Spatial and Temporal Incidence of Ca. Liberibacter in Citrus and Psyllids Detected Using Real Time PCR, December 2010/January 2011. In July 2010, psyllid adults from our HLB negative colony on orange jasmine were caged on new shoots (10-12 adults/shoot/ per tree) that had been caged immediately after trimming and were not exposed to feral psyllid population. On the same tree an additional cage was placed on a previously uncaged shoot that was infested with feral psyllid nymphs. These adults and nymphs remained caged on the shoots for three weeks. Some adults from laboratory reproduced in the cages so when collected there were some nymphs available. Similarly cages with feral populations also had some nymphs and adults at the time of collection. Using PCR, 40 shoots tested HLB negative with average Ct value of 39.8 ‘ 0.07 and 15 shoots tested HLB positive with average Ct value of 27.4 ‘ 0.7. From lab reared adults caged on HLB negative shoots 69 were tested and all were negative. From their nymphal progeny 58 were tested and 1.7% were positive with Ct value 29.8. From the ones caged on HLB positive shoots 51 adults were tested and 12% were positive with average Ct value of 29.6 ‘ 0.7. Among 31 nymphs none were positive. Out of 88 adults that emerged from feral nymphs caged on HLB negative shoots 10% were positive with average Ct value of 28.9 ‘ 0.9. Only 5 nymphs were tested and all were negative. Among 53 adults that developed from feral nymphs caged on HLB positive shoots 9% were positive with average Ct value of 29.6 ‘ 0.9. No nymphs were tested. We are also evaluating the effect of vector management on bacterial titer and fruit yield in the same 12-acre block of a commercial orange grove. The block was divided into 16 plots organized in a randomized block design with eight replicates and four treatments: (1) Nutritional (2) Insecticides (3) Insecticides + Nutritionals and (4) Untreated. Treatments (1) and (4) remained free of insecticides and the vector was allowed to thrive. In treatments (3) and (4), the psyllid population was monitored every 2 weeks and insecticide was sprayed when insect populations surpassed the nominal threshold of 0.5 psyllids per tap sample. To assess the titer values, we selected symptomatic branches on every fifth tree in every row for real-time PCR analysis every four months. Initially CT values and the percentage of PCR positive trees did not differ between treatments. In the last two years, CT values have dropped and most trees eventually tested positive. CT values from insecticide-treated trees became significantly higher in Jan 2010 (25.7 ‘ 0.6), indicating lower bacterial titers, compared with untreated plots (24.0 ‘ 0.1), although no difference between the two treatments in May 2010. Treated trees had significantly higher yields than the untreated trees. These results indicate that mitigation of vector pressure with insecticide decreased alleviated bacterial titer over time and positively affected yield despite the same incidence of HLB. In August 2009, Ct values of psyllids from insecticide treated and untreated trees averaged 38.4’0.9 (0% positive) and 37.5’0.5 (2.4% positive) for adults, 37.8’0.5 (0% positive) and 37.9’0.7 (1.1% positive) for large nymphs and 39.1’0.3 (0%) and 37.6’0.9 (2.5% positive) for small nymphs.
This project has 5 objectives: (1) evaluate efficiency of potential 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 positive response from growers to the extension program funded by this project is illustrated by the adoption of practices such as area wide dormant sprays and ACP monitoring using tap samples. Virtually all commercial groves in the region are participating in cooperative area-wide dormant sprays. More than 70,000 acres were sprayed at least once by air and most of the rest by ground during the past two dormant seasons in the SW Florida region according to data provided by aerial applicators and surveys conducted by Hendry County Extension. More acreage is being sprayed by ground with LV equipment this dormant season, with aerial applications accounting for 27,250 acres to date. Field counts provided by growers and an FDACS-DPI ACP sampling team trained under this project verified effectiveness of the program, and populations have declined steadily over the entire region during the last 3 years, even in untreated plots. The area wide program in SW Florida, now called the ‘Gulf “CHMA” (www.crec.ifas.ufl.edu/extension/chmas/gulf_chma.htm) is conducted in cooperation with Gulf Citrus Growers Association, FDOACS-DPI-CHRP and Hendry County Cooperative Extension (Stansly et al., Citrus Industry, 2009-2010). Its success has stimulated efforts in other citrus growing regions of the state with the help of 10 presentations this quarter to citrus association boards and members (Indian River, Peace River, Gulf Coast) professional societies and other groups. Another successful extension effort is seen in adoption of the “stem tap” sample and other recommended techniques for monitoring ACP populations. Sixty-three percent of growers surveyed in 2010 reported monitoring ACP more than 12 times a year (75% using the tap method) compared to 37% in 2009. An extension (EDIS) document describing ACP sampling techniques with the code ENY857/IN867 is in press. Our present management plan is based on four principles: (1) preemptive insecticidal sprays during winter targeting adult ACP to reduce the population reproducing on spring flush; (2) a rapid and reliable psyllid monitoring system to guide timing of insecticidal control during the growing season; (3) conservation and augmentation of psyllid natural enemies, and (4) testing of insecticides and application technology including low-volume sprayers and timing. More than 90% of surveyed growers in SW Florida responded as either satisfied or very satisfied with their HLB/psyllid management programs. Field and laboratory experiments conducted in 2010 include: (1) A 120 acre trial comparing a standard dormant spray (Mustang), and application of spirotetramat directly to spring flush accompanied or not with applications of aldicarb before or after spring flush. All treatments successfully maintained populations lower than the control until May with treatments that included spirotetramat being effective until July, (3-7) Five field trials evaluating ACP suppression with insecticides, 3 foliar and 2 drench, submitted for publication in Arthropod Management Tests (www.entsoc.org) and placed on our website www.imok.ufl.edu/entomology. Three additional trials of systemic insecticides are being conducted in a newly planted 5 acre block of “Hamlin” orange on ‘US802’ at our Center. Included is a new active ingredient, cyazypyr (cyantraniliprole), from Dupont, that showed excellent potential in lab bioassays and could be an important addition to the arsenal of soil-applied systemic insecticides, presently limited with the loss of Temik to the neonicotinoids imidacloprid and thiamethoxam.
Our purpose is to develop RNA interference in the tomato/potato psyllid (Bactericerca cockerelli) as an effective model for application towards the Asian citrus psyllid, Diaphorina citri. Our hope is that by inducing effective RNAi effects in recipient psyllids, we can help to control psyllid-vectored plant pathogens with the ultimate target being Ca. Liberibacter asiaticus, the causal agent of citrus greening. We originally generated a B. cockerelli normalized cDNA library and have continued to work with this library to identify candidate sequences for RNAi studies. So far less than 100 targets have been sequenced, but this is ongoing . We have used several B. cockerelli sequences for RNAi studies and cloned the D. citri actin, ATPase, Hsp70 and CLIC, D. citri homologs. We have used two primary approaches to evaluate dsRNAs for RNAi activity. Direct intrathoracic injection of dsRNAs into the psyllid hemocoel is done using 200 nl of 100ng/ul dsRNA. This is used as a known control standard to compare against oral delivery of the same dsRNAs. Increased mortality was seen for actin dsRNA when compared with the control GFP dsRNA 3 days after injection. Injection of an ATPase specific dsRNA also consistently suppressed the endogenous ATPase mRNA expression by 30% in independent experiments. We have developed and used an artificial diet system for screening candidate dsRNAs for B .cockerelli . Psyllid acquisition of the dsRNAs was confirmed by end point and quantitative RT-PCR, and by Cy3-labelled dsRNA visualization. We have screened 18 sequences so far by this method, and have confirmed 4 of them as promising candidates for RNA interference. We also cloned C002 and aquaporin (aqp1) homolog genes from B. cockerelli, and are evaluating these sequences now. We see increased mortality of B. cockerelli in a dsRNA concentration-dependent manner for some of our selected candidate sequences. In addition, consistent and specific down-regulation of endogenous target actin and ATPase mRNAs also was shown by quantitative real time PCR.. We also confirmed RNAi activity after a 3 day oral acquisition by using RNA hybridization to detect siRNAs in psyllids. These data clearly demonstrate RNAi activity in psyllids. We are making transgenic plants now to test candidate sequences that have shown promise via in vitro feeding assays. The candidate sequences were cloned in the plant-expression gateway vector pCB2004 and are ready for production of transgenic tomatoes. We are also attempting to rapidly evaluate RNAi effects in plants. We have used a Tobacco mosaic virus (TMV)-based plant expression system to express B. cockerelli sequences in plants, and psyllids are then fed on these plants and monitored for RNAi effects. So far we have seen decreased survival of B. cockerelli for 2 of 6 sequences after 10 days feeding on recombinant TMV-infected plants. However, TMV systemic expression of insert sequences requires approximately 3-4 weeks and resulting infections are non-uniform within tomato plants. These factors likely contribute to the inconsistency seen so far in our experiments. We are working to improve this system and will have additional data by the next reporting period.
This project includes experiments being conducted by USDA-ARS and University of Florida on protecting newly planted citrus trees from Asian citrus psyllid and huanglongbing. This progress report reviews research conducted by University of Florida in Southwest Florida. The original experiment to compare a granular slow release imidacloprid product (Suscon) to the standard liquid formulation was discontinued in spring 2010 due to a poor stand (deer damage) and lack of psyllids during the interval following applications. Three other studies evaluating soil applied products for psyllid control were initiated in spring/summer 2010. The first study included 11 different treatments and an untreated control on 2 year old Hamlin trees and was designed to evaluate the difference between a single application of Admire Pro, Suscon (slow release) Platinum or Cyazapyr versus a split application of these products or the inclusion of Temik and Movento (foliar) as a rotation partner with the Admire Pro (imidacloprid). Initial applications were conducted 25,26 ‘ Feb with the follow up applications on 19,20 ‘ May. Psyllid eggs and small nymphs were found in low numbers on very few flushes on some sample dates and significant differences were found only on 29-Apr when untreated plots contained 8.1 nymphs per flush which was significantly greater than other treatments which did not differ from each other. Nymphs were only found on the low rate of Cyazapyr and two different rates of platinum. Differences in leafminer activity was noted with the split application of Platinum and admire providing greater control of leafminers than the corresponding single application but this trend did not reveal itself with Cyazapyr as the different treatments were not significantly different from each other. Neither Movento or Temik had a significant effect on leafminer incidence. The second study compares three rates of cyazapyr against Admire Pro and Platinum applied on 19,20 Jul to newly planted Hamlin trees. Evaluations were conducted 10,23 ‘ Sep. and no psyllids were found. However, all products have been 100% effective for leafminer control. Leaf samples are being analyzed for residues of all insecticides. Another study is designed to last 3 years with applications of Cyazpyr, Platinum and Admire Pro every three month in rotation with each other. The objective is to assess the ability of these treatments to delay or prevent the onset of HLB in an area of high incidence. Initial treatments were made 29-Jul on newly planted Hamlin trees and evaluations conducted 2,22-Sep with no psyllids being observed although all treatments have been 100% effective in controlling leafminer. Leaf samples are being analyzed for residues of all insecticides.
Ultra low-volume and Aerial Application of Insecticides and Horticultural Mineral to Control Asian Citrus Psyllid in Commercial Orchards. Low volume (LV) aerial sprays have become the principal method of application during the dormant season in SW Florida. More than 70,000 acres have been sprayed at least once during the last two years in the Gulf ‘CHMA’ cooperative dormant spray program, and this is likely to be repeated this season. Meanwhile, we continue our evaluations of LV application of 435 horticultural mineral oil (HMO) which has shown promising results the last 2 years. This year we chose a 40-acre block of mature ‘pineapple’ oranges in Glades County that had not received a dormant spray and consequently was expected to have high psyllid populations. We used a Proptec rotary atomizer P400D spray machine which has provided better results than the modified London Fogger 18-20 and also allowed for higher volumes necessary to combine oil with a foliar nutrient package that is being widely used to mitigate effects of HLB. Applications at 10 gpa were made every two weeks in a randomized complete block (RCB) design with four replications. Asian Citrus Psyllid (ACP) populations were monitored on alternate weeks using stem tap sampling, and leaf samples were submitted to the HLB laboratory at the Southwest Florida Research and Education Center (SWFREC) for PCR analysis. Initial incidence of PCR positive trees was 11%. Contrary to expectations, ACP populations were low, although the accumulated number of adult x days in treated plots was about half (3.4′ 1.1 ACP x day) that in untreated plots (6.3’2.1 ACP x day). In a second trial employing an RCB design with three replicates, the response of ACP populations was evaluated to biweekly applications of oil applied at 2% (v/v) spraying 100 GPA with an aiblast sprayer in an 85-acre block of organic ‘Valencia’ oranges in Charlotte County. However populations were again low and no differences were observed between the treated and the untreated plots. Thus, it seemed that LV at 20% v/v was more effective than HV at 2% v/v the difference in concentration. A third trial evaluating coverage, deposition, and absorption of micronutrients applied at high and low volume was conducted in a 16 acre block of Valencia oranges in Collier County. Comparison was made between the Proptec sprayer spraying @ 10 GPA every two weeks and an air blast sprayer applying the same total amount of material at 100 GPA during summer and falls flush. Leaf samples were collected and are being analyzed at the foliar analysis laboratory at the University of Florida’s Everglades Research and Education Center. Coverage of blue dye on paper targets in the foliage is being analyzed using digitized images and samples are being processed at the Soil and Water Laboratory at the SWFREC to evaluate deposition using a spectrophotometer. In a fourth trial we evaluated psyllid suppression following application of spirotetramat (Movento ‘) at 10 oz/ac applied at 5, 10 GPA with the Proptec or 40, and 120 GPA with a speed sprayer. While sprayer type did not produce clear differences, a trend toward better performance was seen with the greater of the two volumes applied by each sprayer. Stansly, P.A., H. A. Arevalo and M. Zekri. 2010. Area-wide psyllid sprays in Southwest Florida: An update on the cooperative program aimed at controlling the HLB vector. Citrus Industry, 91(10):6-8
Progress on a new special regulatory trap for capturing and preserving citrus psyllids in situ has been advanced enough to develop some prototypes for further field testing and refinement. The capture and preservation components of the trap were developed from laboratory bioassays that examined psyllid behavior in detail. An industry partner has been identified and contracted with to construct the new traps and future work will be focused on evaluating and improving visual and odor components along with the total trap efficiency in the field.
Objectives of this project are to: (1) import, release and evaluate new strains and species of parasitoid specific to D. citri. (2) identify genetic markers that can be used to track T. radiata in the environment, (3) develop efficient methods for mass rearing and release of T. radiata and possibly other species to increase biological control through augmentation of natural populations, and (4) transfer technology to industry clientele. Objectives 1 and 3: Colonies of Tamarixia radiata from Vietnam, China, and Pakistan that we established at DPI- Gainesville as well as the colony at SWFREC of what could now be termed the ‘Florida’ strain continue to provide wasps for release, investigation and distribution. Field releases this quarter of 7,952, 10,839, and 7,354 wasps from the respective Gainesville colonies join 56,031, 60,334, and 45,898, wasps released earlier this year. Wasps from the Pakistani colony continue to be released at the SWFREC while wasps from south China and N. Vietnam were integrated with biweekly sprays of horticultural oil in replicated trials at two commercial groves, in Glades and Charlotte Counties, the latter being organic. Incidence of parasitism in October based on adult emergence from feral nymphs averaged 45% (N=126) and 34% (N=523) from oil-treated and untreated plots at the Glades county site while no nymphs were found at the Charlotte Co. location. Parasitism averaged 35% at SWFREC (N=69) in a release block compared to 0.9% (N=323) in control blocks. An additional 20,000 wasps were produced at SWFREC, increasing the 2010 total to 76,645. These were used to refresh the colony at OrangeCo, conduct laboratory experiments, and release in experimental, organic and conventional groves. We also collaborated with the California Department of Food and Agriculture, USDA-ARS, USDA-APHIS and FDACS-DPI to arrange and conduct a Tamaraxia Workshop this November in Florida, attended by private and public sector researchers from Fl. TX. CA. Mexico, Costa Rica and Belize. Rearing methods are being improved and we hope to see new DPI facilities for mass rearing of Tamarixia in Dundee this year. Objective 2: We have initiated a collaborative follow-up study to (1) with Dr. Evan Braswell of USDA APHIS, Edinburg, TX, aimed at identifying genetic origins parasitoids sampled from release sites and other locations using molecular markers. A. study on the searching behavior of T. radiata was also published (2). Objective 4: An extension document (3) on T. radiata was published at the Cornell Univeristy website “A Guide to Natural Enemies in North America”. Sixty one percent of SW Florida citrus growers surveyed this quarter on what additional coordinated activities they would you like to see applied area wide chose mass release of parasitoids for biological control of ACP. 1) Barr, N.B., D.G. Hall, A. Weathersbee, R. Nguyen, P. A. Stansly, J. A. Qureshi, and D. Flores. 2009. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using ITS and COI DNA sequences. J. Econ Entomol. 102: 2325-2332. 2) Mann, R.S., J. A. Qureshi, P. A. Stansly and L.L. Stelinski. 2010. Behavioral responses of Tamarixia radiata (Hymenoptera: Eulophidae) to volatiles emanating from Diaphorina citri Kuwayama (Hemiptera: Psyllidae) and citrus. Journal of Insect Behavior. 23: 447-458. 3) Qureshi, J. A., and P. A. Stansly. 2010. Tamarixia radiata Waterston [Hymenoptera: Eulophidae], an ectoparasitoid of Diaphorina citri Kuwayama [Hemiptera: Psyllidae]: http://www.nysaes.cornell.edu/ent/biocontrol/parasitoids/Tamarixia.html
Objectives of this project include 1) refinement of sampling methods, 2) testing the influence of adult density and shoot infestation on precision of estimated means and distribution of population within blocks, and 3) evaluation of methods for assessing psyllid density, shoot density, and infestation rates and their integration into a user friendly system accessible to consultants and managers. Accomplishments are described for each objective. 1) We continue to evaluate and refine different sampling methods. Based on our comparisons of stem-tap, sweep-net, and sticky cards and using an amount $ 10 for per hour labor and per day depreciation cost for an ATV, the cost of sampling a 50 acre block of citrus for ACP control averaged $ 17.40 with use of stem-tap or sweep net and $ 213.60 with sticky cards. An additional disadvantage with sticky cards is the delay in obtaining data to make informed decisions treatment decisions or evaluate applications. We found no difference in efficiency between single taps or single sweeps in detecting 0.2-0.3 ‘ 0.1 adults per sample. Below this range, an inordinate number of taps are necessary. However, the net can be used to cover more area than the tap by sweeping multiple times and therefore is able to detect lower ACP populations. On the other hand, sweeping is tiring, collects a lot of trash, and incurs a risk of canker spread. Detailed descriptions with pros and cons of each method are reported in an extension (EDIS, ENY857/IN867) publication which is in press. We have also initiated comparisons of stem-tap with vacuum sampling, which like the sweep net can cover more area than the tap. In a conventional block of ‘Valencia’ oranges adults averaged 0.3 ‘ 0.07 and 0.9 ‘ 0.12 for a sample duration of 6.7 ‘ 0.2 and 8.3 ‘ 0.3 seconds per tap and vacuum sample, respectively. 2) We are working on comparing different sampling methods over large data sets using regression analysis and bootstrapping procedure at precision targets of 0.25 and 0.01 SEM:mean in order to determine the number of samples required using these methods for routine monitoring and analytical modeling respectively. We have already reported some of these estimates in our earlier reports and in several of our presentations and workshops and extension publication mentioned above 3) We recommend 100 tap samples per block divided into 10 stops, five along the perimeter where ACP tends to congregate, and five inside the block. At each stop 10 tap samples are conducted at one per tree, counting ACP, as well as natural enemies and other pests. Then 10 young shoots, each containing ‘feather flush’ are examined with a hand lens to determine if they are infested with psyllid immatures. The search is terminated if 10 young shoots cannot be found after examining 20 trees. The detection ability of this system is more than adequate for most commercial purposes. The system is explained and forms and spreadsheets provided in the EDIS publication and our website: swfrec.ifas.ufl.edu/entlab. We also provide tap sampling kits to growers in our workshops or on demand. In a recent survey, 75% of growers in SW Florida said they used the tap sample and 63% monitored ACP more than 12 time a year, an increase of 26% over last year. We are also working on the development of a website with ZedX Inc. where growers will be able to enter their sampling data on the incidence of pest and disease in order to assess the need and recommendation of appropriate treatments. Arevalo, A., J. Qureshi and P. Stansly. 2011. Sampling Asian citrus psyllid (ACP) in Florida citrus groves. EDIS (In press) Stansly, P., A. Arevalo and J. Qureshi. 2010. Monitoring methods for Asian citrus psyllid. Citrus Industry 91(4) 20-22.
This study monitored Caribbean fruit fly populations counted in approximately 400 McPhail’s traps distributed across a 45 square-mile area receiving both fruit fly bait sprays and Mustang applications for Asian Citrus Psyilld. The study was performed in Vero Beach/Indian River County, north and south of Highway 60 and west of I-95 (also known as St. John’s Marsh area). The approximate distance between trap settings was 1050 feet, and they were checked about every 7 days for a total of approximately 2000 independent observations over the course of the study. The specific subject area included in this analysis encompassed a subset of the 2000 observations in 90 geographically-contiguous designated areas (each designated area is . square mile). The varieties in the groves in which traps were located included White Marsh, Pink Marsh, Star Ruby and Flame grapefruit and Navel, Hamlin, Valencia, Murcott and Temple oranges. This project was a pilot study designed to obtain observational information regarding whether or not Mustang applications applied for Asian Citrus Psyllid control might also suppress fruit fly populations. The construct of the study is that Mustang was applied in the Fall and again in the Spring for psyllid control to all areas monitored in the study. Coincident with that Fall/Spring time frame, Caribbean fruit fly populations were monitored and bait sprays applied as needed to maintain fruit fly certification for the target crops. The result of this arrangement was that all blocks observed in this study received two Mustang applications, but not all areas received bait sprays. The number of bait sprays applied ranged from none to 8, depending on the particular block. Fruit fly counts were monitored starting in August 2009 and continued through March 2010. Despite the considerable effort that went into performing field activities in this study, environmental and other circumstances resulted in very few non-zero values being obtained in the fly count data. This made quantitative statistical analysis difficult. As a result, graphical exploration of observed trends from the subject area was used to interpret these data. Caribbean fruit fly counts obtained from McPhail’s traps totaled across both time and space showed little or no relationship between the number of bait sprays applied and the total number of flies trapped per location. This result is consistent with the experimental supposition that Mustang sprays applied for psyllid control also suppress fruit fly populations. Specifically, fly counts were none to very low in areas both treated and untreated with bait sprays. Therefore, we suggest that factors other than bait sprays were responsible for the low counts observed. These other factors included: 1) Mustang applications applied in the Fall and Spring; 2) weather; and 3) undocumented factors. It is noteworthy that weather conditions over much of the course of this experiment were unfavorable for rapid fruit fly development. The results of this pilot trial do not contradict the premise of the study, but we believe a repetition of this experiment so that more complete numerical data can be obtained may be warranted. Since quantitative support of an experimental hypothesis that there is no difference between fly counts trapped in Mustang-sprayed vs. Mustang-non-sprayed conditions was not possible in this current pilot study, any follow-up experiment should be designed using the following criteria: 1) longer duration; 2) fewer locations; 3) try to get weather data for specific trap locations; 4) try to monitor fly counts in both Mustang-sprayed and Mustang-non-sprayed areas; 4) document other factors that may influence fly counts; and 5) reorganize data structure to facilitate on-going, real-time statistical analysis.
Although a successful trunk cutting, herbicide spray applicator device was constructed and mounted on a small tractor, little industry interest has evolved in its use. BASF Chemicals had expressed willingness to obtain a label for their formulation of the imazapur herbicide (Arsenal), but apparently no further work on labeling has occurred. Chemical Containers Corp had preliminary interest in commercial development of this equipment, but after all of the development and efficacy information was presented to the CRDF Committee the company apparently decided not to pursue development and no further discussions have occurred. This is probably because the foliar nutrition cocktail mixes appear to be maintaining HLB infected trees in a productive state for several years and fewer growers are scouting and removing infected trees. A simplified cutting and application machine was designed and is being constructed. This will be tested at least one time in the late winter in 2011.
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 disease. The models were initially designed for citrus canker but have subsequently been extended to HLB. 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 As previously described, an SEIDR model was developed that uses Markov-chain Monte Carlo methods, and extensive data from South Florida. This model portrays successive snapshots of the occurrence of symptomatic detected trees in known populations of susceptible trees. To date we have been able to estimate the transmission rates and dispersal kernel for HLB by interrogating this prior data center. currently we are working on differential effects of host age on epidemiological parameters and variability across the plantation. this allows us to explore the uncertainty how the predictions which is then incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods. A web based version of the model is in the final stages of development. This web-based model is for non-researcher users you can utilize it as a tool to understand how the disease will increase and spread both residential and commercial situations. It can also be used to test a wide variety of epidemiological and climate/weather variables and their effects on an HLB epidemic as well as various disease control/mitigation parameters to see the effect of these various control strategies. we’ve been using the data from southern gardens citrus that includes both young and mature trees, since host age is likely to be an important consideration for HLB. The model continues to be improved upon to capture more and more of the true features of the data and the disease. The model is being extended to estimate spread in diverse situations, should HLB be introduced into new areas such as TX, CA or AZ. Field plots were established at the USDA, ARS Picos Farm to examine the effects of insecticide control and roguing in various combinations, both to evaluate treatment effects and to collect epidemiological data for model development. These field studies are underway as well however, at the time of this writing there are no detectable statistical differences among various control strategies.
This project was designed to examine the potential disease control of citrus huanglongbing (HLB) by interplanting citrus with guava. In Vietnam guava has been shown to be an effective deterrent to HLB, slowing the disease and keeping plantings alive for up to 15 years that normally succumb in 2-3 year. For all plots and experiments, Guava trees, (Vietnamese white cultivar) were propagated and grown to appropriate size requiring ~1 year. Both nursery and field citrus trees are assayed for HLB every 60 days, and have been assayed multiple times. Psyllid populations are also being monitored continuously every 2 weeks within interplanted plots to document any repulsion of the vector due to guava. Results: Guava vs no guava nurseries: Two nursery sites, a guava protected citrus nursery versus an unprotected nursery, have were established with disease free, PCR-negative citrus trees (2 sweet orange and 1 grapefruit cultivars) in June 2009 and were located in the protected and unprotected plots. The guava trees were grown to appropriate size as indicated in Vietnam prior to outplanintg. To date HLB appears to be progressing more slowly in nursery plots interplanted with guava that non interplanted plots. Citrus/guava interplantings: 3 commercial plantings with multiple replications were established. This required 1 year to grow the guava transplants and a second year to become established in the field before interplanting with citrus per Vietnam protocols. One trial was established in a commercial orchard with collaborators in Southern Gardens Citrus, but two years of freezes and replanting prompted the grower/collaborator to abandon the planting. A second was established in grove in Martin county but the plantation was sold and removed before data could be collected. A third trial planting was established at the USHRL Picos Farm in Fort Pierce. The Picos plot was interplanted with citrus in August 2009. Severe frosts during 2008/2009 and again during Dec 2010 winters continue to affect the USHRL plots and have caused a delay in the experiment. To date, several HLB+ (PCR+) plants have been identified in the USHRL plots after multiple assays. The majority of these positives have occurred as the result of an edge effect. That is, treatment plots closest to an existing HLB+ planting of Valencia were affected. There continues to be no significant difference among the treatments thus far, however this data is insufficient to draw any conclusions at this point. Research using a Y-tube olfactometer to investigate guava volatiles as repellents of the psyllid has been challenging and resulted in no clear psyllid attraction or repulsion. The best response was 1-wk-old psyllids with C. macrophylla shoots, although psyllid attraction to this odor source was weak. Research with white guava (germplasm from Vietnam) did not lead to any improvement. We will continue to investigate repellency of guava alone or in combination with citrus and refined methods to achieve more sensitivity.
Mid Florida Citrus Foundation (MFCF) currently supports the research efforts of scientists from the University of Florida, USDA and private industry. The majority of research projects are conducted by UF scientists at the approximately 112 acre citrus grove. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care cost tend to be higher than a commercial grove due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. The MFCF is in the process of expanding grove to allow more research work to be conducted. This was made possible due to the fact that the MFCF received an EQIP grant from the NRCS. This past summer 13 acres were planted for new research trials, which included an HLB trial, multiple new herbicide trials and new variety evaluations. Additionally windbreaks were established for new and future citrus research blocks. Current plans call for the establishment of 30 additional acres of research citrus trials for the purpose of young tree psyllid control research, evaluation of new psyllid control products, economic study on rooted cuttings, evaluation of miticides and herbicides and large block planting of experimental early orange.
We are still assessing new symptoms appearance in all established experiments. Exp.1 – In the compartments 1 and 2 where citrus plants served as primary source of inoculum of CLas or CLam we are still observing a predominance of infective psyllids and diseased plants with positive conventional PCR for CLas. In average 30.4% of psyllids collected from inoculum source plants and 8.4% collected from test plants were positive for CLas and no one for CLam. In average 12.8% of test plant were positive for CLas and only 0.9% for CLam. From the three Clam citrus infected plants, two had also CLas in a mix infection. Average Ct value for leaves with CLas were 24.7 and for CLam 33.7, indicating a higher titer for CLas than for CLam. In compartment 4, where Murraya exotica served as source of inoculum, 46.0% and 9.4% of psyllids collected respectively in source plants and test plants were PCR positive for CLam, whereas in compartment 3, where Citrus sinensis served as source of inoculum, 6.35 and 4.7% of psyllids collected respectively in source plants and test plants were PCR positive for CLam. 8.2% of test plants were PCR positive for CLam when inoculum source was Murraya exotica and no positive test plant was detected when inoculum source was Citrus. All plants were in other insect-proof screenhouse and will be there for symptoms observation and leaf sampling for PCR analysis at least until December 2011. A new leaf sampling will be done in March to check the presence of both liberibacters in new test plants. A replication of this experiment was started to repeat this experiment. These results will be present in the 2nd International Research Conference on Huanglongbing in Orlando January 10-14th. Exp. 2- All test plants used to HLB-insect transmission are under evaluation for the symptom expression and the presence/concentration of the HLB bacterium by qPCR. Exp. 3 – No HLB-symptoms and infection were observed yet on inoculated trees at different sweet orange varieties and ages that were encaged under insect proof screenhouses. The disease severity (% of symptomatic canopy) of each tree from the alternative experiment under field conditions is being periodically assessed. Because of the heavy defoliation during the drought season and the recovery of tree canopy with new flushes during the spring, the average disease severity in all assessed trees seems to decrease at this time of year. New assessments after leaves maturation will be done.
The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apple psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. The production of greenhouse-grown citrus trees with nutrient deficiency symptoms for both zinc and boron continues. Deficiency symptoms are expected to take a few months to develop. This is especially the case for zinc because of the high mobility of zinc within the plant allowing redistribution of existing zinc to new flush, delaying the development of symptoms. Multiplication of our stocks of HLB infected citrus is also underway to provide a constant source of symptomatic tissue for experimentation once the nutrient deficiency symptoms have developed. We are also interested to determine if strains of Las will have any effect on the attractiveness of trees to D. citri. It has been reported that Las strains have varying levels of virulence and symptomatology (Tsai et al. 2008). We have analyzed DNA samples from HLB positive trees from Polk and Highlands counties as well as the ‘Smoak Grove’ CREC greenhouse strain by PCR and sequencing. Three putative strains of Ca. Liberibacter asiaticus (Las) were found with 5 (CREC greenhouse isolate), 13, and 15 tandem repeats of DNA in the LAPGP locus described by Chen et al. 2010 and have identified sources of budwood. We are currently using the identified budwood sources to acquire greenhouse isolates of the three strains. Cloning and sequencing of loci including the b-operon, OMP (outer membrane protein) gene and phage DNA polymerase to support the differentiation of the three strains is underway (Bastianel et al. 2005; Lin et al. 2008; Okuda et al. 2005; Tomimura et al. 2009).
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