In our previous reports, we provided data showing RNA interference effects in potato/tomato psyllids (B. cockerelli) via standard injection and oral feeding of both dsRNAs and siRNAs. Homologs of actin, ATPase, Hsp70 and CLIC were shown to cause substantial psyllid mortality as compared with GFP dsRNA controls in oral feeding assays. The results led to a manuscript which is now under revision in PLoS ONE. Now we are concentrating our efforts on evaluating anti-psyllid RNAi effects in plants. In the long term this will include transgenic plants, but we are investigating using recombinant plant virus-based expression systems as tools for rapid evaluation. We have now evaluated three different virus vectors and five different host plants. These include Tobacco mosaic virus (TMV), Tobacco rattle virus (TRV) and Potato virus X (PVX) in different host species including tomatoes, tobacco, Datura stramonium, tomatillo and Nicotiana clevelandii. These plants were chosen as they are good hosts for the psyllid, but show differential virus effects. TMV systemic infections in tomatillo, tobacco and N. clevelandii develop very rapidly, within one week, and in tomato after 2-3 weeks. PVX gave consistent but weaker expression in tomatillo, tobacco, D. stramonium, and N. clevelandii, but not in tomatoes. TRV showed weak expression in tobacco and tomatillo, but not in and D. stramonium, N. clevelandii or tomatoes. Tomatillo grows very rapidly, is the good host for B. cockerelli, and systemic expression of TMV gave the highest expression for insert sequences. So, we focused on using the TMV-tomatillo-psyllid system. We used different feeding protocols, including whole plants, leaf cages and stem feeding. For RNA interference effects, knockdown of target mRNAs in dissected gut is more consistent than when using the whole insect samples, as we also observed after in vitro dsRNA oral feeding experiments. Reduced nymph numbers were observed in some experiments suggesting evidence for RNAi activity. We are also attempting to use a leaf-agar system, in which only the infiltrated leaf sections from test plants are transferred to agar plates for psyllid feeding experiments. In summary, our results so far show the possibility of inducing RNAi effects via plant virus expression systems, which could provide practical means to rapidly screen secquences for anti-psyllid RNAi, or could even be used in field conditins.
Our research included both laboratory and field experiments. In the laboratory, we used a controlled droplet generator to investigate the effect of droplet size on the mortality of Asian citrus psyllid (ACP). The technique involved spraying various commercially available formulations (e.g., Danitol, Dibrom, Lorsban) at different droplet sizes on psyllid infested potted trees and counting the number of ACP eggs, nymphs, and adults before spraying and at 3 days and 7 days posttreatment. The results showed that the smaller the droplet size, the greater is the mortality of all life stages of the ACP. For all chemistries, the results suggested up to 80% control of nymphs using sprays with droplet VMD of 40’100 ‘m. Danitol caused greatest toxicity to nymph stage. At day seven, naled was significantly more toxic than chlorpyrifos to eggs and nymphs at all droplet sizes tested. However, some formulations involving suspended particles could not be applied readily with our laboratory apparatus. In the field, we continued the low volume spray trials with insecticides in different chemical classes. The insecticides included insect growth regulators, organophophates, pyrethroids, neonicotinoids and one microbial insecticide. They were sprayed with at the recommended label rates or an experimental rate at spray volumes of ~5 gal/acre. Ambient weather conditions were recorded during applications and up to 28 days posttreatment. The temperature (19.5-38.2 ‘C), relative humidity (68%-95%), mean wind speed at application (0.9 -5.1 mph; 0.4-2.3 m/sec), total solar radiation (203-251 W/m2), rainfall up to day three (0.02″-2.3″; 0.5 mm-58.7 mm) and total rainfall at 28 days (5.0″-11.4″; 128 -290 mm) were measured. From the relative humidity data the delta-T statistic was calculated. The value of delta-T allows for the estimation of the evaporation time of an insecticide droplet and may have a significant impact for low volume applications where the droplet sizes are much smaller than high volume applications. Ideally, the value of delta-T should be between 2-8. Values outside this range indicate marginal spraying conditions and higher than 10 indicate conditions unsuitable for spraying. In our studies, the delta-T values during the applications ranged from 1.0 to1.8. Efficacy of each application was assessed by sampling populations of both adults and nymphs in treated plots compared with untreated control plots on days: three, seven, 14, 21 and 28 in a commercial citrus grove. Unbaited yellow sticky cards and tap counts onto white cards from multiple branches were used to determine the numbers of adults. Populations of nymphs were estimated by examination of individual flush terminals using a ranking scale. Regression analysis was used to determine if there was a relationship between the meteorological conditions measured during the trials and observed mortality of the psyllid nymphs and adults. The efficacy data for day three was correlated with the above canopy wind speed at application. There was a significant positive correlation, indicating that the most effective applications of residual insecticides (organophosphates and pyrethroids) occurred at lower wind speeds but not for insecticides from other classes. No correlation with the value of delta-T and efficacy was shown for all insecticides in all classes tested. The maximum daily temperature for the first three days was positively correlated with increasing efficacy for the residual insecticides tested but not for other classes. The cumulative solar radiation to day 21 post-treatment was negatively correlated with efficacy for the residual insecticides. There was no correlation between cumulative rainfall to day three and efficacy for all insecticides tested. It was unexpected that we found no relationship between rainfall and spray efficacy as other studies have indicated that post-application rainfall can significantly reduce the longevity of an insecticide application. Overall, the results showed that low volume application is effective for psyllid management in citrus.
Experiment 2 plots (within HLB-Wide-Area Management WAM) were harvested in August/11. The yield of this experimental area is still increasing year after year as expected for a 6 yr-old grove. At this harvest, plots without ACP control produced less (46.5 ton/ha) than plots with ACP control (57.2 t/ha for plots with insecticide spray every 28 days, and 58.3 t/ha for plots with insecticide sprays every 14 days). However this difference in yield in non-controlled ACP plots was not only due to the amount of HLB-eliminated trees in those plots, but because the amount of CVC-eliminated trees in those plots. The cumulative incidence of HLB in plots without insecticide was 7.3% and did not differ from 7.1% and 5.3% incidence in plots with insecticide sprays every 28 and 14 days, respectively. The cumulative incidence of CVC in plots without insecticide sprays was 4.8% and was significant higher than 0.6% and 0.5% incidence in plots with insecticide sprays every 28 and 14 days, respectively. The WAM of ACP has kept the ACP population density very low (almost zero) in the last two years even in the plots without ACP control, and the HLB progress rate still low (cumulative incidence from 6.3% in November/10 to 7.3% in October/11 for plots without ACP control; and from 4.5% to 5.3% for plots with ACP control). For Year 3, we will continue collecting data from Experiment 2 under regional management, analyzing the spatio-temporal disease and economics data from both experiments, and publishing the results on scientific journals.
PCR results during the past three years from the three sites where the treatments with the Boyd nutritional cocktail is being studied indicate that the incidence (number of trees infected) of HLB is increasing and that the amount of bacterial present is also increasing (lower ct values). The incidence of infected trees in the Hamlin orange trial at SWFREC has remained at 100% infection since the trial began in 2008. The incidence has increased in the two Valencia trials at grower sites from 79% to 100% and from 40% to 98%. The ct values in the Hamlin trial at SWFREC decreased from 28.01 to 24.99, and from 29.88 to 26.49 and 32.23 to 25.23 respectively, all statistically significant in the two Valencia trials in commercial groves. Yield per unit of tree volume continues to be treatments 2 (complete Boyd cocktail minus the SARs), treatment 9 (complete Boyd cocktail minus hydrogen peroxide, and treatment 1 (complete Boyd cocktail. These three treatments consistently are among the four highest yielding during the past three years. Treatments 4, 5, 6, and 8 which do not contain the foliar applied nutrients Mg, Mn, Zn, Mo, and B are among the lower yielding trees. We are well into the 4th year of a replicated experiment in a 12-acre experiment commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test individual contributions of micro-nutrients + systemic acquired resistance inducers, and insecticidal control on: (1) ACP , (2) on incidence and titer of Can. Libericacter asiaticus (CLas), and (3) fruit yield and quality. While we are continuing with the experiment, results from the first 3 years are being written up for publication. Since our last report we have sprayed insecticides twice in designated plots: Agriflex at a rate of 5oz/ac + 2gal/ac of 435 horticultural oil on 19 July, and Dimethoate 4E (16oz) + 1gal 435 oil on 12 September The nutritional (‘Maury Boyd’) mix was applied on 29 July. Psyllid populations have been significantly lower (P < 0.0001) on insecticide treated trees throughout the growing season (16 June (0.06, 0.44 +/- 0.05 SEM), 30 June (0.04, 0.47 +/- 0.05 SEM), 14 July (0.40, 3.35 +/- 0.29 SEM), 28 July (0.10, 0.62 +/- 0.06 SEM), 11 Aug (0.00, 1.11 +/- 0.08 SEM), 25 August (0.03, 0.87 +/- 0.07 SEM), 8 Sep (0.24, 1.43 +/- 0.12 SEM), 22 September (0.05, 0.80 +/- 0.07 SEM respectively). Tap and flush sampling have been modified to include more complete counts of nymphs (all stages) and eggs on flush. Results of PCR for resets showed 0 and 4 positive HLB trees for January and July assessments, respectively. We are collecting samples from mature and reset trees this month for HLB detection. Leaf samples were collected 20 September from each plot for macro/micro nutrient analysis. A bucket trap was dispensed in the grove (7 October) to identify all leafminer species attracted to the CLM lure, and a fall citrus leafminer damage assessment using a modified Horsfall Barratt scale is planned for later this month when a flushing period is present.
This project has 5 objectives: (1) evaluate efficiency of ACP control techniques in cooperation with growers, (2) develop efficient monitoring methods for ACP, (3) accelerate testing of new chemistries and techniques for ACP management, (4) evaluate the economic component of the comprehensive program, and (5) provide an information bridge between researchers, growers, and industry. The following is an update of ongoing field and laboratory experiments. Experiments are underway to 1) compare selection rates for resistance using mixture (AgriFlex) versus rotation of its two components, and 2) evaluate stability of insecticide resistance and number of generations required to return tolerant populations to susceptibility. Estimates of dose-response relationships of ACP adults to the two insecticides and pre-mix were as follows: commercial grade thiamethoxam (Actara 25WG, LC25 6.48, LC50 15.36) and abamectin (Agri-mek, LC25 1.16, LC50 2.79) and a pre-mix (Agri-flex, LC25 0.26, LC50 0.91). The plan is to evaluate rate of resistance induction in caged psyllids using the LC25 dose, using discriminating dose bioassays to monitor response to 5 treatments: the premix, rotation, each insecticide alone, and a check. Later, insecticides will be withdrawn and return to susceptibility evaluated. Citrus leafminer (CLM) incidence and damage has been increasing, possibly due to increased use of broad spectrum insecticides to control ACP. Canker is also on the rise and growers are demanding assistance with management of CLM, thought to exacerbate the disease. We tested the hypothesis that heavy damage from CLM typically seen in late spring and early summer could be mitigated by controlling the first generation of the year in the spring flush. Aerial applications have not been used often for CLM control, so we also initiated a trial in July with 2 treatments of Intrepid (ground vs. aerial) compared to Delegate applied by air and results are described in the report for project 210. Intrepid (aerial/ground) applications showed the least damage of leaves compared with Delegate or untreated. Finally, we are initiating a colony of CLM to rear the parasitoid Citrastichus phyllocnistoides and support a program for monitoring CLM susceptibility to key insecticides. We began monitoring CLM using pheromone traps dispensed in a natural preserve (Okaloacoochee Slough State Forest) at distances of 2.0mi, 3.0mi, and 4mi (3 traps at each distance) from citrus to determine the furthest distance traps will catch citrus leafminer. We have found what we believe to be 4 different species attracted to the pheromone lure. We are in the process of identifying all the leafminer species caught by CLM pheromone lures in groves and in the slough through use of bucket traps and plant samples. Representative moths will then be keyed to species by a taxonomist and subject to DNA bar coding by a molecular biologist. A botanist has been contacted to identify the host plants. We are collaborating with the DPI CHRP program to evaluate results of the cooperative (area wide) ACP management program, in SW Florida, now the Gulf CHMA which uses a sampling protocol of 50 taps/block. The objective is to provide growers with tabular or graphical representations of results from this area.
Low volume (LV) aerial and ground sprays have become an important method of application in Florida citrus. We continue our evaluations of LV application of 435 horticultural mineral oil (HMO) which has shown promising results the last 3 years. This report documents updated results from the fifth trial begun on February 2011 in a 10.9 acre plot of ‘Valencia’ orange in Lee County. The efficacy of low volume spray spray of 435 horticultural spray oil (HMO) with the Grower Standard and an Untreated control are being compared. This trial is the first with sufficiently high ACP populations to assess the ability of LV oil sprays to provide suppression. Five sprays of 435 oil (13, 29 Jul @ 4gal/ac; 30 Jun 2, 22 Sep @ 2gal/ac) have been completed since the last report. Plots designated for the Grower Standard received 20 oz of Dimethoate + 2gal 435 oil on 30 June and 4oz of Delegate + 4gal 435 oil on 29 July. Mean adult psyllid populations over the last 3 months were 0.35 +/- 0.037,0.19+/-0.024 and 0.031+/-0.009 for the Grower Standard, Oil, and Untreated Check respectively. These values were all statistically different. Thus, the low volume oil treatments are suppressing ACP but not as effectively as the Grower Standard. Canker has worsened in the grove, so we added citrus leafminer (CLM) assessments to determine if treatments are providing some control. Pheromone traps were placed in the grove on 24 May a second damage assessment of leaves using a modified Horsfall Barratt scale will be conducted in late Oct. CLM control requires precise application timing on emerging flush to be effective, so there is considerable interest in aerial application to manage this pest. Therefore, we evaluated ground and aerial applications of the insect growth regulator methoxyfenozide (Intrepid 2F), and a ground application of spinetoram (Delegate WG), an analog of spinosyn A which is derived from actinomycete bacteria. Both products are selective with low vertebrate toxicity and widely used for CLM control. Treatments were applied 7 July in an 94 acre block divided into 12 large plots using a replicated complete block design with 4 treatments and 3 replicates. Pheromone traps were used to monitor CLM populations within all plots before and after application. Intrepid 2F treatments, regardless of application method, showed significantly less damage than the Delegate treatment or the untreated check on 20 July. On 29 July, significantly less damage was observed in response to the Intrepid2F ground and Delegate treatments compared to the untreated check. An assessment 2 months later showed less CLM damage in the Intrepid ground and Delegate plots, with trap captures lowest for Intrepid ground applications. Therefore, significant control was obtained with the aerial application of Intrepid, but not as much or as enduring as with the ground application.
Two 3-year field experiments are being conducted in two commercial orange blocks in Hendry County (southwest Florida). One of the groves is planted with ‘Earlygold’ oranges and the other with ‘Valencia’ oranges. Average HLB incidence estimated in both groves at the beginning of the experiment based on PCR analysis of a random sample of 160 trees was 98% in ‘Earlygold’ and 42% in ‘Valencia’. Experimental design is randomized complete block with 4 replicates and 4 treatments: (1) No insecticide, (2) Calendar applications, in order to drive vector populations close to 0, (3) nominal threshold of 0.2 psyllids per tap, and (4) nominal threshold of 0.7 psyllids per tap. Calendar applications are being applied every month. The insecticides used for these applications in the last three months were abamectine (Agri-Mek SC) at 3.5 fl oz/ac and horticultural mineral oil 2% in July, malathion (Gowan Malathion 8F) at 2.5 pt/ac in August and fenpropathrin (Danitol 2.4 EC) in September at 16 fl oz/acre. No sprays were used for treatments (3) and (4) during this period because nominal thresholds were not reached. No ACP adults have been found in treatment (2) in both blocks along the summer. ACP levels in the other treatments were (0.02 ‘ 0.01, 0.03 ‘ 0.01 and 0.05 ‘ 0.01 ACP adults per tap in treatments (1), (3) and (4) respectively for the ‘Earlygold’ block, and 0.08 ‘ 0.01, 0.010 ‘ 0.004 and 0.04 ‘ 0.01 ACP adults per tap in treatments (1), (3) and (4) respectively for the ‘Valencia’ block). Significant differences among treatments in the ACP cumulative numbers since the beginning of the experiment are being kept in the ‘Earlygold’ block with the lowest ACP populations in treatment (2) (F = 7.21; df = 3, 15; P = 0.005). Due to the low ACP populations in the ‘Valencia’ block, no significant differences have been found yet in the ACP cumulative numbers among treatments (F = 0.71; df = 3, 15; P = 0.5618). To evaluate the incidence that ACP calendar sprays have on the beneficial arthropod fauna two new exclusion experiments on ACP infested flushes were conducted in July and August respectively. As in the first experiment performed last May, mortality due to predation on citrus cohorts was high in July, with no differences between treatments (79.40 ‘ 7.42% and 90.74 ‘ 2.6% in treatments (1) and (2) respectively). In the experiment conducted in August, mortality due to biotic factors was lower. Again, no differences between treatments were found (63.30 ‘ 5.52% and 57.69 ‘ 10.57% in treatments (1) and (2) respectively). On the other hand, a new study has been initiated to evaluate how ACP sprays affect to CLM natural enemies. In this study, during each major flushing period 40 flushes per ACP threshold treatment are selected and the predation and parasitism rates are evaluated. In the first experiment, that was conducted in June-July, predation rates were 94.2 ‘ 1.94%, 92.63 ‘ 3.27%, 91.03 ‘ 2.6287.47 ‘ 5.15% and 87.47 ‘ 5.15% in treatments (1), (2), (3) and (4) respectively. According to these results it doesn’t seem to be yet a negative effect of the ACP sprays on CLM predation. On the other hand, parasitism rates only accounted for 1% of the CLM cohort mortality. The incidence of the ACP sprays on phytoseids abundance is also being evaluated in the two blocks. Significant differences on their cumulative numbers are being found between the calendar spray treatment and the rest of the treatments (t = -2.71; df = 11; P = 0.0204 and t = -2.42; df = 11; P = 0.0338 for the ‘Early’ and ‘Valencia’ blocks respectively). These results would indicate that calendar applications may are having a negative effect on these predatory mites.
Work on this project was already in progress when funding was released in August 2011. Objectives include: 1) scale up production of Tamarixia radiata to levels that will allow better assessment of the potential impact an augmentation program may have on the ACP population and ultimately the spread of HLB, 2) use genetic techniques to identify parasitoids recovered from the field to demonstrate establishment and effectiveness of released strains, and 3) pass on rearing technology to both private and public sectors to encourage production and an adequate supply of these parasitoids into the future. The colony at SWFREC of the previously established strain of T. radiata produced 53,398 wasps this quarter, increasing the 2011 total to 191,570. These wasps were used to maintain the colony and release in treated and untreated plots of commercial experimental blocks in Collier, Lee and Hendry counties. This quarter, 37,300 wasps were released, increasing the number released in 2011 to 124,970. Field releases this quarter of 20,600, 1,700, and 600 wasps from Pakistan, South China and North Vietnam colonies established at DPI, Gainesville join a total of 58,700, 4,185, and 4,700, wasps released this year. Wasps from the Pakistani colony continue to be released at the SWFREC, while wasps from south China and north Vietnam were released in commercial groves in Glades and Charlotte Counties, the latter being organic. Incidence of parasitism based on adult emergence from feral nymphs in the release block at SWFREC (Collier county) averaged 49% (N=565) and 45% (N=93) in July and August, respectively, compared to non release blocks averaging 3% (N=565) and 25% (N=327) in July and August, respectively. Parasitism of feral nymphs collected in August from a release block of a neighboring commercial grove averaged 3% (N = 175). Parasitism of nymphs recovered on sentinel plants placed in the release blocks in August averaged 98% (N=102) at SWFREC and 40% (N=5) in the commercial grove. No nymphs were recovered on sentinel plants placed in the non release blocks at SWFREC but 98% (N=174) were parasitized on sentinel plants placed in the commercial grove, probably indicating movement from the release block. In two sets of sentinel plants placed in September 35-100% (N=31-12) were parasitized at SWFREC and 13-97% (N=13-36) were parasitized in the commercial grove in release blocks. In non release blocks 13-65% (N=23-52) were parasitized at SWFREC and 38-42% (N=89-141) at commercial grove. Feral nymphs were also collected during August and September from experimental groves in Lee and Hendry counties, but less than 1% parasitism was observed. In general feral populations of psyllids were very low in the commercial groves during the report period. The recovered parasitoids are preserved in 95% ETOH and sent to Dr. Evan Braswell of USDA APHIS, Edinburg, TX, to identify genetic origins of these parasitoids using molecular markers. We also initiated the biparental colony of Diaphoencyrtus aligarhensis from Pakistan for release and evaluation at SWFREC. Addionally, we also visited Orange Co. to discuss their production and release system and some collaborative experiments involving release and evaluation of Tamarixia radiata.
Work on this project was already in progress when funding was released in August 2011. Objectives include: 1) scale up production of Tamarixia radiata to levels that will allow better assessment of the potential impact an augmentation program may have on the ACP population and ultimately the spread of HLB, 2) use genetic techniques to identify parasitoids recovered from the field to demonstrate establishment and effectiveness of released strains, and 3) pass on rearing technology to both private and public sectors to encourage production and an adequate supply of these parasitoids into the future. The colony at SWFREC of the previously established strain of T. radiata produced 53,398 wasps this quarter, increasing the 2011 total to 191,570. These wasps were used to maintain the colony and release in treated and untreated plots of commercial experimental blocks in Collier, Lee and Hendry counties. This quarter, 37,300 wasps were released, increasing the number released in 2011 to 124,970. Field releases this quarter of 20,600, 1,700, and 600 wasps from Pakistan, South China and North Vietnam colonies established at DPI, Gainesville join a total of 58,700, 4,185, and 4,700, wasps released this year. Wasps from the Pakistani colony continue to be released at the SWFREC, while wasps from south China and north Vietnam were released in commercial groves in Glades and Charlotte Counties, the latter being organic. Incidence of parasitism based on adult emergence from feral nymphs in the release block at SWFREC (Collier county) averaged 49% (N=565) and 45% (N=93) in July and August, respectively, compared to non release blocks averaging 3% (N=565) and 25% (N=327) in July and August, respectively. Parasitism of feral nymphs collected in August from a release block of a neighboring commercial grove averaged 3% (N = 175). Parasitism of nymphs recovered on sentinel plants placed in the release blocks in August averaged 98% (N=102) at SWFREC and 40% (N=5) in the commercial grove. No nymphs were recovered on sentinel plants placed in the non release blocks at SWFREC but 98% (N=174) were parasitized on sentinel plants placed in the commercial grove, probably indicating movement from the release block. In two sets of sentinel plants placed in September 35-100% (N=31-12) were parasitized at SWFREC and 13-97% (N=13-36) were parasitized in the commercial grove in release blocks. In non release blocks 13-65% (N=23-52) were parasitized at SWFREC and 38-42% (N=89-141) at commercial grove. Feral nymphs were also collected during August and September from experimental groves in Lee and Hendry counties, but less than 1% parasitism was observed. In general feral populations of psyllids were very low in the commercial groves during the report period. The recovered parasitoids are preserved in 95% ETOH and sent to Dr. Evan Braswell of USDA APHIS, Edinburg, TX, to identify genetic origins of these parasitoids using molecular markers. We also initiated the biparental colony of Diaphoencyrtus aligarhensis from Pakistan for release and evaluation at SWFREC. Addionally, we also visited Orange Co. to discuss their production and release system and some collaborative experiments involving release and evaluation of Tamarixia radiata.
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. A subsequent field visit to Brazil plots is planed in late 2011. 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. An electronic glitch was determined in the leaf wetness sensors and new control circuitry was designed and constructed to overcome the glitch. New hydrophobic materials used as the sensor bridge allowing the detection of leaf moisture are being evaluated as well. The newly designed probes with various sensor bridge materials have been tested to determine their viability. A few electronic bugs still remain to work out. Publications: 167. 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).
Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), thought to be primarily a phloem-feeding insect, transmits the Huanglongbing pathogen, Candidatus Liberibacter asiaticus. Because this bacterium is restricted to the phloem and bacterial transmission is the result of psyllid feeding, investigation of the basic feeding behavior of D. citri is needed. In this study, an electrical penetration graph (EPG) monitor was used to: 1) determine whether gender-based differences in feeding behavior exist for D. citri and 2) examine the effects of photophase on D. citri feeding activities. In the first experiment, investigating gender-based differences in D. citri feeding behavior, overall, the number of male D. citri reaching the phloem was 20% higher compared to females. However, the mean duration of phloem ingestion (waveform E2) per insect was significantly higher for female D. citri compared to males. Analysis within treatments (gender) showed that, despite being considered a phloem feeder, the duration of xylem ingestion (waveform G) and phloem ingestion (waveform E2) were not significantly different. Prominent xylem ingestion was probably not caused by desiccation due to trauma, because insects were not anesthetized, starved or excessively handled during wiring. In the second experiment, examining the effects of photophase on D. citri feeding, within-treatment analysis showed that non-probing activities (waveform z and np), phloem penetration and salivation (waveform D and E1, respectively), and xylem ingestion (waveform G) were generally longer in duration per insect during the light photophase. In contrast, stylet pathway activities (waveform C) and phloem ingestion (waveform E2) were longer in duration during the dark photophase. Thus, results suggest that gender and photophase have an influence on D. citri feeding behavior, and are important variables that could affect the outcome of experiments investigating the transmission of Candidatus Liberibacter asiaticus by D. citri. Work has continued examining whether there are differences in the seasonality of psyllid movement of the HLB pathogen. This work has been ongoing for 3 field seasons. While there does appear to be some seasonal effect on the likelihood of psyllids to be carrying the HLB pathogen, our results suggest that the number of psyllids carrying the pathogen increases along with the incidence of diseased trees. At two locations in 2011, the incidence of psyllids testing positive for the HLB pathogen never dropped below 40% regardless of the time of year. This was a dramatic increase in the incidence of HLB positive psyllids from previous years at these locations and coincides with an increased abundance of trees showing symptoms of HLB. These results suggest that once HLB is well established in an area, the presence of any psyllids in an area presents a great risk for HLB spread.
Data collection from the 10-acre young tree protection multi-year trial continues. To date, foliar treatments have been applied as planned on 6 separate dates. Soil-applied neonicotinoids have been applied on three separate dates. Leaf samples from plots have been collected on 11 dates at two week intervals for pesticide residue analysis. These leaf samples have been ground in liquid nitrogen and stored in the freezer for residue analysis at a later date. Since planting, psyllid and leafminer counts have been made on 11 dates, performed every two weeks. Thus far, psyllid populations have remained below detectable levels. However, leafminer damage has occurred with high frequency (up to 90%), particularly in the control and foliar-applied broad spectrum insecticide treatments. Leafminer damage has remained low in the soil-applied neonicotinoid treatments as expected. PCR testing of leaf samples from our plots has failed to detect any Las positive trees in any of the treatments (including controls) to date. This is to be expected since we don’t anticipate getting positive Las test from plants in less than 6 months following infection; the plants have only been in the ground approximately 5 months. A field trial comparing application methods and rates of neonicotinoid insecticides, initiated in May and ended on August 30, has been completed. A trial similar to this one was conducted for the first time in 2010 and was repeated this year to determine if results from year 1 were accurate and repeatable. The primary goal of this trial was to confirm the efficacy of trunk applications compared to soil drench applications and also determine whether repeated trunk applications pose any phytotoxicity risk with regards to overall tree health. The trial consisted of fifteen treatments including varying rates of Admire Pro, Belay 50 WDG and Platinum 75 SG as both trunk applications and soil drenches. The results confirmed that the trunk applications were equivalent to soil drench applications in terms of psyllid control provided. No phytotoxicity was observed. In addition, leaf samples were collected every two weeks, ground in liquid nitrogen and stored in the freezer. These samples will be analyzed at a later date to determine neonic residue levels in leaf tissues and correlate those levels with the duration of control provided. Progress was made in our multi-year field experiment designed to test whether there is any benefit to continuing to control psyllids on trees that are already HLB infected. Inoculations of healthy field grown (caged) trees with selected numbers of presumptive Las(+) and Las(-) psyllids were conducted in May and again in June. Following recovery of the psyllids after a 2-week inoculation access period, psyllids were held in the freezer and then later analyzed using PCR to determine the exact number of Las(+) and Las(-) psyllids that fed and likely inoculated the plants. For the presumptive Las(+) psyllids, approximately 52% were Las(+), whereas all of the presumptive Las(-) psyllids were indeed negative. PCR analysis of leaf samples collected 3 months after the multiple inoculation access periods were Las (-). Growth measurements were also made on trees in each treatment at this time. Work on this objective continues.
A primary function of this grant is to hire a CHMA program assistant within IFAS to facilitate the CHMA outreach activities to further program development. To date, a committee has been formed and a position description drafted. Advertising to begin soon. No funds have been spent from this grant to date. No further action to report.
A total of 6 applications of SPLAT-CLM were made at two locations during 2011: 3 applications each to 90 acres in a grapefruit grove in St. Lucie county (Emerald Grove, The Packers of Indian River) and 82 acres in an orange grove in Charlotte county (TRB Groves). These two field trials represent complementary experiments to determine the potential for reducing the cost of SPLAT-CLM applications by leaving untreated beds (gaps). During the period since our previous report, we continue to collect weekly counts of male leafminers attracted to pheromone-baited traps (a measure of mating disruption) and periodic assessments of mining damage. End of year analysis of results of various gap treatments will attempt to identify coverage patterns that optimize mating disruption and minimize the amount of product applied. The prototype applicator fabricated by International Fly Masters Inc. of Ft. Pierce continues to function well. Final compilation and analysis of data from these field trials will occur at the end of the season when leafminer populations decline to winter levels. A manuscript describing results from SPLAT-CLM trials conducted in 2010 was accepted for publication during the period: Lapointe, S. L. and L. L. Stelinski. 2011. A GPS-guided applicator for high viscosity semiochemical products and optimizing cost of mating disruption for Phyllocnistis citrella with intentional treatment gaps. Entomologia Experimentalis et Applicata (accepted 8/16/2011). Dr. Craig Keathley joined ARS and assumed his position as postdoctoral research entomologist at USHRL, Ft. Pierce on 12 September. Craig has quickly come up to speed on the project and will be taking a greater leadership role in the on-farm validation trials expected to commence in the spring of 2012.
Spatial and Temporal Incidence of Ca. Liberibacter in Citrus and Psyllids Detected Using Real Time PCR, October 2011. Objective 1. Assess seasonal patterns of pathogen incidence in citrus trees and psyllid vector populations in an infected experimental block. Since March 2008, the pathology and entomology have been working at a site located within a commercial grove that initiated nutritional and/or insecticidal sprays on 7-year-old Valencia on Swingle trees. Initially, disease incidence of HLB in trees in the various plots average around 25%. One year later, disease incidence was greater than 80%, and in the nutritional treatment 100%, in the plots. Determining the titer of HLB in symptomatic leaf samples and in collected psyllids was possible by training and resources provided by collaborator, M. Keremane. Citrus leaf samples and psyllids, stored from initiation of the trial and sampled at approximately 6 month intervals, are being processed. Data reveal that while some fluctuations in the titer of bacteria in occur at sampling dates, preliminary conclusions are limited. The data is skewed toward a detectable population of HLB by the selection of symptomatic tissue, therefore it may not be that differences in populations will be detected in symptomatic tissue. Other studies are using other sampling techniques to try to get around this bias. Objective 2. Evaluate the influence of cultural factors that affect incidence and titer of Liberibacter in citrus trees and psyllid populations including tree age, variety, rootstock, block size, surroundings and management practices such as vector control and tree removal. In another location where HLB incidence and tree health is being monitored on grapefruit and Hamlins receiving various treatments, including initially, tree removal, S. Halbert has been conducting trapping of psyllids. Psyllids from the traps are being analyzed for HLB titer by K. Hendricks, SWFREC. Four suction traps were operated at the SW Florida Research & Extension Center from July 2009 to present. These included an 8 meter tall trap and three 2 meter traps. Of the latter, one was in managed citrus, one was in unsprayed citrus, and the other was in an open field. Samples were collected approximately weekly. The psyllids were removed and identified in Gainesville. Beginning in 2011, all Diaphorina citri Kuwayama were tested singly for presence or absence of the HLB pathogen. All three short traps collected D. citri. Both traps located in citrus collected at least occasional D. citri throughout the year, but the trap in the unsprayed citrus collected the most. The trap in the open field showed peak activity in March, coinciding with the spring flush. These collections could indicate that longer distance flights away from the crop occur at that time of the year. Overall, there were five samples positive for Las and three questionable samples in 2011. There were positive samples collected from all three short traps. There was no difference in the numbers of positives by trap. This can be attributed to the fact that citrus greening disease is widespread and common in the Immokalee area. Preliminary data indicates that neither nutritional nor insecticidal sprays impacted the disease progress of HLB because either the treatments were initiated during the long lag time between inoculation/symptom expression or another reason. Yield data from various plots has been collected as a indicator of overall impact of HLB, at one site of these studies.
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