of citrus canker, is necessary to reduce premature fruit drop. 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 (0.25 to 0.50 inches) diameter. 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/ha (1.1 lb/acre) metallic copper significantly reduced incidence of lesions on fruit. Early season fruit disease and cumulative fruit drop were highly correlated among copper treatments (r = 0.95). Although a tropical storm in early August promoted disease on fruit late in the season, late season fruit disease and fruit drop were less well correlated (r = 0.78). In 2008 and 2009, additional 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 the correlation of early season fruit disease was less positively correlated with fruit drop (r = 0.52) compared to 2008. In 2010, disease on fruit and premature drop were not significantly different from the untreated checks although fruit disease and early season infection were still significantly correlated (r = 0.70). Overall, there was little difference in efficacy among copper formulations, although control was reduced for treatments with 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.
The intent of this study is to examine the effect of windbreaks, copper sprays to reduce infection, and leafminer treatments to determine there individual and combined effects on control of citrus canker in Brazilian commercial citrus and the applicability of this strategy to the US commercial citrus industry. A preliminary study was published in Crop Protection 27:807-813, that indicated that copper and insecticide applications significantly reduced canker infection but windbreaks did not have any effect. As described previously, a new series of plots with much more extensive windbreaks were established via a USDA/ARS specific cooperative agreement with the University of Sao Paulo, and the Brazilian cooperator at an IAPAR farm, in Xambr’, Parana state, using, 2-yr-old P’ra on Rangpur lime. Windbreaks were completed and plants were be established in Mid April 2010, but severe winds damaged the windbreaks during two storm events. These windbreaks have been reinforced and rebuilt. This delayed the experiment which is now scheduled to begin in March 2012. The following treatments will then be applied: 1) no sprays (control), 2) Cu++ sprays to reduce citrus canker incidence, and 3) insecticide sprays to inhibit infestations of Asian leafminer (secondary effects). Main effects are windbreak versus no windbreaks. Citrus canker incidence will be estimated on multiple branches on each tree treated as the number of leaves per branch infected. We anticipate running these plots for 2-3 more years to collect all necessary data. The development of the Programmable leaf wetness controller (PLWC) software was written, debugged, is complete, and the control program is working well. New leaf wetness sensors were designed and constructed and calibrated and works well. An electronic glitch was determined in the leaf wetness sensors, new control circuitry was designed, constructed retested, and the glitch overcome. The newly designed probes with various sensor bridge materials are currently being tested. New windbreak trials are being established in Saint Lucie County. Bock, C. H., Graham, J. H., Gottwald, T. R., Cook, A. Z., and Parker, P. E. 2010. Wind speed effects on the quantity of Xanthomonas citri subsp. citri dispersed downwind from canopies of grapefruit tree infected with citrus canker. Plant Di Bock C.H., Graham, J.H., Gottwald, T.R., Cook, A.Z., and Parker, P.E. 2010. Wind speed and wind-associated leaf injury affect severity of citrus canker on Swingle citrumelo. Eur J. Plant Path 128:21-38 Bock, CH, Parker, PE, Cook, AZ, Graham, JH and Gottwald, TR. 2001. Infection and decontamination of citrus canker and inoculated the surfaces. Crop Protection 30:259-264. Hall, D.G., Gottwald, T.R. and C.H. Bock. 2010. Exacerbation of citrus canker by citrus leafminer Phyllocnistis citrella in Florida. Florida Entomologist. Florida Entomologist 93:558-566. Bock, C.H., Gottwald, T.R. and Parker, P.E. 2011. Distribution of canker lesions on the surface of diseased grapefruit. Plant Pathology (Accepted). Bock, C.H., Cook, A.Z., Parker, P.E., Gottwald, T.R., and Graham, J.H. 2011. Some characteristics of the dispersal plume of bacteria of Xanthomonas citri subsp citri in wind-driven splash downwind of canker-infected grapefruit tree canopies. (Plant Disease). Bock C.H., Graham J.H., Cook A. Z., Parker P.E., and Gottwald T.R.. 2012. Predisposition of citrus foliage to infection with Xanthomonas citri subsp. citri. (Submitted to Plant Disease )
The objectives of this study are: 1) to develop a series of flexible stochastic models to predict the temporal increase and spatial spread of citrus HLB and canker. They can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies both in plantations and State-wide. 2) Test various control methods under field conditions to evaluate effects and collect data to parameterize models. SEIDR model. Using Markov-chain Monte Carlo (MCMC) methods, and extensive data from infected areas of South Florida for successive snapshots of the occurrence of symptomatic detected trees in known populations of susceptible trees, we have been able to estimate the transmission rates and dispersal kernel for HLB. A working model has been developed that focuses on the differential effects of host age on epidemiological parameters as well as variability across the plantation and that allows for uncertainty in the parameters as well as variability over time and through space. Our main approach has been the use of Baysian MCMC methods to infer posterior densities on the model parameters. The uncertainty is then incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods. Recently we have developed a second approach known as Approximate Bayesian Computation (ABC) and have used canker and more challengingly HLB as a test bed. In comparing model outputs of the ABC versus the MCMC estimates we determine that the dispersal distances being considered needed to be lengthened to gain the most accurate estimation. This seemingly small difference may become apparent and important when we analyse the differences between control strategies. A web based version of the model (front end) is very far along and now I the final steps of validation testing. Both residential and commercial citrus scenarios are being tested with user selectable and changeable via sliding controls. (See pubs below). The front end allows visualization of the effect of these various control strategies allowing researchers, growers and regulators the ability to compare the results of thousands of simulations for practical management decision making and/or regulatory intervention/strategy building. Via this model we have been able to examine the effects of various controls such as using insecticide applications or not, removing infected trees or not, and the effect of HLB infection in young versus older trees. Model output confirms that controlling secondary infections by diseased tree removal and insecticide applications plus controlling primary infection from new insect immigrations by areawide control strategies, can reduce disease increase to a manageable 2 to 5% increase per year. Industry representatives had indicated that this level of diseases accommodates economically sustainable. This may be quite useful to manage Texas and California outbreaks. A recent trip to Cambridge has moved this front end along significantly and publication/distribution is anticipated soon. Publications: 163. Gottwald, T. R. 2010. Current Epidemiological Understanding of Citrus Huanglongbing. Annu. Rev. Phytopathol. 2010. 48:119’39. S. Parnell, T.R. Gottwald, W.R. Gilks, and F. van den Bosch. 2012. Estimating the incidence of an epidemic when it is first discovered and the design of early detection monitoring. Journal of Theoretical Biology 305: 30-36. M.F. Parry, G.J. Gibson, S. Parnell, T.R. Gottwald, M.S. Irey, T. Gast, and C.A.Gilligan. 2012. Spatiotemporal modelling of the spread of HLB/Citrus Greening on orchard scales. Epidemics (submitted).
The potato/tomato psyllid, Bactericerca cockerelli (B. cockerelli), is a very important plant pest and also vectors of phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum), which is associated with zebra chip disease of potatoes. The B. cockerelli – Ca. L. solanacearum interaction very much resembles that of the Asian citrus psyllid, Diaphorina citri and Ca. L. asiaticus ‘ the latter being the causal agent of HLB. Because the B. cockerelli – Ca. L. solanacearum complex is associated with herbaceous as opposed to woody plants for HLB, we used it as a more facile and simple model system to assess RNA interference potential as a potential strategy for assessing RNAi effects in psyllids. As B. cockerelli sequence information is still limited in accessibility, we took advantage of D. citri EST sequences to amplify homologous sequences from B. cockerelli. Twenty eight sequences were successfully cloned from B. cockerelli using this method. We used hemolymph injection and developed an artificial in vitro oral acquisition feeding assay to deliver RNAi effectors to psyllids. We targeted ubiquitously-expressed and gut-abundant mRNAs by using double-stranded RNAs and siRNAs and were able to induce mortality in recipient psyllids. We also showed knockdown of target mRNAs, and that oral acquisition resulted primarily in mRNA knockdown in the psyllid gut. Concurrent with gene knockdown was the accumulation of target specific ~ 21 nucleotide siRNAs for an abundant mRNA for BC-Actin thereby confirming that effects observed were due to RNAi activity. We also evaluated plant virus-based expression systems as a strategy for more rapid assessment of anti-psyllid RNAi effects after feeding on plants. We showed knockdown of target BC-ATPase mRNAs by using a Tobacco mosaic virus (TMV) vector, but not when using the Tobacco rattle virus (TRV) or Potato virus X (PVX) vectors. We compared using tomato, tomatillo and tobacco plants, and found that like for in vitro feeding, the RNAi effects primarily were observed in the psyllid gut. We also evaluated a leaf disc feeding assay using psyllid nymphs, which resulted in most efficient silencing effects. Feeding psyllids and allowing them to reproduce on TMV-ATPase tomatillo plants also caused a decrease in number of psyllid progeny. These results showed that RNAi can be achieved by plant expression of silencing sequence in psyllids, and the TMV-plant system provided a faster and somewhat convenient method for screening of suitable psyllid RNAi target mRNAs. We have generated transgenic potato plants for hairpin expression of BC-Actin and BC-ATPase by pCB2004B gateway vector, and we are evaluating these plants for silencing effects on the psyllids which feed on the plants. These are 35S promoter based systems and we have now also made constructs using a phloem-specific promoter. In conclusion, we have clearly demonstrated RNAi effects in the tomato psyllid, and used different delivery approaches. We have identified several effective candidate sequences for RNA interference and are moving to evaluating these in transgenic plants.
The goal of this project was to provide a better understanding of the interaction between ACP and Las in order to develop more effective approaches for managing HLB spread. One objective that has been completed was to determine if any seasonality of disease spread occurs. Monthly sampling of psyllid populations (>100,000 ACP total) showed that there is indeed some seasonality to incidence of ACP carrying Las that is likely tied to the presence of new flush from which psyllid acquire the pathogen when feeding for extended durations as nymphs. However, our results suggest that once HLB incidence is high in an area (large number of infected trees) the likelihood of ACP moving the pathogen to nearby non-infected trees is greatly increased such that failure to control ACP at any time of the year will result in additional HLB spread. Two manuscripts relating to this objective are nearing completion for publication this year. A second objective was to examine the factors responsible for transmission of Las by ACP. The first part of this work has been published (Pelz-Stelinski et. al 2010). Briefly this paper demonstrated pathogen acquisition is greatest by nymphs, low rates of transovarial transmission can occur, and rate of successful inoculation of healthy plants with Las increases with increasing vector feeding. Additional studies have continued examining the latency period of Las in ACP nymphs and adults and we are currently analyzing data from this study. We also previously reported that ACP survival decreases when the insects are infected with Las. A series of experiments have been completed to assess the effect of plant infection status on the survival of adult ACP. In experiments to determine the effect of Las on ACP fitness, we have determined that nymphs reared on Las-infected plants develop faster than nymphs reared on healthy plants and that there are no differences in fertility among infected and healthy ACP. A manuscript describing the results of these experiments has been prepared and will be submitted in the next month. Experiments to assess ACP suitability as a host for Las by examining the effect of endosymbionts on ACP survival and pathogen transmission were initiated. Breeding lines of ACP with and without Wolbachia, are being reared to determine whether there is a genetic basis for vector efficiency. Currently, we are testing ACP to confirm endosymbiont infection status in individual ACP lines prior to moving forward with transmission experiments. We continue to explore removal of all endosymbionts from ACP via microinjections of antibiotics, but have had only limited success with a small number of insects using tetracyline. In depth studies of ACP feeding behavior were conducted using an EPG monitor with the goal of developing a better understanding of the biology of psyllid feeding behavior and how insecticides can be used to disrupt pathogen transmission. Most the results from this objective have previously been reported on and have been published in a dissertation by Serikawa 2011. The first of seven manuscripts from this dissertation on the effects of imidacloprid on psyllid feeding behavior has been accepted for publication with additional publications forthcoming.
The overall goal of this two year project was to gain a better understanding of how we can use the tools currently available to protect young trees from becoming HLB infected prior to reaching bearing age. First, in addition to soil-applied imidacloprid, we also demonstrated that thiamethoxam and clothianidin provide the same level of psyllid feeding disruption (prevent HLB transmission). In the field, we examined the duration of control provided by these products when applied as a soil drench versus trunk applications. The initial rationale for testing trunk applications was based on reports from South Africa claiming 6 months control of Trioza erytreae using trunk applications of Confidor (imidacloprid). In our trials using the same product and per tree rate used in SA, we did not observe such a long duration of control, in fact, trunk applications using our labeled products worked better than the Confidor product. Since this initial trial, we conducted three additional trials comparing varying rates of soil vs trunk applications of imidacloprid, thiamethoxam and clothianidin. Results demonstrate equivalent (in some instances slightly better) control provided by the trunk applications. After three successive trunk treatments to the same trees, thus far, no phytotoxicity has been observed. Leaf samples are still being analyzed from these experiments to compare residue levels with observed control in the field. In multi-year trial initiated as part of this grant, we established a 10-acre block of Valencia orange and overlayed different season-long programs to determine which approach would provide the best protection from HLB from the time of planting to bearing age. The treatments compared include use of soil-applied systemics, foliar insecticides and psyllid deterrent compounds. This planting has only been underway 11 months at this point so no conclusions can be made thus far. As part of CRDF funding obtained for the next 3 years, we will be continuing this work which includes assessing psyllid and leafminer populations in these plots, pesticide residue levels, and PCR of trees to compare HLB infection rates between treatments. In laboratory studies, we have demonstrated in choice tests that the deterrent compound reduces psyllid infestation of treated plants by at least 6 fold. Additionally, EPG analysis of psyllid feeding behavior has demonstrated significant feeding disruption resulting in total absence of any phloem feeding behaviors. Our work provided evidence for the likely mechanism for this feeding disruption. This work with additional new objectives will be continued in the subsequent grant funding. We also initiated a multi-year field experiment designed to test whether there is any benefit to continuing to control psyllids on trees that are already HLB infected. Healthy field grown (caged) trees were infested with Las (+) or (-) psyllids. With some plot receiving a one time infestation and others repeated infestation, we will be continuing to evaluate tree decline as it relates to number of HLB inoculation events, thus supporting or refuting the idea that psyllid control will slow the decline of already infected trees. This objective will continue over the next 3 years in subsequent funding. A multi year trial examining the effects of climate (time of year) on the efficacy of pesticide applications was initiated. Results thus far have shown significant differences in duration of psyllid control based on time of year, but little effect of climate in terms of one product performing better than another at differing times of the year. Additional work on this objective will also be continued in the coming year.
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, and field trials conducted in 2011, bark applications of bactericides with or without Pentra-bark were ineffective for uptake of the copper in the formulation and prevention of HLB infection in a field trial of nursery trees in Picos farm with very heavy infectious psyllid pressure. In contrast, preliminary trials conducted with potted nursery and young field trees indicate that soil drench is an effective and consistent application method for increasing copper status of leaves up to several fold over the baseline concentration in leaves. Hence, the trial in Picos Farm, was replanted with Hamlin on Swingle citrumelo trees that were root drenched in late spring with the copper bactericides EXP, Cop-R-Quik, (CQ) Magna-Bon (MB), and copper phosphite (CP) and a zinc phosphite (ZP). Spring flush leaves were collected in July to measure copper and zinc concentration in leaves to test for systemic movement of the metals. Leaf samples for PCR testing were also collected to track Las infection progress. To further evaluate soil drenches of the two commercial bactericides containing chelated copper (CQ and MB) three locations were selected. Two trials in blocks with 1 year old trees that are apparently healthy and a third trial in a block of 4-yr old trees with symptomatic and pre-symptomatic HLB infection as determined by PCR. Hence, the preventative and curative activity of the copper bactericides will be followed pre- and early stage infected trees.
For grapefruit, the objective was to evaluate formulations, rates, duration of residues for copper materials, combinations of copper materials and streptomycin, and application timing for effective canker control on young, fruiting grapefruit trees. Copper applications were made at 21-d intervals starting at spring flush in March until fruit were fully expanded in October. The period of fruit susceptibility to canker was established by applying sprays at 21-d intervals through the fruit growth period, with separate treatments ending spray application at each interval so that duration of protection varied and number of applications ranged from 3 to 11. Copper formulations containing copper hydroxide or basic copper sulfate (metallic rates of 0.67 to 1.12 kg/ha), varied from low to moderately effective for canker control depending on late-season, wind-blown rains. Copper sulfate pentahydrate (Magna-Bon), at a lower metallic copper rate/ha per application, provided equivalent control to other copper formulations. Streptomycin (FireWall, Agrosource, Inc.), alone or with a reduced rate of Kocide 3000, in July and early August gave equivalent control to Kocide 3000 alone. Copper phytotoxicity risk coincided with accelerated fruit growth after summer rains. Time was the most important factor affecting reduction in copper residue per fruit surface area. Th adjuvant Polymer Delivery System (PDS) had no effect on the residue. The objective for a three year trial with Hamlin orange in Hardee County, was to test formulations, rates and number of sprays for control of fruit infection and drop in 6- to 8 year-old ‘Hamlin’ trees. Copper sprays were applied at 21-d intervals after fruit reached 0.5 to 1.0 cm dia. 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 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 season fruit susceptibility did not further reduce canker incidence or fruit drop. In 2009 and 2010, 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). 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 copper sulfate pentahydrate at lower rates of metallic copper provided less control. 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. A major concern is that excessive use of copper as a bactericide may lead to development of copper-resistant strains of Xcc. Streptomycin was tested as an alternative to copper bactericides. A semi-selective medium was developed 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. No copper- or streptomycin-resistant strains of Xcc were isolated after three seasons, however, the frequency of citrus epiphytic bacteria resistant to these chemicals increased. Improved canker management technology was transferred via updates of the recommendations in the Florida Citrus Pest Management Guide, reports in Citrus Industry magazine and annual presentations at the Florida Citrus Show and multi-county extension meetings.
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 (MB) (Copper sulfate pentahydrate), Cop-R-Quik (copper nitrate), a copper phosphite (CP), an experimental copper (EXP) or oxytetracycline (OTC) mixed with 0.1% Pentra-Bark or left non-treated. After for 2-3 weeks, the leaves were observed for phytotoxicity. No phytotoxicity was observed and systemic bactericidal activity in the plants was assayed with detached immature leaves inoculated with Xanthomonas citri subsp. citri (Xcc) as a Gram negative bacterial surrogate for non-culturable Canidatus Liberibacter asiaticus (Las). The canker disease control effect was measured as the number of the canker lesions in a detached leaf in vitro assay. Canker lesion no. per leaf was reduced for trees treated with EXP, but not OTC, CP or MB. When the canker assay was repeated on a second set of plants treated with EXP or MB and compared to another soluble copper Cop-R-Quik, EXP did not show canker control activity but Cop-R-Quik reduced lesions in the assay. Objective 2: In July 2011, four of the bactericides OTC, CP, MB and EXP were evaluated in a field trial by painting 1-2 yr-old nursery trees with a mixed with 0.1% Pentrabark. This planting, located at USDA-ARS Picos Farm in Ft. Pierce is highly exposed to infectious psyllids based on a previous study evaluating SARs located there (NAS-86). The trees were assayed for HLB status in January 2012 and mean Ct values for all treatments were between 22 and 23, i.e. all trees were highly infected with Las. Preliminary trials conducted with potted nursery and young filed trees indicate that soil drench is a more effective and consistent application method for increasing copper status of leaves than trunk painting. Henceforth at Picos Farm, replanted Hamlin on Swingle citrumelo trees will be root drenched. The bactericides chosen for further evaluation include EXP, Cop-R-Quik, MB, and a novel copper phosphite (CP) and zinc phosphite (ZP). These formulations will be applied as a root drench in the same volume of water used for application of neo-nicotinoid insecticides (250 ml). Terminal leaves will be collected periodically to measure copper or zinc concentration to test for systemic movement of the metals. Leaf samples for PCR testing will be collected at 3 month intervals to track Las infection progress.
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. Four 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. The 2011 harvest experienced a 27% yield increase 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. 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 in tree health across treatments. After pruning trees at 6 MPI for canopy management, sampling at 7 months post inoculation (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 has been inoculated and monthly samplings of leaf and root tissue are underway. Root samples are split for qPCR Las quantification and starch analysis for a quantitative measure 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. 67 of the 75 inoculated trees are now positive with disease symptoms developing. No treatment effects have been observed, except that phosphite alone results in higher than expected infection of roots within 4 months of inoculation, and the effect is still present at 6 MPI. Now that leaf symptoms have developed and roots are consistently positive, sampling for microscopy will begin at the end of this month.
Under Objective 1 and 2: Soil applications of SAR inducers at various rates and application frequencies were evaluated for control of canker in field trial of 3- or 4-yr old ‘Ray Ruby’ grapefruit trees in southeastern Florida. Reduction of foliar incidence of canker produced by one, two or four soil applications of the neonicotinoids, IMID and thiamethoxam (THIA), or acibenzolar-S-methyl (ASM, Actigard) was compared with 11 foliar sprays of copper hydroxide and streptomycin applied at 21-day intervals. In 2011 crop season, all treatments significantly reduced the incidence of foliar canker on the combined Spring-Summer-Fall flushes compared to the untreated control, depending on rate, frequency and timing of application on young grapefruit trees under canker epidemic conditions. Soil application of systemic neo-nicotinoid insecticides has also been demonstrated to induce SAR and provide canker control for bearing trees but use of higher rates of these systemic insecticides for young fruiting trees is currently restricted. A preliminary trial in 2011 with 5 yr-old fruiting grapefruit trees trees demonstrated the efficacy of trunk application for IMID, THIA and ASM at 3.5X the label rate per season to compensate for the larger tree volume. Trunk application was as effective for canker control on foliage as soil application and control matched that of 11 sprays of copper. The objective for current field research is to develop more effective suppression of fruit infection using trunk applications of neonicotinoids and non-insecticidal SAR inducers (e.g. ASM). . Under Objective 3, the integrated use of ASM, THIA and IMID soil applications was evaluated 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 was in the non-treated 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 (Belay) 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. In 2012 spring, field trials the rates and and timing of soil drenches and trunk treatment with neonicotinoids are congruent with recommendations for use of neonicotinods for systemic control of psyllids . Novel non-neonicotinoid insecticides and fungicides with demonstrated systemic activity against canker in greenhouse trials are under evaluation in addition to ASM for integration with neonicotinoids and foliar copper sprays to optimize canker control on non-bearing and young bearing trees.
This report is for July/2011. 1) Evaluation of screens impregnated with insecticide barriers. As explained in previous reports, the experiment is being conducted in two farms located in the Sao Paulo State. On the farm located in Sao Manuel 14 evaluations were performed. In the area with screen impregnated with insecticide barriers 2 insects were collected, and in the area without screen nothing was collected. In Descalvado 12 evaluations were performed, in a total of 2 and 1 psyllids collected in areas with and without barrier, respectively.
1) Evaluation of screens impregnated with insecticide barriers. This experiment has already been completed (see annual report sent on 2/2/12). 2) Evaluate the impact of treatment of plants with systemic insecticides on the transmission of Ca. L. asiaticus by starved psyllids. The transmission trials have already been completed. The test plants are being evaluated by PCR. 3) Determination of the concentration of pesticides present in the sap of the xylem and phloem of citrus plants and the lethal concentration to D. citri This experiment is delayed because the equipment for the extraction of plant sap is being imported. The device has already been bought, however the manufacturer has not delivered it yet. The experimental area has been selected and the experiment must be started in September (after Brazilian winter).
Exp.1 ‘ All test plants from the first trial were collected and submitted to real time PCR for the last time in January/2012. In the compartments where citrus plant were used as inoculum source of Las and Lam, the test plants were naturally more infected with Las than with Lam (0 and 3.8% of test plants infected with Lam and 9.6 and 19.2% of tested plants infected with Las). In the compartment with Murraya as source of inoculum of Lam, 8.2% of test plants got infected. The second trial is ongoing but there is no result yet. Also we had an infestation of Tamarixia radiata in our ACP breeding plants that completely devastated it and postponed the new releases of ACP into the compartments. We needed to restart our ACP HLB-free breeding. Exp. 2- Acquisition Period for Las and Lam. Different acquisition periods (AP) (30 min, 1h, 3h, 6h, 12h, 24h, 3 days, 7 days, 10 days and 14 days were tested. After these APs in sweet orange trees as source of inoculum, adults ACP were transferred to Rangpur lime seedlings for 21 days and after tested by PCR for Las and Lam. Both Lam and Las were detected by PCR in adult ACP after 1 hour AP (6.6%). For Las the efficiency of acquisition increased with the AP up to 7 days (16.6%) and decreased to 3.3% with 14 days AP. For Lam the efficiency of acquisition increased to 30% with 1 days AP and decreased after that to 3.3%. Inoculation Period for Las and Lam. Different inoculation periods (IP) were tested (30 min, 1h, 3h, 6h, 12h, 24h, 3 days, 7 days, 10 days and 14 days). Adults ACP had 21 days of AP in sweet orange as source of Las and Lam. After, they were put on test plants for different IPs. After 6 and 12 months, shoot samples were analyzed by PCR and all samples were negative for both Las and Lam. Exp. 3 ‘ Regardless the inoculation of trees at the screenhouses, some encaged trees showed symptoms just after building the screenhouses in December 2008 (probably they were already infected before be protected) and the severity progress of symptoms were assessed in those trees. About three year after that the average severity was 46.2 and 63.7%, respectively % for Hamlin/Rangpur lime planted in 1999 (6 trees) and planted in 2004 (2 trees). For Valencia/Rangpur lime planted in 1999 (5trees), the severity was 63.7% and for Pera/Rangpur lime planted in 2005 (1 tree) it was 37.5%. In the alternative experiment, comparing the disease severity on plants of Val’ncia/Swingle from January/March 2011 to January March 2012, the youngest trees (planted in 2007) showed the highest initial severity and also the highest final severity than older trees planted in 1999 and 1995. Comparing Valencia sweet orange at the same age but in different rootstocks, the severity increasing at the same period in trees grafted in Volkamerian lemon were double than in trees grafted in Swingle citrumelo. Comparing different scion varieties grafted on Swingle and at the same age, the severity increase were higher in Hamlin than in Valencia.
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