During the first quarter of funding, Core Citrus Transformation Facility (CCTF) continued to process the orders for transgenic Citrus material. Demand for genetically transformed citrus plants stayed high resulting in influx of new orders listed here by names of genes of interest or plasmids: p6; p33; p7; p10; pMOG8000; pAS7; pAS13*; pNAC1; pMKK7; pMOD1; and pSucNPR1. The work also continued on the old orders that were previously partially completed. Considering that transgenic Duncan plants carrying NPR1 gene exhibited significant resistance to Citrus canker, CCTF received order for production of commercially important Flame grapefruit cultivar transformed with the same NPR1 gene (order completed-NPR1 gene: 10 plants). A gene thought to be superior to NPR1 (so-called superNPR1) was introduced into Duncan grapefruit (superNPR1 gene: 12 plants). Introduction of NPR1 into Hamlin orange cultivar and superNPR1 into Flame is half completed. Work on the order pAS7 that is associated with HLB tolerance/resistance is half done (5 Duncan plants). Order that included use of pLC plasmid is also completed (8 Hamlin orange plants). Ten Mexican limes transformed with gene in pHK plasmid were produced, but satisfaction of this order will include production of more plants per client’s request. Five plants of Mexican lime transformed with p33 gene were also produced. CCTF produced more plants for the old orders: N1* gene: 3 Duncan; C5*: 3 Duncan; CL1 gene: 2 Valencia, 3 Duncan; CL2 gene: 1 Duncan; PiTA gene: 1 Valencia; CIT108p: 1 Flame. About thirty more soil-adapted plants will be submitted to the PCR testing as a secondary proof of their transgenicity before delivery to clients. Some of the funds from the grant were used to hire additional help through the summer resulting in seasonal increase of CCTF capabilities. Please be informed that the person directly managing the CCTF (and co-PI) is Dr. Vladimir Orbovic.
The objective of this project was to investigate three questions: 1) what is the seasonal pattern of Ca. Liberibacter asiaticus prevalence in leaf tissue on a grove scale; 2) what are the flushing patterns of citrus and whether these flushing patterns affect the prevalence of Ca. Liberibacter asiaticus in Diaphorina citri or citrus leaves; and 3) what is the prevalence of Diaphorina citri carrying Ca. Liberibacter asiaticus on a grove scale and how does it compare the results from the citrus trees in the same grove. In 2008 and 2009 Ebert and Rogers demonstrated that the prevalence of Ca. Liberibacter asiaticus in the Asian citrus psyllid varied seasonally but the pattern between seasons was not consistent. It was suggested that perhaps the reason for the differences between the years related to the flushing patterns of citrus and the prevalence of the bacterium in the leaves where psyllids are feeding. This project aims to determine if there is a relationship between the frequency of disease on branches and psyllids Three locations of low to high disease levels have been selected. Leaves and psyllids are being collected from the sites. Samples are being processed for disease detection via qPCR. The post-doc that will be working on this project has been hired and is expected to begin working at the beginning of September.
Citrus extension agents have been actively involved in assisting Florida citrus growers and others in increasing their knowledge to adopt practices for maximizing the control of the Asian citrus psyllid and management of citrus greening or HLB. The citrus extension agents have used a variety of programs to achieve these educational goals. Major programs conducted by citrus agents are as follows: 1) ‘Low Volume Technology for Citrus Pests’ program series was conducted in 6 locations to discuss application methods and pesticides for optimum control of psyllids. This series reached over 300 participants state wide. 2) ‘Low Volume Sprayer Calibration Rodeo’ events were conducted in 3 locations to determine if low volume equipment currently being used meets the 90 micron requirements being required by some pesticide labels. These events evaluated more than 70 different sprayers. This series increased the awareness in the citrus grower community of the need for equipment to spray at the 90 micron particle size. 3) ‘2010 Florida Citrus Growers’ Institute’ reached over 370 participants with timely information on greening, psyllid management and citrus genomics. Twenty speakers from local, state, national and international research and businesses made presentations during the 7-hour program. Archived web presentations of numerous programs conducted by the citrus agents are available on the web at www.citrusagents.ifas.ufl.edu . This site hosts presentations from the Citrus Growers’ Institute (2008, 09 and 10) and citrus agents program series related to low volume spray programs. Total web hits using Urchin 5 web statistics exceed 709,900 for the site and included 3,034 for the 2010 Florida Citrus Growers’ Institute, 9,254 for the Low Volume Application series and 207,495 for all citrus agents’ newsletters. Providing web-based format allows program information to be readily available for those who could not attend programs and serves as a reference source for all to use. Field visits have always been an important aspect of grower education in Florida by the Extension Service. Annually, more than 500 grower visits are conducted by agents to assist growers in making timely decisions, increase knowledge and choose appropriate production practices to achieve necessary control of specific pests/diseases. Citrus newsletters are offered by agents and are distributed electronically or in a printed version. These newsletters reached over 2,500 growers, citrus employees or others involved in the Florida citrus industry. Timely and unbiased information is essential to maximize production and enhance pest control strategies. Polycom broadcasts have been received in selected counties to ensure citrus related seminars and special programs are offered to ‘local’ growers without having to drive to a single site. These programs save considerable time and money while providing timely information to all growers. Polycom broadcasts included: citrus black spot and seminars broadcast from research and education centers on timely topics focused at effective and timely information dissemination. Urban audiences and Master Gardener trainings have been conducted to ensure that these groups are aware of the risks of growing citrus as well as disease and insect issues. Over the grant period, more than 500 Master Gardeners and urban clientele have participated in one or more of the citrus agent instructed programs. While many of the programs have been in central and south Florida, programs have also been offered in Okaloosa and Jackson Counties in west Florida. Presentations have also been provided by citrus agents at regional, national and international meetings to effectively communicate the latest production and field findings to as wide of an audience as possible. When battling greening and psyllids, agents must reach beyond their local regional area to gain and present information.
The citrus extension agents have been actively involved in educating citrus growers and homeowners about greening (HLB) and other disease issues (citrus black spot) around the state. Major programs where citrus agents were involved include: Citrus Growers Institute ‘ this program was conducted by the citrus extension agents in Avon Park at the South Florida Community College campus. A total of 376 participants attended the full-day program where 20 speakers presented timely information related to greening and/or psyllid management. Program speakers included international, national and in-state experts. The program was free to attend with some program costs being offset by the FCPRAC grant and lunch was sponsored by 7 chemical companies. All presentations were recorded and currently available on the citrus agent’s web site at www.citrusagents.ifas.ufl.edu . The site received 3,034 hits to access presentation information in May and June of 2010. This site also has the presentations from the 2008 and 2009 Institute programs that discussed various aspects of greening. To view or download the presentations, the above site is accessible to anyone, regardless of location around the world and provides timely and historical information in combatting this major disease. Citrus Black Spot ‘ A program was hosted by various county offices or research centers around the state to provide timely information related to citrus black spot. By hosting the polycom broadcast, growers were able to receive timely information with little travel time as compared to attending a central state-wide meeting. Master Gardener Trainings ‘ programs were conducted in Collier, Okaloosa and Jackson counties where over 220 participants received information on citrus and its diseases, including greening and citrus canker. Educating urban audiences is also important in combatting citrus diseases state-wide. Presentation of research findings on the use of Remedy (triclopyr) to control citrus sprouts was presented at the Florida State Horticultural Society annual meeting. Citrus sprout control is essential when removing greening infected trees as a large percentage (up to 80%) of sprouts emerging from stumps of removed trees have been shown to be positive for the disease within 9 months of tree removal. Grove visits by agents during this quarter exceeded 79 field visits in addition to 31 office visits to address greening issues. In addition to field and office visits, agents received a significant number of phone calls and have highlighted greening management topics and issues in their citrus newsletters. Grower educational programs were also conducted or assisted with by agents in Arcadia, Avalon, Dade City, Ft. Pierce, Immokalee, Myakka City, Sebring and Wauchula. These programs highlighted greening, citrus black spot and/or area-wide management areas.
Two experiments were initiated at the Southwest Florida Research and Education Center (SWFREC)T to assess impacts of insecticidal control of ACP on non target pests and beneficial insects. The experimental block consisted of 15-yr-old sweet orange ‘Valencia’ trees planted on double-row raised beds at a density of 132 trees/acre. For first experiment, trees were hedged so new shoots attractive to ACP and citrus leafminer (CLM) and beneficial insects were plentiful. Nine treatments (435 Oil, Movento 240 SC + 435 Oil, NAI-2302 + 435 Oil, Agriflex (MK 936) + 435 Oil, Micromite 80 WGS + 435 Oil, low and high rates of Belt 4SC + Induce, M-Pede + Addit, Portal 0.4EC) all intended to control ACP and an untreated control were randomly distributed across 4 replicates in a RCB design. Treatments were applied on 14 May using a Durand Wayland 3P-10C-32 air blast speed sprayer operating at 1500 RPM with 2 stainless steel T-Jet # 5 nozzles per side delivering 65 gpa and evaluated at 3, 10 and 17 days after treatment (DAT). Ten randomly selected shoots per plot were collected and examined under a stereomicroscope in the laboratory to count ACP nymphs. Three fully expanded leaves on each shoot were examined to count CLM larvae and mines. Density of ACP adults and beneficial insects was estimated from three central trees in each five-tree plot by counting the insects falling on a clipboard covered with an 8 . . 11 inch laminated white sheet placed under randomly chosen branches which were then struck 3 times with the PVC pipe to make a count for one ‘tap’ sample. Four tap samples were conducted per tree. All treatments provided significant reduction in psyllid populations compared to control for up to 17 days. Significant reduction in number of CLM larvae compared to the control was observed in all treatments through 10 DAT except 435 Oil alone at 3 DAT, Portal at 10 DAT and M-Pede + Addit at both observations. CLM larvae or mines were not significantly less than the control in any treatment except for Movento + 435 Oil, Micromite + 435 Oil, Portal and M-Pede + Addit, an effect not observed against ACP. Such increase in CLM population could be due to reduced number of CLM natural enemies in those treatments. No spiders were observed in the Micromite treatment and most were observed in the 435 oil alone treatment. Numbers in the treated and untreated trees did not differ statistically except 435 oil alone and control. Only 9 adults or larvae of Olla v-nigrum and Harmonia axyridis ladybeetles were observed in samples, 4 in the control, 3 in low rate of Belt and one each in 435 oil alone and M-Pede + Addit. In the 2nd experiment treatments of 435 Oil alone, Delegate 25 WG + 435 Oil, Provado 1.6 F + Induce, Movento 240 SC + 435 Oil and three rates of an experimental product from Bayer CropScience with or with 435 Oil or NIS were applied on 12 July and evaluated at 3, 10, 17 and 24 DAT. Application and evaluation procedures were same as above except application volume of 150 gpa. Compared to control all treatments provided significant reduction in ACP populations through 24 DAT. CLM larvae were reduced by all treatments at 3 and 10 DAT but only Movento, Delegate, and low and medium rates of experimental product with 435 Oil were effective at 17 DAT. CLM mines did not differ between treated and untreated trees at 3 DAT, but were less at 10 DAT in treatments of 435 Oil alone, Delegate + 435 Oil, and experimental product + 435 Oil or NIS. At 17 DAT all treatments except Provado and experimental product alone had fewer mines than control. CLM larvae or mines did not increase in any treatment compared to control. Spiders were the only beneficial arthropods observed during sampling and treatment effect was not significant. Overall, treatment effects against CLM were less marked and more short lived than against ACP. Increase in CLM populations in some treatments indicate possible negative impacts on their natural enemies. This year survey of grove managers by SWFREC and IFAS extension was distributed. So far, increase in citrus leafminer, mites, and scales under current ACP management was reported by 50%, 30% and 20%, respectively.
Progress to enhance biological control of ACP in Florida is described here by objective number. 1) A total of 24,213, 13,165 and 9,466 Tamarixia radiata were released from colonies at DPI-Gainesville initiated with wasps imported from Pakistan, south China and North Vietnam. Wasps from Pakistan were released at the SWFREC experimental grove and from China and N. Vietnam at two commercial groves, in Glades and Charlotte Counties, respectively, the latter being organic. Releases in commercial groves were integrated with biweekly sprays of horticultural oil. Nymphs recovered on sentinel plants and from grove trees were determined to be mummified or not and reared in the laboratory for adult emergence. Parasitism by T. radiata on sentinel plants in the release block at SWFREC grove averaged 77% and 61% in April and June, respectively, compared to 0-14% and 0-11%, respectively, in blocks where parasitoids were not released. Very few nymphs were recovered on sentinel plants placed at the same locations in July, presumably due to predation, and none were parasitized, although parasitism averaged 31% and 0% among feral nymphs from release and non-release blocks, respectively. Parasitism on sentinel plants placed in five blocks of a neighboring conventional grove averaged 22%, 11%, and 4% in April, June, and July, respectively. No releases of the Pakistani strain were made at this location and parasitoids from previously established strain being reared at SWFREC had been released through March only. Parasitism on sentinel plants placed in oil-sprayed and untreated plots in April averaged 14% and 25%, respectively, at the Glades Co. location and 5% and 15%, respectively, in the organic grove. In Glades Co, 50% parasitism was also observed on sentinel plants placed in a neighboring block where parasitoids were not released. No nymphs were recovered in June from sentinel plants placed in the control plots at either of these two locations. Few were recovered on sentinel plants placed in oil-treated plots and 75% and 28% were parasitized at Glades and Charlotte, respectively. 2) A collaborative study with Dr. Norman Barr, USDA-ARS Mission TX, and others on the genetic characterization of T. radiata from these colonies, as well as from Florida and the Caribbean was completed and published. Further research will be conducted to develop genetic markers for T. radiata to track establishment and performance of different strains in the field. 3) The colony at SWFREC of the previously established strain of T. radiata produced 44,790 wasps between January and July 2010 that were used to help maintain the colony at OrangeCo, conduct laboratory experiments, and release in experimental, organic and conventional groves including the ones mentioned under objective 1. Nymphs collected from an organic grove in Lake Wales in April and June and reared in the laboratory were not parasitized. Releases at this location were made through March only. A collaborative study with CREC entomologists to understand behavioral response T. radiata to volatiles emanating from ACP, citrus and opposite sex conspecifics was recently accepted for publication in the Journal of Insect Behavior. OrangeCo started its own production of T. radiata and through our collaboration initiated a large scale field study to test integration of T. radiata releases into a judicious management program (dormant sprays followed by oil sprays based on scouting) and conventional chemical control for ACP. We also tested an Insta-Clip’ 4-Sided Screen House (13′ x 11′) to cage pruned citrus trees in the field in order to multiply and release T. radiata in large numbers early in the season. About 400 psyllids and 200 Tamarixia adults were released in each cage. Shoot density averaged 28/38.9 dm3/tree with average infestation rate of 56% and 22 nymphs per shoot. Parasitism in the cages averaged 74%. 4) We made several statewide and national presentations and published reports to reach the target clientele in addition to assisting Orange Co. and DPI to establish their mass rearing facilities and start field testing,
We reported (FDACS contract 00070950) that insecticidal treatments of HLB infected trees decreased bacterial titer and coupled with nutrient treatments increased yield compared to infected trees that were not treated with insecticides. This may indicate that an important portion of the Liberibacter population in the system resides in psyllids, that weaken the tree’s ability to resist the disease by continuous inoculation with bacteria. These results indicate that insecticides are still a necessary component of an HLB management program, even for infected trees. The goal of the present project is to establish economic thresholds under different juice price scenarios that optimize returns on investment when a nutrient/SAR package is being applied in groves with moderate to high incidence of HLB. Dr. Cesar Monzo Ferrer, graduate of the Agricultural Research Institute of Valencia (Spain) has been employed as a Post Doctoral Associate under supervision of P. Stansly to undertake these studies. Two 3-year field experiments have been initiated in commercial orange blocks in Hendry County (southwest Florida). One block consists of 30 acres in 32 rows of 10 year old ‘Early Gold’ on ‘Swingle’ citrumelo rootstock. Average HLB incidence estimated in this grove based on symptoms is 74% and randomized samples for PCR analysis are being processed. The second block consists of 12 acres of ‘Valencia’ orange also on ‘Swingle’ and appears to have similar incidence of positive trees which will also be confirmed by PCR. 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 will be applied approximately every 1-2 months and consist of a rotation insecticides recommended for managing this pest. Because the dormant season has been previously demonstrated to be the most effective time to treat with insecticides, treatments 3 and 4 will receive one or two ‘dormant’ sprays in February and in December and February respectively without considering any threshold. Adult psyllid populations are being monitored every two weeks by tap sampling. Flushing patterns and flush infestation is being estimated by assessing the the number of new shoots per tree and evaluating the proportion infested with psyllids. Negative incidence of spray applications on natural enemy populations will be evaluated by counting beneficials in tap samples and flush observations. Economic thresholds will be calculated using the following parameters: treatment costs (pesticides and foliar nutrient applications), fruit price, insecticide efficacy, yield loss, and a pest density-dependent function. Annual yield will be determined as the average kg of fruit per tree obtained in each treatment after harvest. Yield loss will be calculated as the proportional difference between the yield obtained by trees receiving the ‘Calendar Applications’ treatment and the yield from each of the remaining treatments. Yield loss will be related by non-linear regression with the accumulated number of adults/tap obtained from biweekly monitoring in each of the treatments as well as the incidence of HLB and the average bacterial titer in each plot as estimated using Q-PCR. At the beginning of the experiment average psyllid infestation in the ‘Early Gold’ block was estimated at of 0.26 ‘ 0.04 adults/tap. On July 30, treatments (2) and (3) were sprayed using the spinosyn Delegate at 4 oz/ac plus 5% 435 horticultural mineral oil. The treatment drove psyllid populations in sprayed plots down to 0.01 ‘ 0.01 adults/tap in treatment (2), and 0.03 ‘ 0.01 in treatment (3). Comparisons between sprayed and unsprayed treatments have revealed no negative effect yet of the spray on beneficial insects.
This is an update of the annual report submitted in April 2010. Objective 1: Identify genes positively regulating SA-mediated defense in citrus We continue to clone additional SA genes from citrus. We are focusing on three well-characterized SA regulators, EDS1, SID2, and WIN3. Recent developments in the cloning part include: 1) We obtained both 5′ and 3′ end RACE sequences for ctEDS1 and are in the process of cloning the full length ctEDS1 gene; 2) We obtained 3′ end RACE sequence for ctWIN3 and 5′ end RACE sequence for ctSID2 and we are now trying to use alternative methods to obtain 5′ end sequence for ctWIN3 and 3′ end sequence for ctSID2. We had problems before in obtaining transgenic plants expressing ctEDS5/pBINplusARS. We reasoned that the transformation rate with this gene might be low. So we re-did plant transformation, starting with several flats of T0 plants for both Col and the eds5-1 mutant in order to increase the chance of obtaining transformants. Now we have several independent Arabidopsis lines in both Col and eds5-1 background expressing ctEDS5. We will further study these plants for their defense phenotypes in the next generation. We also transiently expressed ctEDS5 in tobacco and found that overexpression of this gene did not cause obvious cell death and other damages to tobacco cells. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues Developments on other transgenic plants include obtaining homozygous T2 seeds for the following transgenic plants by plate selection with antibiotics: 1) Col and npr1 expressing ctNPR1/pBINplusARS; 2) Col and pad4-1 expressing ctPAD4/pBINplusARS; and 3) Col and ndr1 expressing ctNDR1/pBINplusARS. We already planted some of these lines for defense phenotype assays.
We continue to clone additional SA genes from citrus. We are focusing on three well-characterized SA regulators, EDS1, SID2, and WIN3. Recent developments in the cloning part include: 1) We obtained both 5′ and 3′ end RACE sequences for ctEDS1 and are in the process of cloning the full length ctEDS1 gene; 2) We obtained 3′ end RACE sequence for ctWIN3 and 5′ end RACE sequence for ctSID2 and we are now trying to use alternative methods to obtain 5′ end sequence for ctWIN3 and 3′ end sequence for ctSID2. We had problems before in obtaining transgenic plants expressing ctEDS5/pBINplusARS. We reasoned that the transformation rate with this gene might be low. So we re-did plant transformation, starting with several flats of T0 plants for both Col and the eds5-1 mutant in order to increase the chance of obtaining transformants. Now we have several independent Arabidopsis lines in both Col and eds5-1 background expressing ctEDS5. We will further study these plants for their defense phenotypes in the next generation. We also transiently expressed ctEDS5 in tobacco and found that overexpression of this gene did not cause obvious cell death and other damages to tobacco cells. Developments on other transgenic plants include obtaining homozygous T2 seeds for the following transgenic plants by plate selection with antibiotics: 1) Col and npr1 expressing ctNPR1/pBINplusARS; 2) Col and pad4-1 expressing ctPAD4/pBINplusARS; and 3) Col and ndr1 expressing ctNDR1/pBINplusARS. We already planted some of these lines for defense phenotype assays.
Since the end of April and the completion of the first year of our grant, we have pursued the following: – We have continued with population and grapefruit leaf disease/resistance studies to examine the effect of transiently expressed Bs3 promoter constructs on the growth of an expanded range of X. citri strains. These studies concur with the preliminary results showing that the constructs limit X. citri growth and produce HR against a number of strains. – We have continued to grow out stably transformed Duncan grapefruit lines and identified positive transgenics by PCR. – We have had some discussions with parties in Argentina about their interest in this genetic approach to controlling citrus canker. – We are selecting tobacco plants with the Bs3 promoter constructs driving reporter genes to aid in our investigation of the interaction of various X. citri TAL effectors with the promoter elements.
Mesa Tech International (MTI) has developed low cost heating systems employing microcontroller-based temperature control. Circuit boards supporting heating elements and microcontroller interfaces have been designed, fabricated and tested. These systems have been found to support isothermal amplification in low cost plastic housings. Sample preparation methods based on novel passive buffer exchange technologies developed at Los Alamos National Laboratory and refined for field deployment and application to crude citrus tissue extracts at MTI, provide an effective means of isolating amplification ready nucleic acids without complex user interventions or external instrumentation. The integration of this sample preparation technology with amplification and detection sub-systems will result in an easily used molecular diagnostic device suitable for use under field conditions with no need for additional specialized equipment. In this period, MTI has developed an integration strategy for incorporating passive buffer exchange-based sample preparation with isothermal amplification and lateral flow detection. Development of the strategy has prioritized methods that can be realized without moving parts such as valves or pumps, yet accomplish the required fluidic manipulations to transport amplification reactions from a temperature controlled chamber to a lateral flow detection strip. In addition to eliminating any need for automated moving parts, the strategy employs low cost components that are suitable for incorporation into a device designed for disposal after a single use. Prototypes are fabricated from laser cut sheet plastic using prototyping equipment available in-house at MTI, reducing prototype development costs and turn around times. Initial test systems have been fabricated to experimentally determine physical design parameters and to allow testing of material properties. These breadboard integration test platforms are now being refined to incorporate circuit boards supporting both temperature control and fluidic control mechanisms. To assure designs are relevant to applications most needed in the citrus community, MTI’s efforts are focused on the development of a functional first generation prototype that will target the etiologic agent of citrus greening or HLB (Candidatus Liberibacter). Toward this goal we are currently assessing assay chemistries for their utility in the detection of C. Liberibacter nucleic acids both from laboratory purified material and from infected plant tissue processed with MTI’s passive buffer exchange sample preparation system. A small number of greenhouse infected tree samples were processed using established RNA purification methods in Norm Schaad’s laboratory at the USDA Fort Detrick facility. These RNA preparations were assayed at MTI using MTI designed primers and probes targeting sequences identified by Schaad’s group as useful in the detection of C. Liberibacter. These initial studies demonstrated that MTI’s primer sets were capable of amplifying C. Liberibacter derived targets and detecting the resulting amplicons on lateral flow detection strips. More recently, a larger number of citrus nucleic acid samples were obtained from Mike Irey (United States Sugar Corporation). These samples consisted of 20 field samples collected in Florida and found by Irey’s team to be positive for HLB by real-time PCR, 10 greenhouse samples positive for HLB and 20 control samples found to test negative in Irey’s laboratory. We have conducted an initial screening of these samples with a DNA-based assay and have found near perfect agreement between our assay results and those anticipated on the basis of real-time PCR tests conducted in Irey’s facility. One sample negative by real-time PCR exhibited borderline positive test results in our assay.
Under Objective 1 (define rates and formulations of copper sprays for more effective control) one trial with Red grapefruit has been set up in Ft. Pierce, FL and one trial with Hamlin orange in Hardee County, each with 14 treatments of formulations, rates and combinations with other chemical treatments of interest. This season, trials have low to moderate disease epidemics as a result of a periodically wet spring, moderately wet summer and, so far, an absence of tropical storm events. Under Objective 2 (establish the period of fruit susceptibility for grapefruit and oranges), 2008 and 2009 trials established that season long sprays are necessary for control on fresh grapefruit, and sprays through mid-July are required to prevent fruit drop of Hamlin. These results are being summarized for publication in 2010. Under Objective 3 ( evaluate the use of streptomycin [Firewall]) two trials, one non-bearing and one bearing, in grapefruit have been initiated to evaluate streptomycin for canker control. The process of preparing the package for Firewall to obtain an EPA Section 18 Emergency Exemption has been initiated based on data from Florida trials in 2007-2009. Under Objective 4 (To define risk for development of bacterial resistance to copper [Cu] and streptomycin [Sm] in FL citrus groves) a number of factors favorable for the development of copper resistance in Xcc were identified, but further investigation is necessary to fully assess the risk for streptomycin resistance. Although no CuR strains of Xcc were detected in Florida , many strains of Xanthomonas alfalfae subsp. citrumelonis (Xac), the casual agent of citrus bacterial spot in Florida, were resistant to copper. This is the first time copper resistance has been reported in Xac and since Xac and Xcc share the same host and thrive under similar environmental conditions, the concern is that copper resistance may be horizontally transferred from Xac to Xcc. This concern is supported by experiments that showed that copper resistance genes can be conjugated among different species of Xanthomonas including Xcc and Xac. Moreover, although no CuR or SmR strain of Xcc was isolated from citrus trees repeatedly sprayed with copper or streptomycin for 3 consecutive seasons, the frequent sprays caused an increase of the population of endemic bacteria with resistance to these chemicals. The intensive use of these bactericides may consequently increase the risks for acquisition by Xcc of copper or streptomycin resistance genes from epiphytic bacteria. This possibility is confirmed by the presence of Xcc copper resistance gene homologues in bacteria from the citrus tree canopy which are able to confer resistance to copper sensitive strains of Xanthomonas. Cloning and characterization of copper resistance genes in Xcc revealed copL, copA and copB as the major determinants of resistance. Homologues of these genes with identity higher than 90% occurred in CuR strains of several other species of Xanthomonas and other bacterial species, indicating that these copper resistance determinants are widespread and may be transferable into Xcc populations under repeated use of copper for citrus canker management. Under Objective 5 (rapid transfer improved canker management technology to the Florida citrus industry), the 2010-11 canker management recommendations have been published in the Florida Citrus Pest Management Guide and Citrus Industry Magazine. Oral presentations have been delivered to the Florida Citrus Production Managers and at county extension meetings.
Objective 1: Potential for soil application of the neonicotinoids, Admire (imidacloprid, IMID)and Platinum (thiamethoxam, THIA), and Actigard (acibenzolar-S-methyl, ASM), to provide long-lived SAR control of canker was evaluated. In the field trial of 3- and 4-year-old ‘Ray Ruby’ grapefruit trees in Ft. Pierce, Florida, soil drenches of IMID, THIA, and ASM were compared with contact activity of Kocide 3000 (copper hydroxide, CH) and/or Firewall (streptomycin, STREP) applied as a foliar spray at 21-day interval for canker control on foliage. Canker on each set of vegetative flushes was assessed as the percentage of leaves with lesions. In 2008, despite above average rainfall and a tropical storm event, all treatments significantly reduced incidence of foliar canker. Spray of CH was the most effective treatment. As a group, soil drenches of SAR inducers reduced foliar disease incidence depending on rate and frequency of application. In 2009, all treatments significantly reduced incidence of foliar canker compared to the untreated check. IMID, THIA, and ASM as soil drenches were each effective for sustained control of canker on young trees under epidemic conditions. Control with SAR inducers was highest for four applications of ASM in 2008 and 2009 seasons which demonstrated the value for maintenance of SAR with repeated soil applications. Objective 2 Integration of soil applied IMID with foliar applications of copper sprays for control of canker. IMID applied once at the beginning of the season followed by 11 CH sprays gave the bst control in the 2009 trial. This suggests that SAR and copper could be used in an integrated program for augmenting canker control for young fruiting trees. In 2010, a trial integrating ASM at different frequency of soil application with THIA and CH sprays at 21 day interval was set up on 1-yr-old Vernia sweet orange in Ashland, FL. Incidence of newly infected trees is just starting to increase in the various treatments this summer. This highest incidence of disease trees and leaves is in the nontreated check. Objective 3 is to evaluate of the complementary use of ASM, THIA and IMID soil applications to increase and/or extend canker control in 2-yr-old grapefruit trees. Canker was first observed in the trial area in September 2009 after a very high rainfall period in August (17 inches). The pattern of disease spread was across the trial area from southwest to northeast. Incidence of trees with canker was 56% in the non-treated check trees, whereas in most of the SAR treatment combinations the incidence was less than 10%. Incidence of canker in this trial continues to increase in 2010. Trials with soil applied inducers in Parana, Brazil will be evaluated in September with the collaborator.
The objective is to test the effect of soil applied neonicotinoid insecticides used for systemic psyllid control as inducers of SAR to determine, if possible, the direct effect of SAR on HLB disease progress in newly planted citrus trees subjected to psyllid mediated infection or budwood-inoculated infection. Hamlin trees were planted in May 2009 at the USDA-ARS, Picos Farm in Ft. Pierce FL and treated as follows: 1) untreated check, 2) foliar insecticide to control psyllids, 3) soil applied imidacloprid/thiamethoxam to induce SAR, 4) soil applied imidacloprid/thiamethoxam plus the foliar insecticide to induce SAR and control psyllids, 5) HLB bud graft-inoculated untreated check, 6) HLB bud graft-inoculated with soil applied imidacloprid/thiamethoxam to induce SAR. In 2009, the effect of SAR induction on HLB infection progress was inconclusive perhaps because the soil applied imidacloprid/thiamethoxam control of the psyllid may have caused an uncontrolled interaction with psyllid transmission. In 2010, the trial utilized soil-applied SAR inducing Actigard instead of imidacloprid/thiamethoxam in treatments 3,4 and 6. In December 2009 (6 months post-HLB inoculum challenge), 34 of 300 trees in the experiment were PCR positive (11%). In July 2010, 49 trees or 16% are PCR positive. Most of the PCR positive trees are located on the west side of the trial area which is proximal to pruned, heavily HLB infected citrus, and few infected trees on the east side bordered by citrus seed (trifoliate hybrids) and pine flatwoods. The highest number of PCR positive trees (12) occur in the untreated checks with or without graft inoculation, treatments 1 and 5, and in the graft-inoculated treated trees with soil applied SAR inducers, treatment 6; 7 positives are detected in the soil applied SAR inducer, treatment 3, 4 positives occur in the foliar insecticide,treatment 2, and 2 positives in the foliar insecticide plus soil applied SAR inducers, treatment 4. There are many new visually positive trees that cannot be confirmed as positive by PCR analysis due to the seasonally low bacterial titer in HLB affected trees throughout the state in mid summer. If the visually positive trees is are assumed to eventually be PCR positive, the effect of SAR induction on HLB infection progress remains inconclusive. Two HLB-SAR experiments of similar design are set up in Parana, Brazil. One is HLB graft-inoculated and the other is proximal to an unmanaged farm with HLB. Each of these trials utilizes soil applied Actigard for the SAR treatments. The HLB status of the Brazil trials will be assessed in September 2010 by the collaborators.
Objective 1: Survey and confirmation of HLB in seedlings from HLB-affected trees. In 2009, 500 seedlings grown from seed extracted out of mild to severely HLB-affected fruit from Pineapple orange and Murcott tangor groves in Hendry Co. were assayed for HLB detection. All seedlings were negative for HLB detection by PCR in repeated assays in July and Nov. 2009. Objective 2: Thermotherapy of HLB-affected seed for the same seed sources under Objective 1 were treated at 125F, 130F and 135F to test for the effect of heat treatment on HLB detection in seedlings. Thermal treatments were lethal to Murcott, but not Pineapple seed. The emergent seedlings at 125F (142), 130F (98) and 135F (97) were PCR tested. All seedlings were negative for HLB in repeated PCR assay in July and Nov. 2009. Objective 3: In October 2009, seed source trees in two Florida nurseries were found positive for HLB by FDACS-DPI (Nursery 1) or Southern Gardens Diagnostic Lab (Nursery 2). Discovery of infected seed source trees in two Florida citrus nurseries identifies a potential (but unconfirmed) risk of outdoor seed source trees acting as a source of inoculum for introduction into nursery propagations. In Nursery 1, seed was collected from symptomatic branches of two sources of Swingle citrumelo, four sources of ‘Kuharske’ Carrizo citrange and one source of Cleopatra mandarin. In Nursery 2, seed was collected from one source of Sekwasha mandarin. From 189 to 278 seedlings from each source provide enough leaves for PCR testing in February 2010 and June 2010. The June test yielded a single questionable PCR detection for Cleopatra mandarin. Follow-up testing of this seedling from two separate midrib DNA extractions resulted in inconsistent positive results from 16S qPCR analysis with a mean detected Las titer of 6.9 and 4.3 16S copies/reaction (76.7 and 47.8 bacteria/midrib or 3652 and 2020 bacteria/g of midrib) for the two DNA extractions. Even in this case, detection by qPCR only occurred in only 75 and 33 percent of all 16S qPCR runs for the two DNA extractions. Las was not detected using beta-operon qPCR primers in either DNA extraction even after multiple runs. False amplification at very high Ct’s, 43-50 (5 to 0.001 copies/rxn by standard curve), occurs about 33 percent of the time with the 16S primer/probe system from water and healthy controls, whereas false amplification has not been observed with the beta-operon primer/probe system. Because the positive detections are within the range of false amplifications in water and healthy DNA extracts, we are currently using nested PCR in the 16S rDNA, beta-operon gene, and OMP gene to attempt confirmation of Las presence by sequence analysis.
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