Based on results from the previous Contest Project (CRDF#400), eleven compounds have been screened by the contest committee as candidates for further research to determine their efficacy for control of HLB based on their ability to substantially reduce the titers of the bacterium Candidatus Liberibacter in our grafted citrus assay, lack of phytotoxicity to citrus and potential for registration. After first evaluating combinations of these molecules using the graft-based chemotherapy method under a separate agreement, this project extension will evaluate these 11 compounds as control agents for the HLB bacterium individually and in combination using infected, container-grown citrus and HLB-affected scions. The objective of this project is to determine an optimum chemical formulation that may be registered for field control of HLB. In this quarter (Sep to Dec, 2013), 80 treatments from 11 compounds or combinations have been applied to HLB-affected potted-plants by soil-soaking coupled with three heat treatments (Tm=40, 42 and 45). Las bacterial titers in all these treatments will be tested in every 2 months. In this quarter, the second test has been done. The preliminary results showed that heat treatment promoted the growth of new flushes from the seriously HLB-affected citrus. No bacterium was detected in the new leaves but present in the old leaves two months after treatment with some of the compounds coupled with the heat treatments. No great differences in Las bacteria were found among the heat-treatments (Tm=40, 42 and 45). Four months later, several treatments were effective in reducing the Las bacterium both in the new and the old leaves, especially SDX, AMP. The research is ongoing.
The objective of this project is to optimize attractancy of visual targets and to determine what segment of the ACP population is responding to traps to better interpret surveillance data and to better target collection of dispersing psyllids. Knowledge of the factors that enhance psyllid attraction to a target as well as enhance detection by psyllids of the target against the background will result in optimization of the design and placement of a visually targeted trap. A series of experiments will evaluate the role of UV reflectance, translucence, color contrast patterns, background contrast, polarization and color saturation. Laboratory studies have been initiated on attraction of ACP to transmitted yellow or green light of different wavelengths to determine the optimal colors for use in field trials. Data are being collected in the Materials for constructing traps (acrylic sheets, colored films, paints, tangletrap, polarization filters) have been or are being purchased. Paint mixtures with different levels of UV reflectance are being formulated and reflectance characterized with a radiometer. These will be evaluated in laboratory assays and then in the field. Levels of transmitted light through colored films have also been characterized prior to use in laboratory assays.
Progress on development of an acoustic trap to capture male Asian citrus psyllids (ACP) searching for females on branches of citrus trees continued in the fourth quarter of 2013. New versions of the BugPhone trap were constructed for field testing. The Bugphone is an Arduino Uno microcontroller device that detects male vibrational calls and plays back female replies, attracting the males to a sticky cone attached to the tree. The operation of the Arduino was described in a paper published in the December 2013 issue of Florida Entomologist. One of the devices is being tested in the laboratory in preparation for use when ACP populations begin building up in early Spring. Some revisions have been included in the new BugPhone, including a more noise-free signal amplifier, lower power usage, and an option to include a memory chip so that signals obtained during a 1-2-week test period can be retained and examined for comparison with trap catches. In a new phase of this study, we have identified suppliers of robust devices that can produce that can produce signals to confuse duetting male and female ACP in field environments and have begun a search for a postdoctoral associate to assist in development of mating disruption techniques.
The objective of this research is to evaluate non-neurotoxic insecticides against Asian citrus psyllid (ACP), and provide growers with additional tools for management of ACP. We are investigating those non-neurotoxic insecticides that have shown promise against insect pests similar to ACP. These additional tools may not only prove effective against ACP, but also could assist in ACP resistance management programs as needed tools for effective rotation with current insecticides. In the past quarter, we have been evaluating methoprene and diofenolan in the laboratory. Methoprene and diofenolan are juvenile hormone (JH) analogs that acts as growth regulators. We evaluated developmental inhibition of 1st, 3rd and 5th nymphal instar ACP to the subsequent developmental stage by application of methoprene. We treated 1st, 3rd and 5th nymphal instars with six different concentrations of methoprene ranging from 0 – 256 ‘g/ml. For 1st nymphal instars, we observed 87, 51, 41, 33, 30 and 13 percent development into second instars for concentrations of 0, 16, 32, 64, 128, 256 ‘g/ml, respectively. Similarly, concentrations of 0, 16, 32, 64, 128, 256 ‘g/ml resulted in 84, 71, 65, 53, 46 and 22 percent survival of third instars into fourth instars. When 5th nymphal instars were treated with concentrations of 0, 16, 32, 64, 128, 256 ‘g/ml, percent survivals were 84, 71, 70, 62, 59, 47%, respectively. These results indicate that treatment with methoprene successfully reduced the survival of the nymphal stages of ACP. We also investigated the residual contact toxicity of methoprene on eggs laid on plants after treatment. The plants treated with concentrations of 0, 20, 40, 80, 160 and 320 ‘g/ml resulted in egg hatch inhibition of 5, 14, 42, 63, 86 and 93 percent. Settling behavior of ACP on treated plants was also investigated under laboratory conditions. ACP showed no preference between the treated plants with various concentrations of methoprene ranging from 0-256 ‘g/ml. Currently we are investigating feeding behavior of ACP adults on plants treated with methoprene and the effect of methoprene on ACP reproduction. Diofenolan was evaluated for its ability to inhibit egg hatch of 0-48 hr aged eggs. Percent egg hatch inhibitions of 8, 27, 33, 39, 46, 68, and 96 percent were observed when eggs were treated with concentrations of 0, 10, 20, 40, 80, 160 and 360 ‘g/ml. Currently we are investigating the effect of diofenolan on 48-72 old ACP eggs. We will also investigate the effect of diofenolan on nymphal survival and ACP reproduction. Following completion of evaluations of methoprene and diofenolan (JH analogs), we will begin an evaluation of tebufenozide and methoxyfenozide (ecdysteriod agonists). The results of our research thus far suggest that several non-neurotoxic insecticides are effective in disrupting growth of the immature stages of Asian citrus psyllid.
Management of huanglongbing (HLB) is linked with effective management of the vector, the Asian citrus psyllid (ACP). Recently, tree nutrition supplement programs have been developed that may maintain the health and productivity of HLB-infected citrus trees; however, their impact on ACP biology, pathogen transmission, and insecticide management efficacy are either unknown or there is limited information. The objective of this study is to determine how nutritional supplement programs may affect ACP biology. The outputs are important because nutritional supplement programs are currently implemented by many growers and removal of HLB-infected trees is no longer a popular practice. Outputs for this research may be improved by adding a mature citrus grove experimental field site. Also, we have initiated additional laboratory experiments with other nutrients based on our current progress and available information. Currently, we are focusing most of our field research on one highly replicated experiment at the Mid-Florida Citrus Foundation Grove at Conserv II. This experimental is being conducted within 2-year old resets and allows us to address the question of how nutritional programs affect ACP and pathogen transmission within replanted groves and young tree care. We are in the process of initiating a similar experiment within a planting of mature citrus trees with a mix of HLB-infected and non-infected trees to enhance this investigation. This new field experiment will be composed of mature uninfected and HLB-infected trees. The treatments will be the KeyPlex program versus a non-supplemented control. Before establishing the plots, we are beginning to perform qPCR on approximately 200 trees to determine baseline HLB infection. Also, ACP adult density monitoring is beginning with weekly tap sampling, and nymph density will be monitored with bi-weekly flush examination. We will perform a bi-annual qPCR of the 200 trees, and an annual leaf sample nutrient analysis. The expected output of this additional field experiment should be a better understanding of how nutritional supplement programs affect psyllid population dynamics and pathogen transmission in mature trees. We have begun purchasing materials, including: ‘ qPCR on 200 trees 4 times= total of 1000 qPCR ‘ Nutritional analyses 80 trees, 2 times= total of 160 nutritional analysis ‘ From March to September at least 40 trips to the field ‘ Cost of field maintenance (irrigation, fertilizer) Laboratory experiment materials: ‘ 40 Valencia plants
Over the past few years, we have conducted several investigations focusing on the behavior of Asian citrus psyllid (ACP) with the goal of identifying specific behavior modifying chemicals. The purpose is to formulate these chemicals into controlled release devices for slow release in the field. The approach has focused on three areas: 1) attractants, 2) repellents, and 3) chemicals that prevent ACP from identifying pathogen-infected trees. Throughout these investigations, we have collaborated with industry, working with three companies: ISCA Technologies, Alpha Scents, and Chemtica. With each company, we have either developed new prototype devices or we have evaluated exiting promising technologies. From this work, two of the companies have made significant progress with developing ACP attractants and the next step goal is to bring to market more effective monitoring traps and attract-and-kill devices. Also, two repellent formulations are being pursued by two separate companies; one of which is in the later stages of development. Finally, we have evaluated one tool for preventing psyllids from finding infected trees with promising results. We have initiated development of a novel insect behavior-modifying product to control the ACP. Decoy is a biodegradable emulsion capable of releasing methyl salicylate (MeSA) at rates sufficient to disrupt ACP behavior and transmission of HLB, while attracting ACP insect predators and parasites to treated plots for three months. The formulation is under development by ISCA Technologies. The repellency of Decoy prototype formulations will be determined in laboratory-based cage studies. We have also begun increasing experiments examining new possible ACP repellents by investigating extracts from noxious plants that have been previously identified as insect repellents in the studies given below: G’k’e, A., L. L. Stelinski, R. Isaacs, and M. E. Whalon. 2006. Behavioral and electrophysiological responses of grape berry moth (Lepidoptera: Tortricidae) to selected plant extracts. Journal of Applied Entomology 130: 509-514. G’k’e, A., L. L. Stelinski, and M. E. Whalon. 2005. Behavioral and electrophysiological responses of leafroller moths to selected plant extracts. Environmental Entomology 34: 1426-1432.
Currently we are evaluating insecticide resistance in the Asian citrus psyllid (ACP) against insecticides with different modes of action across the state of Florida. Starting in 2009, an increase in resistance against the neonicotinoid, imidacloprid, was noted. Recently, we determined that this rise in resistance is primarily mediated by an increase in enzymatic activity of a class of enzymes known as cytochrome P450 monooxygenases. In that same study, it was noted that several populations of ACP had increased resistance against another insecticide, cypermethrin, which is a pyrethroid. Pyrethroids mediate their effects by modulating the kinetics of sodium channels within the nervous system called voltage-gated sodium ion channels. This increase in resistance could be due to increased enzymatic activity as found for neonicotinoids, or could be due to target-site mutations of the channel, rendering it insensitive to the insecticide. The course of action to mitigate the rise in insecticide resistance will depend on which mechanism confers resistance. It is particularly important to determine if the resistance is enzymatically based due to the fact that enzymes typically metabolize insecticides across different classes, e.g., cytochrome P450 monooxygenases can break down organophosphates, neonicotinoids, pyrethroids, along with others. Therefore, continued use of a particular insecticide may drive resistance through a population that will affect the efficacy of not only that particular insecticide, but those from other classes. We have begun to expand current work to include the determination if increased resistance to cypermethrin is due to enzymatic activity, target-site insensitivity, or a combination thereof. A better understanding of the underlying mechanism conferring resistance against this compound and other compounds leads to better management to delay resistance against currently utilized insecticides. We have collected insects ACP from field populations where cypermethrin had been used. All experiments with field populations of ACP are being compared against our susceptible laboratory strain. Two types of assays are being developed to determine the basis for resistance against this compound, topical and enzymatic. Topical assays with different sub-classes of pyrethroids and DDT are being developed to determine if resistance is mediated by target-site insensitivity. Enzymatic assays will be used to determine if there is an increase in enzymatic activity of the three major classes of enzymes known to mediate insecticide resistance, esterases, cytochrome P450 monooxygenases, and S-glutathione transferases. Protocols for these assays have already been established and were those used in our studies to elucidate the mechanism behind imidacloprid resistance. For enzymatic studies, insects will be tested under normal and induced conditions to determine response of enzymes to insecticidal exposure. Response of enzymatic pathways to insecticidal exposure will be evaluated with qPCR to measure changes in mRNA abundance levels and biochemical assays to detect changes in enzyme levels. We have also begun purchasing necessary materials such as: ‘ Substrates and standards for biochemical enzymatic assays ‘ Insecticides ‘ mRNA isolation and cDNA synthesis kits
Our objective for this project has been to evaluate botanical compounds as repellents for Asian citrus psyllid (ACP) with the purpose of developing possible repellent formulations for use in the field. We are working through a list of non-neurotoxic products and determining their effects on psyllid population development including effects on development, fecundity, fertility, and life table development as a result of each chemical. As a result of this research, we have identified some non-neurotoxic chemicals that are effective against ACP. Some of these were previously registered for use on tree fruit, some have gained alternative application use registration, and some are possible compounds that may be useful in the future. Since beginning our work, we have nearly completed research with some of these, such as Novaluron, for example. This compound may be a tool for ACP control. We have conducted both laboratory and greenhouse studies to determine the efficacy of novaluron against various life-stages of ACP. Rimon’ is a commercial blend of novaluron, which is an insect growth regulator used widely in insect management, but is not registered for citrus. The compound was tested on two stages of ACP–fifth instar nymphs and eggs, at three field rates used for similar insect pests, 10, 15 and 20 fl oz/acre. Flush with approximately 15 fifth instar nymphs were dipped into the three preparations of insecticide and mortality was determined 72 hours post-treatment. The three field rates of 10, 15 and 20 fl oz/acre induced 30, 60 and 62% mortality, respectively. For effects on egg hatch, flush with an average of 25 eggs between 0-3 days old were dipped into the insecticide dilutions and the number of live nymphs was determined every 24 hours for eight days. At the 96 hour time point when eggs began to hatch, controls had 1.8-fold more live nymphs over the 10 fl oz/acre treatment and 2.2-fold over 15 and 20 fl oz/acre treatments. Bioassays are also underway to evaluate the viability of eggs from treated females. The initial data suggest that the non-neurotoxic insecticide novaluron is effective against the immature stages of ACP in laboratory assays, showing similar effects to other known insect growth regulators against this pest. We have initiated expanded the above research at a faster pace with other insect growth regulators, including allostatins and ecdysteroid agonists. We have begun purchasing materials including: ‘ Chemicals ‘ Plants for Insect colonies ‘ Plants for experiments ‘ Pots ‘ Soil ‘ Fertilizer
We have initiated this study with two laboratory experiments. The objective of the first experiment was to understand how communication between plants may affect psyllid host acceptance. We showed that ACP adults were more attracted to plants exposed to volatiles from HLB-infected plants than plants exposed to volatiles from other uninfected plants. Our next objective is to understand if this communication between plants is mediated through methyl salicylate. We will submit uninfected plants to an air flow contaminated with methyl salicylate, and control plants to a clean air flow. We hypothesize that plants submitted to an air flow of methyl salicylate will be more attractive than plants submitted to clean air. The second experiment aims to test the effect of drought stress on the recruitment of ACP by HLB-infected citrus. We previously demonstrated that drought stressed plants were not attractive to natural enemies, despite the presence of psyllids feeding on them. Our next objective is to test if drought stressed HLB-infected citrus plants will be less attractive to ACP compared to drought-stressed uninfected citrus plants. To complement these two laboratory experiments, we are now working to identify the gene(s) involved in methyl-salyclate production in citrus. The Citrus Genome Database was screened for putative salicylate methyltranferase (SAMT) amino acid sequences from Citrus sinensis using a reiterative homology-based searching strategy with SAMT sequences from other plant species. Both genomic and transcriptomic databases were searched, producing 21 hits with high sequence identity. These sequences were subjected to phylogenetic analyses with representative amino acid sequences from other plant species to identify the likely SAMT amino acid sequence(s), which resulted in the identification of one sequence as the likely candidate designated as CsSAMT. The nucleic acid coding sequence was retrieved from the above database, and intron/exon splice regions were identified. Quantitative PCR primers were designed and tested, and a qPCR protocol to investigate the response of CsSAMT mRNA abundance to herbivory, CLas infection and abiotic stress is being developed. Regarding the field portion of the project, we compared one grove comprised of only reset trees with a grove where resets were replanted only to replace HLB-infected trees. ACP adult counts were significantly higher in the grove composed only of resets than on resets present within a grove that was made up of a mixture of mature and reset trees. In addition, we found that 5 out of 7 resets trees in the totally replanted field were HLB-infected, whereas all of the 7 resets trees tested in the ‘mixed’ grove were all uninfected.
Our objective for this project has been to evaluate botanical compounds as repellents for Asian citrus psyllid (ACP) with the purpose of developing possible repellent formulations for use in the field. Plant extracts containing compounds such as terpenes, steroids, alkaloids, phenolics and cardiac glycosides are known to affect insect behavior and can function as deterrents to insect pests. In the current investigation, we tested extracts of several plants known to produce such secondary compounds that affect behavioral responses of plant pests. Therefore, this series of experiments aimed to determine the response of Asian citrus psyllid, a specialist crop pest, to the same extracts that have been found effective in previous investigations. Four non-host plant extracts from Bifora radians, Arctium lappa, Humulus lupulus and Xanthium strumarium, were tested against ACP adults for their potential as repellents or oviposition deterrents. A multi-arm olfactometer was used to assay the behavioral response of ACP to these treatments and was compared against a cuticular hydrocarbon positive control, which may act as an ACP sex pheromone. Male ACP responded significantly to H. lupulus extract, although their strongest response was to the putative female pheromone (45.0%). Female ACP showed no behavioral response to the cuticular pheromone (as observed in previously published investigations), attracting only 5.0% of ACP, but female ACP were attracted to extracts of H. lupulus, X. strumarium, and A. lappa. Choice tests were conducted to compare oviposition by ACP on untreated citrus and on citrus treated with one of the extracts. Despite the attraction in the olfactometer assays,, H. lupulus extract significantly reduced egg laying; only 5% of the total deposited eggs were laid on citrus leaf flush treated with this extract. The extract of B. radians provided nearly complete (97%) protection against oviposition by ACP in these laboratory assays for up to 7 days after application. Current investigations are focusing on a longer time course of effectiveness of these extracts and subsequent field testing.
The objective of this study is to determine how enhanced nutrition of citrus plants may affect Asian citrus psyllid (ACP) biology. We have initiated this study with complementary field and laboratory experiments. Regarding the field experiment, last season we found significantly more ACP adults on nutritionally supplemented trees than on control trees during two sampling dates, and more ACP nymphs on nutritionally supplemented trees than on control trees during one sampling date. We also assessed the HLB infection status of trees with qPCR after one year of nutritional supplement application. We found no difference in the proportion of HLB-infected trees between control and nutritionally supplemented trees. These results tend to indicate that nutritional supplements do not protect citrus trees against HLB infection but also do not increase the risk of infection either. To confirm these results, we are performing an experiment, under laboratory conditions, where citrus resets are placed within an enclosed Las-infected ACP laboratory colony (55% of the psyllid had been confirmed Las positive). Half of these citrus trees are nutritionally supplemented and the other half are sprayed with water (control). We will follow the infection status of these trees over the course of 1 year to observe if the plants treated with the nutritional supplements are more or less protected against HLB infection. We are also investigating the effect of nutrient sprays on Las acquisition by ACP. To this end, we are performing the experiment under both field and laboratory conditions. Under field conditions, we did not find significant differences in Las acquisition by psyllid when nymphs or adults were exposed to HLB infected trees treated with nutritional supplement versus a water (control). In laboratory experiments, we found a significant reduction of Las acquisition by ACP adults after 10 days of exposure on nutrient supplemented trees as compared with control trees. We did not find a difference in pathogen acquisition between supplemented trees and control trees 28 days after exposure. In summary, our data suggest, thus far, that nutritional supplements only slightly increase ACP populations compared to untreated control trees and these supplements do not impact transmission of the Las pathogen.
We are evaluating the efficacy of the new neonicotinoid, flupyradifurone (Sivanto’, Bayer CropSciences), against the Asian citrus psyllid (ACP). This new chemical was included in the panel of insecticides evaluated in our annual survey for insecticide resistance in field populations of ACP. Through this survey, it was noted that the LD50 (dose required to kill 50% of the insects) for this chemical was approximately ten-fold higher than that of the currently used neonicotinoids, imidacloprid and thiamethoxam. We have confirmed this observation, and have begun testing this insecticide in other assays including contact vial assays where it was also found that a ten-fold higher concentration of flupyradifurone was required to induce the same level of mortality as imidacloprid and thiamethoxam. Experiments are planned to determine if the difference in efficacy is limited to topical exposure or also includes oral exposure (ingestion) of the insecticide. Additionally, preliminary experiments show that flupyradifurone imparts an anti-feeding effect on ACP, but not as effectively as imidacloprid, again requiring higher doses to impart the same level of efficacy. All experiments thus far have been conducted on adults, but given their importance in the transmission process of Candidatus liberibacter asiaticus, equivalent experiments will be conducted with nymphs. We are nearing completion of the molecular characterization the ACP sodium voltage-gated ion channel, the target site of pyrethroids. In previous field surveys, ACP resistance to pyrethroids was observed. Within insects, there are several naturally-occurring allelic forms of the channel that confer resistance to pyrethroids, known as knock-down resistance (kdr). Changes in the allelic frequencies of the channel in favor of those that confer resistance, driven by pyrethroid exposure, has accounted for 80% of the cases of pyrethroid resistance in insects. We characterized the channel to determine its conservation with those from other insects, and have found it to be highly conserved. This suggests that natural populations of ACP likely possess the same allelic variation as observed in other insects and therefore will likely respond similarly to selection pressure from pyrethroid exposure. Because pyrethroids are an important insecticide class that is used in rotation with neonicotinoids for ACP population control, understanding and predicting the molecular mechanisms that confer resistance is imperative to prolong the usefulness of this insecticide class. We have collected 100 field samples from four different locations across the state of Florida, and we will be identifying the allelic forms of the channel in field populations of ACP, and their associated frequencies. From this, we plan to develop a high-throughput qPCR-based assay to detect these differences so that allelic frequencies can be correlated with insecticide response to pyrethroids. This method will allow us to quickly assess field populations for allele frequency so that suggestions can be made concerning insecticide treatment for optimized rotation of insecticide modes of action.
Update for 12/31/13 During this quarter, field trials were initiated to examine the effects of soil-applied systemic insecticide applications in terms of both psyllid control and possible future presence in floral nectar in the spring of 2014. The purpose behind nectar examination was to determine whether these insecticides applied to the soil would translocate to nectar and if so, determine the amount of time needed between application and bloom to minimize any potential risk to non-target organisms feeding on the nectar. In this study, treatments were made to replicated plots on a monthly basis beginning in late September. Each plot was treated only once in order to establish plots with different time intervals between application and bloom. Data on psyllid populations was also collected to demonstrate that treatments were providing effective control of psyllid populations. Monthly applications to new sets of plots will continue through February or March, based on the initiation of bloom in the experimental block. Once bloom is initiated, nectar will be collected from plots and analyzed using LC-MS-MS.
Update for 12/31/13 During this quarter, field trials were initiated to examine the effects of soil-applied systemic insecticide applications in terms of both psyllid control and possible future presence in floral nectar in the spring of 2014. The purpose behind nectar examination was to determine whether these insecticides applied to the soil would translocate to nectar and if so, determine the amount of time needed between application and bloom to minimize any potential risk to non-target organisms feeding on the nectar. In this study, treatments were made to replicated plots on a monthly basis beginning in late September. Each plot was treated only once in order to establish plots with different time intervals between application and bloom. Data on psyllid populations was also collected to demonstrate that treatments were providing effective control of psyllid populations. Monthly applications to new sets of plots will continue through February or March, based on the initiation of bloom in the experimental block. Once bloom is initiated, nectar will be collected from plots and analyzed using LC-MS-MS.
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. Visual tests demonstrated ACP was attracted to leaves when given the option between a leaf or nothing. It did not matter what the nutrient status of the leaves was (P < 0.05). There was no difference in response between an HLB symptomatic and healthy leaf but ACP were significantly more attracted to non-symptomatic HLB+ leaves than symptomatic HLB+ leaves (P < 0.05). There was also no difference in visual response to the Zinc or Boron deficient leaves compared to healthy leaves. In the feeding tests, ACP preferred to feed on the nutrient deficient leaves, either zinc or boron but there was not a significant difference. The majority of the insects died before the fecundity could be measured in any treatment. We were not able to perform the olfactometer tests, including the HLB strains, as the equipment was malfunctioning when our material was ready to test. Some experimental controls were performed but the results were highly biased to one blank treatment compared to the other. By the time the problem was located in the apparatus our material was no longer available. HLB positive trees from Polk and Highland Counties were analysed 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. 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 was completed. Results from sequence analysis clearly defined two strains based on conserved mutations in the b-operon sequence, matching strains from Japan and Vietnam and a strain from Vietnam for the northern and southern Florida strains, respectively. We are working on confirming our results of our experiments but the trees needed to have the nutrients rebalanced and they needed to be allowed to grow without harvest as they are very weak from the nutrient deficiencies.
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