The main focus of our experiments on the time-course of movement of Ca. Liberibacter through the psyllid anatomy is dependent upon establishing a quarantined, high-titer plant/psyllids rearing system so that ÐqPCR psyllids can be introduced and tracked by dissections and microscopy through their ontogeny. This rearing system is under construction and excellent progress has been made and some adult and immature as well as plant samples have been collected for qPCR analysis (in progress). At the same time, we have studied the insect/plant/bacteria epidemiology in the local orchards, and from these trees have collected and preserved the first specimens for electron microscopy and FISH technique optimization. As a consequence of our pilot studies and communications with other laboratories, we have affected a paradigm shift from the earlier understanding that flush was the source of greatest infectivity for the psyllids, to a new understanding that older leaves contain the highest titer. Psyllids oviposit preferentially on the flush, and it appears that they accumulate HLB by maintaining their position on the growing leaves during their ontogeny. Evidence is accumulating that HLB may reproduce in the psyllid body also. These new concepts and other newly arriving information are constantly steering and revising our ongoing methodology. As we continue to build our rearing system to generate large numbers of specimens guaranteed to have baseline high titers, several specimen lines were collected and processed in different ways to yield different sets of information at this early stage in our study: 1) We have adapted a procedure for rapid, gross, localization of whitefly endosymbionts to the psyllid so that we can quickly and roughly determine degree of infectivity in the general areas we found organs of transmission to occur. +qPCR and -qPCR specimens were preserved for this technique. The results thus far (May collections) tindicated high variability of signal present in free orchard psyllids vis-ˆ-vis a baseline from non-infective psyllids from quarantine colonies. The qPCR reports (Ct values) for 25 psyllids sampled randomly from orchard (left) and quarantine ÐqPCR colonies (lowest value of 20 indicates high HLB titer, highest value of 40 indicates no HLB titer. Mean = 32.965, SD = 5.835). 2) The same lines of specimens were also processed for high quality ultrastructural detail using the electron microscope. These lines will be compared to determine the morphology of the pathogen and the tissues that harbor it. Refereed Publication: Cicero, J., Brown, J.K., Roberts, P. D., and Stansly, P.A. 2009. The digestive system of Diaphorina citri (Kuwayama) and Bactericera cockerelli (Sulc) (Homoptera: Psyllidae). Ann. Entomol. Soc. Am. 102:650-665.
Funds were received in April of 2009. Since that time citrus and other host plants of Candidatus Liberibacter asiaticus were established, propagated and infected with Liberibacter asiaticus at Beltsville for use in this project. A half time student worker was hired to assist in greenhouse duties. Plants were confirmed to be positive for the pathogen by real time PCR assays. Colonies of the insect vector of the pathogen were also established and maintained at Lake Alfred, Florida and Ft Detrick, Maryland. Test shipments of insects were sent to Beltsville to confirm shipping procedures and to perform initial extracts for testing. Many administrative tasks were completed, including transfer of funds to subcontractors at the University of Florida, USDA ARS, Ft Detrick, MD and Agdia, Elkhardt, IN. Material transfer agreements were negotiated with Sigma-tau Pharmaceutical Company, Rome, Italy to acquire the specialized vector for phage display technologies and with Agdia, Elkhardt IN for exchange of materials. Visiting scientists were recruited internationally, identified in China and Italy and arrangements were made for their study visits in Beltsville, to begin August 10 and October 20, 2009. Funding arrangements were made with the USDA Foreign Agriculture Service to support these visitors on this projectÕs funds.
A procedure of shallow cuts in the tree trunk sprayed with some effective herbicides usually killed HLB infected trees in trials in commercials groves. The most effective herbicides found so far contain Imazapyr. As little as 0.33 oz. of a 1 to 4 diluted product was effective at all times except during the spring flush, when apparently a one to one dilution was needed. A vehicle attachment to cut the trunk and spray apply the herbicide is now being developed. This will be mounted on a 4 wheel drive utility vehicle and tested under commercial conditions. Further tests of herbicide dilution, other products and the possible need for higher concentrations in the spring-summer period are planned also. Some chemicals still to be tried include: Velpar (hexazinone) and Ally (metsulfuron) and Clearstand (imazapyr+metsulfuron). Tree killing using Midas (Iodomethane) continue with another trial scheduled for July. This trial will evaluate deeper placement in the soil and a surface spray on barrier to retard volatile loss of the chemical. Trials of aerial applications of herbicides to kill abandoned citrus trees are being monitored. These trials were applied the week of 6 July by the South Florida Water Management District (SFWMD) in areas near Ft. Pierce, FL. About 600 acres were sprayed for kill of an area to be reverted to watershed. Another area was divided into 6 acre plots to test chemicals and rate. All applications were made using a helicopter. It is planned to make the first evaluation of the plots the second week of August. The results of that test which included Arsenal and Clearstand will be in the next report. Other chemicals will be tested for tree kill under abandonment conditions using a simulated aerial spray. A Curtec or Proptec sprayer will be set up to spray from the upper register only, droplet size will be adjusted and volume will match aerial applications of 5 to 10 gal/acre. Materials to be tested will include Velpar (hexazinone) and Ally (metsulfuron) and Clearstand (imazapyr+metsulfuron). The Imazapyr products will be the standards to compare other products to. The effectiveness of the Imazapyr products in the SFWMD tests will provide a guide for how well the simulation test did compared to the aerial test.
Primary: Cultural Practices to prolong productive Life of HLB Infected Trees The objective of duplicating the positive results obtained by citrus grower Maury Boyd with his nutrient/SAR foliar spray program appears to be well on the way to achievement. The foliar spray mixture was broken into component groups of micronutrients (Mg, Zn, Mn, Mo) SARs (Salicylic acid, Serenade Max WP), Phosphorus acid, Hydrogen peroxide, and other (macronutrients, citrus spray oil). Three trial sites, randomized & replicated, of 6 to 7-yr-old Valencia orange on Swingle rootstock were established; 1) two acres 100% infected at IFAS Immokalee, 2) six acres 50-60% infected commercial grove, and 30 acres 15-20% infected commercial grove. The trials were started at the beginning of 2008 with all combinations of the component groups being applied to the 3 flushes in 2008 and spring flush 2009. In 2008 trees were all symptomatic of HLB and confirmed positive with PCR. Trees had dieback and poor growth. Results at the end of 2008 showed treated trees varied in their response, most decreased in tree rating and the full treatment maintained trees in many plots as compared to controls and untreated trees. Winter 2008 and spring 2009 saw mild freeze damage followed by extreme drought stress. Tree yield was to low to harvest at sites 1 and 2. A dramatic recovery was apparent in many of the treatment group with the 2009 spring flush. Trees grew vigorous shoots with normal to long internodes and full size leaves. Many trees flowered and set a heavy crop load for young trees. Most treatments improved but the best results are being seen with the complete mixture. Monitoring BoydÕs grove has recorded a fourth year of fruit set and vigorous growth. Canopy density, shoot length, and leaf size is normal. HLB symptoms remain evident on foliage and new vigorous shoots initiate from infected stems. Yields the past three years have maintained or increased. Fruit drop was observed on HLB infected trees at fruit maturity. Juice quality is high and equal to uninfected trees. Root samples have shown no root deterioration or sloughing. Primary cost estimates of HLB Foliar Spray program are that net production costs increase by less than $200 per acre. The threshold for removing HLB infected trees could by less than 2% per year if nutritional and SAR treatments preserve production of infected trees for at least 10 years. Subcontract: Evaluation of Systemic Acquired Resistance Inducers Combined with Psyllid Control to Manage Greening in Infected groves The trial began on march 19, 2008 and occupies 12 acres of ÔValenciaÕ orange on ÔSwingleÕ citrumelo, divided into 16 plots in an RCB design with 4 replicates and 4 treatments: ± a micronutrient SAR cocktail and ± insecticide applications as needed to control psyllids. We have been able to maintain low populations in the sprayed plots 0.7 ± 0.2 adults per 10 tap samples compared with 9.9 ± 1.4 adults per 10 tap in neighboring non-treated plots. Trees receiving the cocktail are visibly in better condition but differences in production after 1 year were not significant although trends were favorable: (1.30±0.13 for treated plots vs 0.98±0.1 boxes for untreated). The treatments are not yet having a measurable effect on the incidence of greening. Instead, a geostatistical analysis of the distribution of infected plants indicates edge effects.
The objectives of the research during this period is to (1) perform genome-wide sequence analysis to identify Simple Sequence Repeat (SSR) loci from genomic sequences of “Candidatus Liberibacter asiaticus” (Las). (2) Design and develop PCR-based SSR molecular markers for detection and genetic analysis. We carried out research experiments and successfully met milestone of the objectives. Followings are the summary of research activities and accomplishments 1. Genome wide sequencing analysis to identify Simple Sequence Repeat loci We conducted genome sequence analysis to identify simple sequence repeat (SSR) loci. A complete ÒCandidatus LiberibacterÓ asiaticusÓ (CLas) genome sequences has been obtained by pyrosequence technology using the DNA extracted from a single Las-infected psyllid (Duan, et al., 2009). The bacterial has genome size 1.23 M base pairs represented in a circular chromosome with average 36.5% GC content. The software, ÒTandem Repeat FinderÓ was used to search SSR loci genome wide. These searches included mono, di-, tri-, tetra-, penta-, hex- type of perfects, in-perfect and compound types of simple repeats. The criteria to identify SSR loci for designing primer were as followings: 1) for each type of repeat, we selected loci containing at least 5 or more of SSR motifs. 2) each locus has one copy per genome. These researches led to the identification of 123 loci that met the criteria and were potentially useful to design SSR markers. 2.In silico CLas sequence validation. To ensure that sequence loci selected for SSR marker development are unique to CLas, in-silico sequencing analyses were carried out using BLAST to compare the 123 SSR candidate loci with all the available microbe sequences in the NCBI databases. Next, selected SSR sequences were aligned by CAP3 program to remove any duplicate loci. Ten of SSR loci were found to be duplicate and therefore were removed from the list. About 200 bp up and down stream of flanking sequences of each selected loci were then selected for SSR primer designs. 3. SSR primer design. ÒMolecular BeaconÓ software (V. 7.0) was used for primer design. As much as possible, the same parameters were applied for all SSR primer designed, which included, ~50% GC, Tm=55oC, primer length Å 20bp, self dimer/cross dimer .G= -5 kcal/mol., and amplicon sizes ranging from 150 to 350 bp (Lin et al., 2005). Under these criteria, all designed primers will work in the same PCR conditions thereby facilitating SSR primer validation. Of 113 SSR selected loci, 45 of them passed the requirements for SSR primer design. To further evaluation multiplex detection, computational algorithms were used to search possible multiplex primer sets that had the least formation of self and cross primer dimmer under multiplex conditions. Using above criteria, 24 SSR primers had been designed. 4. PCR validation. To evaluate the usage of SSR markers, PCR assays were conducted using multiple CLas strains collected from Florida, Brazil, China and India. Specificity assays also performed to check if these SSR primers interact other citrus related pathogens such as Xylella fastidiosa 9a5c, Xanthomonas axonopodis pv. Citri, Citrus tristeza virus, ÒCa Liberibacter africanus, ÒCa Liberibacter americanusÓ and ÒCa Liberibacter solanacearumÓ. PCR products were then resolved by a 5% polyacrylamide gel and visualized by the silver staining. Of 24 primers we designed, 20 SSR primers showed clean and expected fragment sizes. Seven SSR primers are capable of differentiating CLas strains among various collections. (1)Duan et al., 2009. Complete genome sequence of citrus huanglongbing bacterium, ÒCandidatus Liberibacter asiaticusÓ obtained through metagenomics. Molecular Plant-Microbe Interaction. 22:1011-1020. (2) Lin, et al., . 2005. Multilocus Simple Sequence Repeat Markers for Differentiating Strains and Evaluating Genetic Diversity of Xylella fastidiosa. Applied and Environmental Microbiology 71:4888-4892.
The objectives of the research during this period is to (1) perform genome-wide sequence analysis to identify Simple Sequence Repeat (SSR) loci from genomic sequences of “Candidatus Liberibacter asiaticus” (Las). (2) Design and develop PCR-based SSR molecular markers for detection and genetic analysis. We carried out research experiments and successfully met milestone of the objectives. Followings are the summary of research activities and accomplishments 1. Genome wide sequencing analysis to identify Simple Sequence Repeat loci We conducted genome sequence analysis to identify simple sequence repeat (SSR) loci. A complete ÒCandidatus LiberibacterÓ asiaticusÓ (CLas) genome sequences has been obtained by pyrosequence technology using the DNA extracted from a single Las-infected psyllid (Duan, et al., 2009). The bacterial has genome size 1.23 M base pairs represented in a circular chromosome with average 36.5% GC content. The software, ÒTandem Repeat FinderÓ was used to search SSR loci genome wide. These searches included mono, di-, tri-, tetra-, penta-, hex- type of perfects, in-perfect and compound types of simple repeats. The criteria to identify SSR loci for designing primer were as followings: 1) for each type of repeat, we selected loci containing at least 5 or more of SSR motifs. 2) each locus has one copy per genome. These researches led to the identification of 123 loci that met the criteria and were potentially useful to design SSR markers. 2.In silico CLas sequence validation. To ensure that sequence loci selected for SSR marker development are unique to CLas, in-silico sequencing analyses were carried out using BLAST to compare the 123 SSR candidate loci with all the available microbe sequences in the NCBI databases. Next, selected SSR sequences were aligned by CAP3 program to remove any duplicate loci. Ten of SSR loci were found to be duplicate and therefore were removed from the list. About 200 bp up and down stream of flanking sequences of each selected loci were then selected for SSR primer designs. 3. SSR primer design. ÒMolecular BeaconÓ software (V. 7.0) was used for primer design. As much as possible, the same parameters were applied for all SSR primer designed, which included, ~50% GC, Tm=55oC, primer length Å 20bp, self dimer/cross dimer .G= -5 kcal/mol., and amplicon sizes ranging from 150 to 350 bp (Lin et al., 2005). Under these criteria, all designed primers will work in the same PCR conditions thereby facilitating SSR primer validation. Of 113 SSR selected loci, 45 of them passed the requirements for SSR primer design. To further evaluation multiplex detection, computational algorithms were used to search possible multiplex primer sets that had the least formation of self and cross primer dimmer under multiplex conditions. Using above criteria, 24 SSR primers had been designed. 4. PCR validation. To evaluate the usage of SSR markers, PCR assays were conducted using multiple CLas strains collected from Florida, Brazil, China and India. Specificity assays also performed to check if these SSR primers interact other citrus related pathogens such as Xylella fastidiosa 9a5c, Xanthomonas axonopodis pv. Citri, Citrus tristeza virus, ÒCa Liberibacter africanus, ÒCa Liberibacter americanusÓ and ÒCa Liberibacter solanacearumÓ. PCR products were then resolved by a 5% polyacrylamide gel and visualized by the silver staining. Of 24 primers we designed, 20 SSR primers showed clean and expected fragment sizes. Seven SSR primers are capable of differentiating CLas strains among various collections. (1)Duan et al., 2009. Complete genome sequence of citrus huanglongbing bacterium, ÒCandidatus Liberibacter asiaticusÓ obtained through metagenomics. Molecular Plant-Microbe Interaction. 22:1011-1020. (2) Lin, et al., . 2005. Multilocus Simple Sequence Repeat Markers for Differentiating Strains and Evaluating Genetic Diversity of Xylella fastidiosa. Applied and Environmental Microbiology 71:4888-4892.
The objective of this project is 1) to complete Las genome sequence and to conduct comparative genomics of the Liberibacter species; 2) to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development; 3) to confirm if Las bacteria are seed-transmissible and their role in HLB development. A complete circular genome of Candidatus Liberibacter asiaticus has been obtained using multiple displacement amplification and 454 pyrosequencing technologies from a single Las-borne Asian citrus psyllid (Diaphorina citri). Annotation of the 1.23 Mb genome sequence uncovered 1136 predicted coding sequences (CDS), and all but 300 of these CDS could be assigned to one of the defined Clusters of Orthologous Groups of proteins (COGs). Phylogenetic analysis of the full genome sequence has shown that within the Rhizobizles, Las is closely associated with members of the Rhizobiaceae family. Completion of this genome has also revealed that Las has a limited capacity for aerobic respiration, is likely auxotrophic for at least five amino acids, lacks Type III and IV secretion systems although the Type I secretion machinery appears to be present and 40 ATP-binding cassettes (ABCs). With such a large number of ABCs in relation to the small size of the Las genome and the nature of ABCs, it is quite possible that some of these transporters affect virulence, host range and/ or symptom elicitation. A more extensive discussion can be found in our paper entitled ÒComplete genome sequence of the citrus huanglongbing bacterium, ÔCandidatus Liberibacter asiaticusÕ obtained through metagenomics, which is published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin in USDA-ARS, Parlier, California, we have obtained a nearly complete genome of Ca. L. psyllaurous with 34X coverage. We have also obtained the draft genome (approximately 70%) of Ca. L. americanus using multiple displacement amplification and 454 pyrosequencing technologies. We are currently confirming the sequence of these contigs both in the psyllids and host plants. The information from our genome sequence allowed us to design new primers and probes that target various regions of the bacterial genome. Using these new primers and probes, we revealed the genetic diversity of Candidatus Liberibacter asiaticus (Las) collected from Florida, Brazil, China and Japan. The relationship between the diversity and disease phenotypes were partially correlated. A putative insect transmission determinant gene was identified. The role of this gene is being investigated. The seed transmission of Las is tested in grapefruit, sweet orange and trifoliate orange. Relative high titer of Las detected from seed coat and inner seed coat of the seeds collected from HLB-affected citrus plants. Very low titer of Las was detected from the seedlings, ranging from 3 to 42% using nested PCR. Most, if not all the seedlings did not have typical HLB symptoms and the threshold of the bacterial titer for HLB, even in the three year old seedlings. The results indicated that the seed-transmitted Las could not cause HLB by themselves. The role(s) of these seed-transmitted Las is under investigation.
Funds for the second year of this study were received on April 3, 2009. So the bulk of the funds will be used on experiments next season. However, we did conduct several additional experiments this season before the project’s approval, but the data has not yet been fully evaluated. Most recently, we have just completed a month of harvests after applying foliar K, Mg, and an antitranspirant to late season Valencia oranges. Research results from the first season were successfully completed, the results reported at the annual Florida State Horticultural Society (FSHS) meetings in June 2008 and at Packinghouse Day and the Indian River Postharvest Workshop in Sept. 2008, a manuscript published in the FSHS proceedings (http://www.fshs.org/), and results reported in previous FCPRAC progress reports. To summarize, preharvest treatments included up to three foliar potassium (K) sprays (March, October, and January) or withholding irrigation for up to two months before harvest. In one commercial block, foliar mono-potassium phosphate (MKP) was applied at 23.5 lb MKP per acre (0-52-34; 8 lb K2O per acre) with 4 lb per acre low-biuret urea (46-0-0) applied at a total volume of 125 gal per acre. In another block, the grower applied 3 gal per acre of a commercial 3-18-18 formulation at a total volume of 250 gal per acre. Postharvest treatments included holding fruit for three days at 30%, 55%, or 100% (including wetting the fruit) RH before washing and storing at 70 oF under ambient RH. In the first grapefruit block, preharvest foliar MKP treatments applied 2 months or more before harvest did not significantly reduce peel breakdown. However, peel breakdown was significantly reduced in the second block with foliar 3-18-18 applied 2, 3, or 4 weeks before harvest. Blocking irrigation and rainfall for 49 days before harvest increased peel breakdown, whereas wetting the fruit after harvest and maintaining high postharvest RH reduced peel breakdown compared to exposing fruit to lower RH for even only 3 d before washing. While data is still being collect from this year’s tests and collected data has not been completely analyzed, the results agree with last season’s results. In addition, application of an antitranspirant also resulted in a significant reduction in peel breakdown after harvest, and the application of foliar Mg (Epsom salt) showed a trend that, while not significant, is worth repeating. For the current 2009-10 season we will set up at least two field plots in two different commercial grapefruit groves and apply preharvest foliar K, antitranspirants, and Mg (for which research presented at the International Citrus Congress meetings in China suggest may also reduce peel disorders). We will also test combinations of these materials as well as other treatments that might be suggested by research and/or grower reports.
Funds for this study were received on March 18, 2009. Since these experiments must take place early in the season when the fruit require degreened to meet market requirements, the bulk of the funds will be used on experiments in the fall and early winter when late rains result in pin-point lesions and washing the fruit may inhibit degreening operations. As part of another project, however, we have just built a research grading line that we will also use for this study that will allow evaluation of a number of washing treatments and other variables to develop optimum conditions for grading fruit for canker lesions. Furthermore, preliminary results that supported the current project were presented at the FSHS meetings in June 2009 and a manuscript submitted for publication in the FSHS proceedings. These results showed that inhibition of degreening was significantly less when fruit were washed for only ~15 seconds on a high-pressure washer with brushing compared to longer (1 to 2 min) brush washing treatments. With this information, we will test that ability of even shorter wash treatments or wash treatments without brushing (i.e., only sprays of high pressure water) to clean fresh citrus enough for adequate grading for defects and citrus canker lesions. The variables that will be evaluated include different methods of washing, coloration of the harvested fruit, required degreening time, cleanliness of the fruit, presence of other peel markings (i.e., windscar, melanose, etc.), speed of fruit travel, lighting conditions, the color of the rollers, width of the grading area, and number of graders per unit area.
This is a continuing project for which funding was released this year on 27 April and a comprehensive management scheme has taken shape and been documented: (a) sampling plans for monitoring psyllid populations, (b) preemptive dormant sprays of broad-spectrum insecticides targeting adult psyllids, (c) wide choice of selective and broad-spectrum insecticides to use in the growing season, and (d) strategies for conservation and augmentation of biological control. Objectives and some of the corresponding achievements during this quarter are listed below. 1) Assist growers with planning and evaluation of field tests to develop site specific season-long psyllid control on young and mature trees including active ingredients, timing, application methods, rates etc. – SAR inducers and micro-nutrients did not statistically improve the quantity or quality of the harvest after one year although trends are positive (see Report FCPRAC # 0179) – Aerial application of broad spectrum insecticides reduced ACP adults although ground sprays (speed sprayer) were more effective. LV application of 435 Oil every 2 or 3 weeks at 2 gal./ac. during growing season appear to be have good effects although a single such application with spinetoram or dimethoate was not effective (see Report FCPRAC # 0210). – Area wide dormant spray: 80,000 acres were treated by air and many more by ground in a voluntary program last season promoted by Gulf Citrus Growers Association. A field survey coordinated by SWFREC-Entomology and DPI-CHRP documented greatly reduced psyllid populations compared with the few blocks left untreated. A survey of grower responses is being conducted in coordination with Multi-County Cooperative Extension and plans are to coordinate two dormant sprays this season. 2) Assist in development of efficient monitoring methods to make and evaluate control decisions. Tap samples provide timely information and are 14 times faster than sticky traps. Tap sample and sweep nets gave similar results and took about the same time although taps may be more convenient and less fatiguing (see Report FCPRAC # 0164) 3) Accelerate testing of new chemical tools for psyllid suppression: (a) Laboratory bioassays with new products from Dupont and Gowen are continuing, (b) Foliar application of spirotetramat, and fenproximate with 435 Oil or NIS (spirotetramat) during bloom 2009 suppressed psyllids for 2 months, (c) foliar and soil-applied field testing continues this summer. All field test results are published in Arthropod Management Tests and on our website http://www.imok.ufl.edu/entlab/. 4) Evaluate the economic feasibility of psyllid management strategies. The cost of two aerial dormant sprays is less than $25/ac each and could be as low as $15/acre earch depending on material, volume and distance from the airstrip. The cost of monitoring psyllids using the tap is about $25 per block up to 100 acres so could be done 20 times per year for as little as $5/ac total, depending on block size and would avoid unnecessary sprays during the growing season that could cost up to $50/ac. Details on website. 5) Provide an information bridge between growers and researchers: (a) Conducted 2 scouting workshops, 1 management workshop, and 2 talks given at the Florida State Horticultural Society annual meeting. (b) Published: Stansly, P. A., J. A. Qureshi, and Arevalo, H. A. 2009. Why, when and how to monitor and manage Asian citrus psyllid. Citrus Industry. March 2009. Other publications available at http://swfrec.ifas.ufl.edu/entlab/ and at http://swfrec.ifas.ufl.edu/hlb/database/ (see Report FCPRAC # 0004)
This project is in its second year and has already has provided a comprehensive management strategy that is being used by many growers as we continue to make improvements. Tactics being employed include: (a) sampling plans for monitoring psyllid populations, (b) preemptive dormant sprays of broad-spectrum insecticides targeting adult psyllids, (c) systemic insecticides for young trees (d) a wide choice of tested, selective and broad-spectrum insecticides for use during the growing season based on scouting results, and (d) strategies for conservation and augmentation of biological control. Objectives and some of the corresponding achievements during this quarter are listed below. 1) Assist growers with planning and evaluation of field tests to develop site-specific season-long psyllid control on young and mature trees including active ingredients, timing, application methods, rates etc. – SAR inducers and micro-nutrients did not statistically improve the quantity or quality of the harvest after one year although yields increased by 30% and visual differences of overall tree health were observed (see Report FCPRAC # 0179) – Aerial application of broad spectrum insecticides reduced ACP adults although ground sprays with a speed sprayer were more effective. LV application of 435 Oil every 3 to 4 weeks at 2 gal./ac. during growing season appears to be working although populations were low. A single such application with spinetoram or dimethoate using LV ever other row was much less effective at reducing ACP populations than a speed sprayer running every row (see Report FCPRAC # 0210). – Voluntary area wide spray: 80,000 acres were treated by air and many more by ground in a voluntary program last season promoted by Gulf Citrus Growers Association. A field survey coordinated by SWFREC-Entomology and DPI-CHRP documented greatly reduced psyllid populations compared with the few blocks left untreated. A survey was conducted in coordination with the Multi-County Cooperative Extension to evaluate pest control practices and attitudes regarding area wide control was responded to by 27 growers, managing groves of from 42 to 18,000 acres for a total of 107,000 acres of citrus in SW Florida. Results indicated wide adoption of the comprehensive strategy, with 97% of growers are planning preemptive dormant sprays, 97% using some type of sampling of which 95% used the tap sample, 76% flush observation, 14% Sticky traps, and 9% sweep net, with 85% of growers sampling > 3 times a year. 3) Accelerate testing of new chemical tools for psyllid suppression: (a) Laboratory bioassays with new products from DuPont and Gowen were finalized and some promising products under development, (b) Foliar application of spirotetramat, and fenproximate with 435 Oil or NIS (spirotetramat) during bloom 2009 suppressed psyllids for 2 months, (c) foliar and soil-applied field testing continued this summer. All field test results were published in Arthropod Management Tests and on our website http://www.imok.ufl.edu/entlab/. 4) Evaluate the economic feasibility of psyllid management strategies. The cost of two aerial dormant sprays is less than $25/ac each and could be as low as $15/acre earch depending on material, volume and distance from the airstrip. The cost of monitoring psyllids using the tap is about $25 per block up to 100 acres so could be done 20 times per year for as little as $5/ac total, depending on block size and would avoid unnecessary sprays during the growing season that could cost up to $50/ac. Details on website. 5) Provide an information bridge between growers and researchers: Since June 2009 we have (a) Conducted 2 scouting workshops, given 2 talks given at the Florida Entomological Society, and 5 grower talks, published an article in Citrus Industry and increased the volume of citations for the HLB database to > 1,600 references (see Report FCPRAC # 0004).
This is a continuing project for which funding was released this year on 13 April. For our first objective, we obtained 3 new colonies of Tamarixia radiata, a species specific parasitoid, from south China, North Vietnam and Pakistan last year and now have secured approval from USDA-APHIS-PPQ to release these into the environment. Also, one of us (RN) made another trip to a different part of China although no new colonies were obtained. For our second objective, we have completed a study with the help of Dr. Norman Barr, USDA-ARS Mission TX, to characterize these and other strains genetically so that they can be tracked in the environment. The study confirmed that the different colonies shared the same internal transcribed spacer (ITS) region of nuclear ribosomal DNA, showing that the collections represent a single species. This together with a host range study conducted by Ru Nguyen in quarantine was used to obtain the release permit. The mitochondrial cytochrome c oxidase I (COI) marker was used distinguished the northern Vietnam and Pakistan colonies from each other and from the other colonies. Comparison of COI sequences from field collected populations of Puerto Rico, Guadeloupe, and Texas indicated that Florida is not a likely source of the introduced T. radiata in Puerto Rico but is a likely source of the introduction into Texas. We had already begun to ramp up our colonies of psyllid and the strain of Tamarixia radiata (Objective 1) in preparation for release and evaluation this spring and summer (objective 2). We have over 200 Murraya paniculata plants in our new screen house for this purpose, a new air-conditioned glasshouse for the psyllid colony and another for the parasitoid colony with a backup in a climate-controlled insectary. Parasitoids are being produced at the rate of 600-800 per week. So far this year, 7,050 of these wasps have been released in both conventional and organic commercial citrus groves in Florida. We are also providing information and starter colonies to assist a commercial grove in Florida to mass rear the parasitoid for their use. Parasitism rates are being monitored by rearing field collected nymphs of D. citri and exposing infested sentinel plants in the field to compare with published studies (see below). Nymphs recovered on sentinel plants in the conventional grove were 52% parasitized by T. radiata. Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), an endoparasitoid of D. citri from southern China is also being reared and released throughout the state. A total of 2400, 1325, and 3550 wasps were released during 2007, 2008, and 2009, respectively, in conventional and organic citrus groves and dooryard orange jasmine (Muraya paniculata). A parasitism rate of 6-19% was calculated based on adult emergence from nymphs reared from M. paniculata in May 2008. For objective 3, we released over 0.5 million predatory mites (Amblyseius swirskii) on mature orange trees in a 20 acre organic grove in Lake Wales, FL and are monitoring the populations of mites and psyllids in the grove. Thus, substantial progress was made on all objectives this quarter. Publications (Objective 4) 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. Journal of Economic Entomology (submitted). 2. Qureshi, J. A., M. E. Rogers, D. G. Hall, and P. A. Stansly. 2009. Incidence of invasive Diaphorina citri (Hemiptera: Psyllidae) and its introduced parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in Florida citrus. Journal of Economic Entomology. 102: 247-256. 3. Qureshi, J.A., and Stansly P.A. 2009. Exclusion techniques reveal significant biotic mortality suffered by Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50: 129’136.
(1) Screening of chemical compounds that eliminate or suppress the Las bacteria in periwinkle using the optimized regeneration system. Antibiotics (penicillin G and streptomycin, oxytetracyclin), a biocide (DBNPA), a fungicide (zineb) and two peptides (D4E1 and D2A21) were tested for their ability to eliminate or suppress Las bacteria and to promote the growth of severe HLB-affected cuttings using the optimized regeneration system. More than 75% of plants could be regenerated from the severe HLB-affected periwinkle cuttings treated with a combination of penicillin and streptomycin (PS). All regenerated plants from the HLB-affected cuttings treated with PS tested negative for Las, even by nested PCR. The Las bacteria were undetected in these regenerated plants with average Ct value of 39.33. When treated with oxytetracyclin or peptides (D4E1 or D2A21), the regenerated plants also tested negative by nested PCR or qPCR with Ct values over 32. However, the regeneration percentage was low (30%). The regenerated control plants treated with water tested positive for Las by PCR and qPCR with low Ct values <26.0, indicating that the bacteria titers were at least 100-fold higher than those in the antibiotic or peptide-treated, regenerated periwinkle. DBNPA can also suppress the Las bacteria. The severe HLB-affected cuttings had a regeneration rate of 33.1 %. The Ct values of the regenerated plants ranged from 27 to 30. The fungicide zineb was not effective in controlling Las bacteria. Whether treated with zineb or not, the Las bacteria can keep reproducing. The Ct value was lower in the zineb-treated, regenerated plants than those treated with antibiotics or peptides, and similar to the water control. Zineb is not very soluable. The water solubility is only 10 mg/L. (HLB-affected periwinkle was treated using PS, oxytetracyclin or DBNPA three times a week by foliar spray (previous treatments were by root soaking). The results were same as for the HLB-affected cuttings treated by root-soaking. Antibiotics PS and oxytetracyclin effectively suppressed or inhibited Las bacteria, and DBNPA partially suppressed Las bacteria. (2) Effect of screened chemical compounds on HLB-affected citrus. Nutrition and HLB development in citrus. The nutrients K, Zn, N, and Ca were tested for their ability to suppress HLB symptoms in citrus. Potassium and Zn, but not N or Ca, delayed HLB symptoms. Neither treatment affected Las bacterium after 9 months. K and Zn reduced titers of Las at 5 months after treatment. Effect of SAR-inducer on HLB development. Seven months after treatment with SAR-inducer (SA and antiguard), grafted, HLB-affected citrus tested positive for Las by PCR with an average Ct value of lower than 28, indicating SAR was not effective in suppressing or inhibiting the Las bacterium. This had also been the case with a perwinkle host. Effect of antibiotics on HLB-affected citrus by root soaking. HLB-affected citrus was soaked in the PS or oxytetracyclin solutions three times a week. One month post-treatment, the HLB-affected citrus tested negative for Las by nested PCR, and undetected by real-time Q-PCR, Oxytetracyclin and water control treatments were positive for Las. (3) Conclusions Antibiotics PS (penicillin and streptomycin) and oxytetracyclin are very effective in control of liberibacter, especially PS. Peptides are also effective, but expensive for field use. Transgenic citrus expressing these peptides may provide effective HLB control. DBNPA can reduce Las titers. Zineb is ineffective.
Funds for this project were not released until 9 June although work commenced at the beginning of April 2009. We are evaluating tap sampling against sticky traps, and sweep net methods for the first objective. A 12-acre commercial block of Valencia oranges was divided into 16 plots, eight treated with insecticides and 8 left untreated. Three sampling methods were assessed: the ‘tap’ sample, the ‘Yellow Corn Rootworm Trap’ (YCRW), and the ‘ACP Trap’. For the tap sample a randomly chosen limb was struck 3 times and fallen psyllids counted from a laminated white sheet of paper held 1 ft below the foliage. Each plot contained six sampling stations located at random, each consisting of a tree with two types of traps hung in the canopy. Traps were collected after 2 weeks, at which time two tap samples were taken from the same tree. The YCRW trap captured significantly more adults per trap per tree during the two-week period (9.3 ‘ 1.2 vs. 1.8 ‘ 0.27 in untreated and treated plot respectively) than did the ‘ACP trap’ (4.78 ‘ 0.59 per trap in untreated plots, and 1.1 ‘ 0.02 in the treated plots). ACP adults counted with the tap sample averaged 0.86 ‘ 0.11 per tree in untreated plots and 0.17 + 0.09 in the treated plots. A bootstrap procedure, which resampled from the database with replacement was used to obtain robust estimates of the standard error of the mean (SEM) and confidence intervals for the mean. Under high density, the SEM:mean ratio was estimated at 8.3% for the tap sample and 8.0%, 8.3% for the YCRW and ACP traps respectively for a sample of 100. Under low density SEM:mean ratios were 34, 10 and 14% for the tap sample, YCRW and ACP Traps respectively. Sticky traps cost approximately $1.00 each, and their deployment, collection and reading takes 14 times longer than the tap sample: an average of 7 minutes per trap (hanging, collecting and reading) compared with 30 seconds per tap sample (average of sampling and recording data from 10 non-continuous trees). On the other hand, they caught about 10 times more psyllids, although this was over a 2 week period whereas data from the tap sample data is available immediately. Tap sampling was also compared with sweep net sampling in eight 2-8 acre blocks of untreated and treated citrus in June at Southwest Florida Research and Extension Center. Two tap or sweep net samples were conducted on the bed and swale sides of each of four trees per eight locations per block. In the untreated block, psyllids averaged 0.6 ‘ 0.1 and 0.8 ‘ 0.2 per two taps or sweeps per tree, respectively. Average time required to conduct two taps or two sweeps was 7 seconds. Psyllid density in the treated blocks was much lower compared to the untreated block and averaged 0.2 ‘ 0.03 and 0.1 ‘ 0.02 adults per two taps or sweeps, respectively, and the mean time required to conduct either sample was 6 seconds. Both tap and sweep net seem to be equally effective for psyllid counts and time at both high and low densities, although average density was less than one adult per two taps or sweeps. However, the sweep net is more work, could be difficult to count at high density, and could spread citrus canker. Technology transfer (third objective): Stansly, P. A., J. A. Qureshi, and Arevalo, H. A. 2009. Why, when and how to monitor and manage Asian citrus psyllid. Citrus Industry. 90 (3): 24-26. Arevalo, H. A., P. A. Stansly, A. B. Fraulo, J. A. Qureshi and L. J. Buss. 2009. Sampling for Asian citrus Psyllid. Field Sheet. SWFREC- University of Florida, http://swfrec.ifas.ufl.edu/entlab/pdf/extension/ACP_sampling_english.pdf. Stansly, P. A., H. A. Arevalo, and J. A. Qureshi. 2009. Scouting citrus for pests and beneficials. . Extension presentation (M. Zekri Organizer). March 19. SWFREC/ Multi-County extension. Stansly, P. A., H. A. Arevalo, and J. A. Qureshi. 2009. Monitoreando Plagas e insectos beneficos en citricos. Extension presentation (M. Zekri Organizer). April 28. SWFREC/ Multi-County extension.
Spatial and Temporal Incidence of Ca. Liberibacter in Citrus and Psyllids Detected Using Real Time PCR Funds for this project were released on 19 March although research began earlier in anticipation of funding. In order to assess seasonal patterns of the pathogen in citrus trees and psyllids in regions of high HLB incidence, a 12 acre block of ‘Valencia’ orange trees was selected at a commercial grove in 2008. The block was divided into 16 plots, half of which were treated with insecticides as needed to suppress psyllid populations while the other 8 plots were left untreated. A sample of psyllid adults collected in the block in November 2008 and analyzed at USDA-ARS Riverside labs contained 21% HLB positive psyllids. Samples of the most symptomatic leaves from every fifth tree were taken on 13 Nov 2008 and analyzed for the presence of Ca. Liberibacter asiaticus at the Huanglongbing Diagnostic Laboratory, Southwest Florida Research and Education Center (SWFREC), Immokalee, FL. DNA was isolated using a commercially available kit (Qiagen; Valencia, Califorina, USA) for plant (DNeasy Plant kit) and psyllids (DNeasy Blood and Tissue kit). Quantitative PCR analysis was conducted using specific TaqMan primer/probe assays for plant (Li et al. 2006) and psyllid (Manjuanth et al. 2008). Thirty-four percent of the samples tested PCR positive. A tree determined to be PCR positive and one PCR negative tree in each plot was trimmed to induce new flush. On 3 June, 10 psyllid adults from our HLB negative colony on orange jasmine (Murraya paniculata) were released on a new shoot that had been caged immediately after trimming. An additional cage was placed on a previously uncaged and psyllid-infested shoot on the same tree. All cages along with branches and psyllids were collected 2 weeks later for PCR analysis. Nineteen percent of shoots caged with psyllids from the HLB negative colony tested positive for HLB on trees, regardless of whether trees had previously tested HLB positive or negative. In contrast, shoots that were naturally infested with psyllids when caged were 63% and 56% positive on previously HLB positive and negative trees, respectively. It would appear that infestation with infected psyllids from the field was over 2.5 times more likely to result in a positive plant sample, whereas the previous history of testing had little bearing. We are processing the adult psyllid samples taken at the same time. We are also caging infested shoots on symptomatic trees at SWFREC and commercial groves and collecting monthly samples of psyllid adults found. Adults emerging from shoots on HLB symptomatic trees caged with negative psyllids during Feb-March 2009 and analyzed at US Sugar and SWFREC were found to be 5% HLB positive by both labs, although psyllids collected at large at the same time and location were 14% positive. Psyllids that were caged on these trees during July-August 2008 were 27% positive according to the USDA-ARS Riverside lab. Therefore, psyllid origin and time of collection are major sources of variation on PCR results that we will need much more data to sort out. As our methods for addressing the first objective are refined as described above, the second objective is being address by the identification of additional sites to survey for psyllids which differ with respect to tree age, variety, and rootstock, and block size as described in grant proposal. A survey to administer to growers for tracking HLB and grove management tactics in the selected sites is in preparation. (Refs: Li, W., J.S. Hartung, and L.Levy. 2006. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. Journal of Microbiological Methods. 66:104-115. Manjuanth, K.L., S.E. Halbert, C. Ramadugu, S. Webb, and R.F. Lee. 2008. Detection of Candidatus Liberibacter asiaticus in Diaphornia citri and its importance in the Management of Citrus Huanglongbing in Florida. Phytopathology. 98(4):387-396.)
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