Sensory impacts in unpasteurized and pasteurized juice from HLB-affected fruit: Valencia fruit samples from April and May 2009 were evaluated in a consumer sensory panel. Objectives were to assess consumer acceptability of symptomatic, asymptomatic, and healthy fruit. Three treatment groups for each harvest sample date were hand-extracted to yield 6 total juice treatments, which were further split into thermal treatment types. Consumer sensory testing indicated that symptomatic samples produced juice that was less accepted, less sweet and less flavorful that either control or asymptomatic products. Pasteurization does not appear to affect acceptance in any sample except for the symptomatic product from the April Valencia harvest date, where pasteurization impacted the overall acceptance, sweetness and flavor. Flavor and aroma changes in HLB fruit: Analysis of juice samples from the previous season continued. Esters responsible for the fruity, sweet aroma in OJ were significantly lower in symptomatic juice. Esters such as ethyl butyrate and ethyl-2-methyl butyrate were 20-50% lower in symptomatic compared to control juice. Important aldehydes such as hexanal were 50% lower in symptomatic fruit. Alcohols were generally higher in symptomatic fruit. Linalool, responsible for the fruity, citrus aroma in OJ, was 45% higher in symptomatic fruit. Although there was little difference in flavor volatiles between juice from healthy or asymptomatic fruit, aroma volatiles in juice from symptomatic fruit were out of balance compared to that of healthy juice. Phytohormone changes in HLB fruit: Ethylene production, ACC content and expression of ethylene biosynthesis genes were analyzed in isolated juice vesicle tissue from early season Valencia fruit. In contrast lower biosynthesis-related gene expression and lower production in symptomatic whole fruit, ethylene production increased in juice vesicles when compared with asymptomatic or healthy ones. To examine the spatial role of phytohormones in HLB disease symptom development, IAA and ABA contents were evaluated in flavedo removed from the stem-end, mid-section, and stylar-end of healthy, asymptomatic, and symptomatic fruit. IAA content was highest in the mid-section, lower in the stylar end, and lowest in the stem-end in all fruit. However, IAA was 2-fold higher in mid-section but 2-fold lower in the stylar-end of symptomatic fruit. Although little difference in ABA content was measured in whole fruit, spatial analysis indicated that flavedo from symptomatic fruit had 3-fold higher ABA content in the mid-section and 3-fold lower content in the stylar-end. Results indicate increased stress in HLB-affected fruit and suggest a role for temporal and spatial redistribution of ethylene production, IAA and ABA contents in premature fruit abscission, fruit shape and seed abortion. Yield, peel color, fruit size and seed abortion in HLB fruit: Accumulation of chlorophyll a and b and total chlorophyll were 3-fold higher, while b-carotenoid content was 2-fold lower in flavedo of symptomatic fruit compared with healthy tissue. Pigment accumulation in asymptomatic flavedo was less compared with symptomatic flavedo but higher when compared with healthy fruit. Starch content was reduced in symptomatic and asymptomatic flavedo compared with healthy tissue. In symptomatic and asymptomatic flavedo, iodine-stained starch grains were sparse or absent when viewed with light microscopy, however, starch grains were abundant throughout the flavedo of healthy fruit. Iodine staining confirmed light microscopy observations. The use of iodine staining as a tool for early detection of HLB-affected in asymptomatic fruit in the field will be explored this coming season. Extension and education: Four members of our group made oral presentations at the International Citrus and Beverage Conference. A tabletop extension display was also prepared and presented, and written materials distributed. Similar displays are planned at upcoming industry meetings.
Sensory impacts in unpasteurized and pasteurized juice from HLB-affected fruit: Valencia fruit samples from April and May 2009 were evaluated in a consumer sensory panel. Objectives were to assess consumer acceptability of symptomatic, asymptomatic, and healthy fruit. Three treatment groups for each harvest sample date were hand-extracted to yield 6 total juice treatments, which were further split into thermal treatment types. Consumer sensory testing indicated that symptomatic samples produced juice that was less accepted, less sweet and less flavorful that either control or asymptomatic products. Pasteurization does not appear to affect acceptance in any sample except for the symptomatic product from the April Valencia harvest date, where pasteurization impacted the overall acceptance, sweetness and flavor. Flavor and aroma changes in HLB fruit: Analysis of juice samples from the previous season continued. Esters responsible for the fruity, sweet aroma in OJ were significantly lower in symptomatic juice. Esters such as ethyl butyrate and ethyl-2-methyl butyrate were 20-50% lower in symptomatic compared to control juice. Important aldehydes such as hexanal were 50% lower in symptomatic fruit. Alcohols were generally higher in symptomatic fruit. Linalool, responsible for the fruity, citrus aroma in OJ, was 45% higher in symptomatic fruit. Although there was little difference in flavor volatiles between juice from healthy or asymptomatic fruit, aroma volatiles in juice from symptomatic fruit were out of balance compared to that of healthy juice. Phytohormone changes in HLB fruit: Ethylene production, ACC content and expression of ethylene biosynthesis genes were analyzed in isolated juice vesicle tissue from early season Valencia fruit. In contrast lower biosynthesis-related gene expression and lower production in symptomatic whole fruit, ethylene production increased in juice vesicles when compared with asymptomatic or healthy ones. To examine the spatial role of phytohormones in HLB disease symptom development, IAA and ABA contents were evaluated in flavedo removed from the stem-end, mid-section, and stylar-end of healthy, asymptomatic, and symptomatic fruit. IAA content was highest in the mid-section, lower in the stylar end, and lowest in the stem-end in all fruit. However, IAA was 2-fold higher in mid-section but 2-fold lower in the stylar-end of symptomatic fruit. Although little difference in ABA content was measured in whole fruit, spatial analysis indicated that flavedo from symptomatic fruit had 3-fold higher ABA content in the mid-section and 3-fold lower content in the stylar-end. Results indicate increased stress in HLB-affected fruit and suggest a role for temporal and spatial redistribution of ethylene production, IAA and ABA contents in premature fruit abscission, fruit shape and seed abortion. Yield, peel color, fruit size and seed abortion in HLB fruit: Accumulation of chlorophyll a and b and total chlorophyll were 3-fold higher, while b-carotenoid content was 2-fold lower in flavedo of symptomatic fruit compared with healthy tissue. Pigment accumulation in asymptomatic flavedo was less compared with symptomatic flavedo but higher when compared with healthy fruit. Starch content was reduced in symptomatic and asymptomatic flavedo compared with healthy tissue. In symptomatic and asymptomatic flavedo, iodine-stained starch grains were sparse or absent when viewed with light microscopy, however, starch grains were abundant throughout the flavedo of healthy fruit. Iodine staining confirmed light microscopy observations. The use of iodine staining as a tool for early detection of HLB-affected in asymptomatic fruit in the field will be explored this coming season. Extension and education: Four members of our group made oral presentations at the International Citrus and Beverage Conference. A tabletop extension display was also prepared and presented, and written materials distributed. Similar displays are planned at upcoming industry meetings.
A manuscript reporting the sequence of symptom development and the identification of callose and phloem protein 2 (PP2) as the amorphous and filamentous plugging materials, respectively, has been prepared and is in the final stages of review for submission to Plant Physiology. The upset of normal phloem translocation of carbohydrates to other parts of the plant and ultimately the starvation of the root system (Etxeberria et al, 2009, accepted) may be the main reason for tree decline in response to HLB infection. >Field samples were collected and prepared to determine the relative amounts of amorphous versus filamentous plugs in order to evaluate if one type is more important than the other in disrupting phloem sap flow. Samples include sweet orange and grapefruit collected in the Indian River District or southern flatwoods. Additionally, samples from sweet orange were collected near Lake Placid and Murcott in the southern region. Examination of these samples has begun. >Earlier work by a colleague has indicated that two of our common rootstocks do not express HLB symptoms when grown as seedlings in a greenhouse or growth chamber and infected with the HLB organism. These are being examined for possible suppression of the phloem plugging mechanism. More importantly, field grown seed source trees have been found with + PCR reactions for HLB. These will be tested for phloem plugging and HLB tolerance. >Work to understand the mechanism by which the bacterial infection leads to this phloem plugging is underway as insufficient bacteria are present in the phloem to directly plug the phloem. To determine how the causal bacteria elicits the over expression of phloem plugging materials, one Agilent microarray has been designed based on the genome sequence of Candidatus Liberibacter asiaticus. Bioinformatics analysis was performed to identify potential virulence factors. Six potential virulence factors were cloned into pGEMT-easy vector. The insertion was confirmed using PCR with gene specific primers, and will be sequenced for further confirmation. Those virulence factors are being expressed in tobacco and citrus to study their potential roles in virulence. >Transgenic approaches to achieve over-expression of the citrus §-1,3-glucanase gene using different promoters, though protoplast/GFP co-transformation and Agrobacterium-mediated transformation are underway in order to minimize HLB associated callose-plugging. The citrus §-1,3-glucanases gene from Valencia embryogenic callus and young leaves (McCollum et al., 1999) was cloned. Citrus .-1,3 glucanase cDNA (GenBank accession number AJ000081) was synthesized from Valencia leaf and embryogenic callus. A 1011 bp citrus .-1,3 glucanase gene fragment was amplified. To obtain the suitable restriction site and additional cMyc tag (to facilitate subsequent western analysis) on the cDNA, another PCR reaction was performed using a new primer set with a different restriction site including the cMyc tag. The final PCR product (1059bp, designated BG3) was purified, cloned into the pGEM T-Easy vector (Promega) and sequenced. The cloned BG3 fragment was ligated into a vector named pUCLON between the 35S promoter and 35S terminator, and was transformed into host E coli DH5. cells. Plasmid DNA was isolated using 5prime kit and checked through restriction digestion and also PCR analysis. The re-ligated plasmid was named pUBG3. The HindIII fragment from pUBG3 was excised and ligated into the HindIII site on another vector pCIT101 holding GFP/NPTII fusion gene on it. The final vector was designated as pCITBG3 and was transformed into Agrobacterium. It is now ready for co-transformation experiments. Plasmid vector pARS108 with the ER-targeted GFP gene was also used to make a new construct for efficient protoplast transformation. Another vector, pGASS, was constructed to target BG3 expression in the phloem tissues only. Two new sweet orange callus lines are available to carry out these and other transformations.
Primary: Cultural Practices to prolong productive Life of HLB Infected Trees The first objective of the project has been accomplished. This objective was to duplicate at another site the positive results using nutrient/SAR foliar sprays to rejuvenate HLB infected trees seen in citrus grower Maury Boyd grove. We have seen a rejuvenation of heavily infected HLB trees using the foliar spray mixture of micronutrients (Mg, Zn, Mn, Mo), SARs (Salicylic acid, Serenade Max WP), Phosphorous acid, Hydrogen peroxide, and other macronutrients applied with citrus spray oil. These results have been seen in three randomized & replicated trial sites of 6 to 7-yr-old Valencia orange trees. Initial infection rates among the trials ranged from 100% infection at IFAS Immokalee site, to 50-60% infection in one commercial grove, and 15-20% infection in another commercial grove. In 2009 all possible combinations of the component groups were applied to the spring, summer, and fall vegetative flushes at each site with similar positive results. After applications to the 3 flushes in 2008, and during the drought in the spring of 2009, the trees produced vigorous flushes with normal internodes and large leaves. Flowering resulted in set fruit with a normal to large crop on trees that did not produce a harvestable crop in 2008. To date the crop has held on the trees with very little fruit drop. Although the trees are symptomatic of HLB they have continued to flush vigorously through the summer. New growth has regenerate from below the dieback on twigs with dieback. A dramatic recovery has been apparent in many of the treatments. Most treatments improved tree condition and the best results are apparent with the complete mixture. The macro and micro nutrients seem to be the most important. Fruiting appears to be enhanced with the addition of the Phosphorous acid and SARs. Yield from the 2008 Valencia crop from 7-yr-old trees harvested in summer 2009 at the 30-acre-commercial showed yield highest in the treatment containing macro & micro nutrients, Phosphorus acid, and hydrogen peroxide (0.80 box/tree). Yield was least in the treatment containing macro nutrients and hydrogen peroxide (0.43 box/tree). A spreadsheet has been developed and will be posted on the SWFREC website that calculates the net present value of grove returns over 10 years assuming production and price assumptions that the grower can input. The spreadsheet allows a grower to determine the threshold rate of tree removal after which an alternative nutrient management program for greening infected trees would return a higher net present value. Subcontract: Evaluation of Systemic Acquired Resistance Inducers Combined with Psyllid Control to Manage Greening in Infected groves In a 12-acre commercial block of Valencia oranges 7-years-old trees we evaluated the effect of micronutrients + systemic acquired resistance (SAR) inducers, and their interaction with psyllid chemical control. With 4 replicates in an RCB design for a 2×2 factorial experiment with two main effects: 1) 3 applications a year of micro-nutrients + SAR at two levels (Yes or No) and 2) insecticide applications based on scouting at two levels (Yes or No). Plant tissue, ACP adults, and nymphs (when available) are collected for PCR analysis every 4 months and results plotted and evaluated using quantitative geostatistical analysis. Incidence of positive trees was 33% in November 2008 and April 2009 and their distribution did not change over the two dates. With respect to the ACP, our results indicate significant differences in populations between insecticide-treated and untreated trees throughout the year, which maintained differences up to 26X despite close proximity. In contrast, the micro+SAR treatments are not having an effect on psyllid populations.
Specific hybridization technology using non-radioactive digoxigenin-labeled probes specific for HLB pathogen to detect the onset of HLB infection and its progression in citrus trees in the field is being developed. As outlined in the previous quarterly report, several regions of the HLB pathogen were identified, based on the sequence information of the HLB pathogen and were used to design specific primer pairs for the detection of HLB in infected citrus trees and psyllids. The amplified products were highly specific for the HLB DNA from HLB infected citrus plants. Probes were designed to these regions. Digoxigenin labeled single stranded RNA probes and PCR based DNA probes were generated and used in dot blot hybridization against nucleic acids isolated from the HLB infected citrus and Psyllids carrying the HLB pathogen. A linear relationship of the signal was observed in the dot blots with increasing amounts of DNA used. However, in the tissue blots from HLB positive citrus plants (from the green house) the hybridizations are not consistent. Tissues blots from some sections were positive but some were not. This probably is because of the uneven distribution of Las that is normally observed with HLB infection and the low titer of the pathogen. We are currently optimizing the tissue blot hybridization using high specific activity probes and multiple probes if necessary. The procedure for isolating the nucleic acid from single psyllids were optimized, and we have been able to amplify Liberibacter specific amplicons from single Infected psyllids using pairs of Las specific primers. The primer pair designed based on the tufB region of the Las genome was observed to be very specific and gave consistent amplification from the psyllids. We plan to use this primer pair to generate non-radioactive probes for the detection of Las in psyllid populations under laboratory and field conditions. Current work: Optimization of the citrus tissue blot hybridization on nylon membranes using Las specific non-radioactive DNA and RNA probes. Develop similar technology with psyllids.
The first year summer tests are concluded with mixed results. Over 300 trees were treated with the herbicide formulas Arsenal (Powerline) or Clearstand using 1 to 6 or 1 to 7.5 dilutions. These rates had a very poor percentage kill and required retreatment. For summer use, trunk applications of these herbicides will have to be in the 1 to 1 or at most 1 to 3 dilution rates. Next summer 1to 1, 1 to 2 and 1 to 3 dilutions will be tested. Fall tests are showing good results again. Alternatives to 1 1/2 inch blade cuts are being tested. Manual power saw blade cuts to different depths with two different herbicide dilutions have been made for evaluation. An electro-hydraulic mechanized tree trunk incision clamp system was developed and tested on a 4 X 4 ATV. Three problems were identified in the initial tests. First, the systemÕs gross weight proved too much for the ATVÕs front end suspension. Second, the systemÕs hydraulic motor was damaged, therefore resulting in erratic actuator operating pressures. Third, the clamping mechanism design was too vulnerable to operating stresses: The main actuator was not able to produce enough compression pressure. The incision blades and their corresponding compression angles were not adequate to reach the required trunk incision depth. To help resolve these issues, the entire system was transferred onto a utility tractor, which its front end suspension is capable of enduring the systemÕs gross weight. The system shares the same hydraulic pump as the tractor uses for its end loader operations, therefore eliminating erratic actuator operating pressures. A larger bore cylinder and a revised clamping mechanism design, including incision blade gaps and reduced compression angles, improved the trunk incision depth, which should be adequate for the herbicide spray application. This system should be completed and tested with herbicide sprays in November. Aerial sprays applied by the South Florida Water Management District to 600 acres of abandoned citrus were evaluated about 60 days after treatment this summer. Many trees were successfully killed, but in two or three blocks where scion and rootstocks differed between different ages of trees not all of the trees were killed. There small plot spray tests were not as successful as their earlier test last winter. Again, it appears that sprays during active growth are not as successful. There are 40 acres remaining in this test area and plans call for applying at least 4 treatments with 2 replications (5 acre plots) in order to evaluate some surfactants to improve efficacy. Also, additional chemicals will be tested.
Objective (2) Employ qPCR to detect Ca. Liberibacter presence (or absence) in the experimental adult psyllid cohorts given a range of acquisition-access periods as immatures or adults. Objective (3) Employ qPCR to quantify Liberibacter titer in psyllids and dissected organs, and in subsamples of the ÔtiteredÕ cohort, define the gross association of Ca. Liberibacter in thick sections by fluorescent in situ hybridization to develop a Ôgross anatomical road mapÕ of Ca. Liberibacter accumulation in key organs, tissues, and cells. With the completion of our new quarantine greenhouse, we are now maintaining 12 colonies of infected psyllids, 8 on budded Citrus, and 4 on Citrus infected by psyllids collected in the field. These colonies allow for a baseline of high HLB titer when sampling all instars as needed. New information from a recent publication (Inoue et al 2009) indicates that the best approach for determining organ-to-organ movement of C. Liberibacter in the host body is to work with 4th and 5th instars transplanted from noninfected plants to infected plants. To accommodate this finding, single branches were selected with enough leaves to allow for removing some for qPCR while at the same time hosting adjacent, transplanted larvae. These larvae were confined to the branches in such a way as to maintain control and vigilance during their AAP. Cohorts were given AAP on post-flush leaves for 1 hr to 24 hrs, then processed for qPCR and both flourescence and electron microscopy. Accompanying this approach, cohorts of 2nd, 3rd, 4th, 5th and adult instars were sampled directly off of different portions of infected plants and processed the same way. This series of samples will best represent actual proliferation profiles, since, in the field, larval mobility is negligible, and late instars are theoretically preinoculated from their early instar ontogeny. This inherited inoculum would build up to high titers along with the inoculum they imbibe themselves. We expect that amidst such a broad series of confined, transplanted larvae, incipient infiltration of bacteria into the digestive system will be seen, and will allow us to determine the organ-to-organ, or organ section, sequence of proliferation. We expect that in samples coming directly off of infected plants, the bacteria will have had time to build up to high titers inside the host, and the routes bacteria took during proliferation will be obfuscated using the current FISH labeling system. Twenty specimens of leaves and psyllids were sampled for qPCR from areas of the host plants from which the study specimen lots came. Eleven lots of psyllids were collected for microscopy, with each lot divided into two sublots, one for FISH, the other for TEM. The first TEM investigation of guts and glands of infected 5th instar larvae is in progress at this time. Inoue, H., Ohnishi, J., Ito, T., Tomimura, K., Miyata, S., Iwanami, T. and Ashihara, W. 2009. Enhanced proliferation and efficient transmission of Candidatus Liberibacter asiaticus by adult Diaphorina citri after acquisition feeding in the nymphal stage. Annals of Applied Biology 155:29-36.
Objective 1: We conducted tests with nutrient analyses of leaf samples collected from HLB-infected and healthy trees to establish relationships which could be used for (early) diagnosis of HLB. A protocol was established to correct the distortion of leaf nutrient data caused by fluctuations in leaf dry weight (DW). Starch accumulation in symptomatic leaves can significantly alter the interpretation of nutrient status when diagnosed on a DW concentration basis. Conversion of leaf nutrient data to a leaf area basis is one accepted approach which we are using for correcting the influence of DW fluctuation. The Diagnosis and Recommendation Integrated System (DRIS) is another approach to leaf nutrient diagnosis which eliminates any erroneous background noise due to changes in DW. In DRIS, the DW values simply cancel out in a series of nutrient concentration ratios. Thus nutrient interpretation with DRIS considerably reduces the bias from undesirable nutrient concentration or dilution effects due to uncontrollable changes in leaf tissue DW. Furthermore, versions of the DRIS computations are available which can estimate an index of the leaf DW (DWI). In our studies with HLB-infected blotchy mottled leaves where starch accumulation caused DW to increase, the DWI calculated by DRIS was correlated with measured DW. Analytical labs do not routinely report DW for leaf samples, but by using DRIS, we are able to calculate DWI and obtain an estimate of undesirable DW changes which we need to be aware of. Furthermore, DWI may be valuable for diagnosing early onset of HLB in asymptomatic leaves even before PCR methods can detect the Candidatus Liberibacter spp. DNA. Objective 2: An HLB infected commercial citrus grove near Haines City, Florida has been located for field trials to determine the effects of remedial applications of plant nutrients on HLB. The experimental design for this trial has been established and includes standard dry fertilization with microsprinklers, and drip irrigation with continuous liquid fertilizer injection. Overlaid on these primary treatments is a series of micronutrient foliar spray applications. The fertigation treatments have been established for approximately 6 weeks and are functioning well. The foliar treatments have been decided upon and the first application is set to be made the week of October 19. Objective 3: The greenhouse for the hydroponics experiment has been retrofitted to meet IFAS HLB requirements. The hydroponics systems has been designed and is in place in the greenhouse. Plant material has been received from a commercial citrus nursery and the plants to be infected are currently being inoculated with HLB. Once the infected buds are established in the trees, all of the plants will be transferred to the hydroponics system and established on a complete nutrient solution until symptom development takes place on the inoculated plants, at which time the various nutrient-altered treatments will be imposed.
Reducing the amount of vegetative growth produced annually by citrus trees in Florida would reduce the opportunities for Asian citrus psyllid reproduction and thereby the spread of Hunaglongbing. This could be done in citrus without detrimental effects on yield because citrus trees in Florida produce an excess of leaves above that required to support maximum fruit yield. Excess tree growth is routinely controlled through hedging, but little research has been done to examine the effects of hedging on vegetative growth. Recent research in Florida has shown that branch re-growth can be reduced when hedging is performed in fall under Florida conditions because of the onset of cool temperatures. Additionally, research in Florida indicates that late-summer hedging may be able to synchronize a final late-season flush and thus, reduce new flush leaves present during the winter to support over-wintering psyllids. In other fruit crops, such as apple, where excessive vegetative growth can be problematic plant growth regulators (PGRs) are routinely used. However, the use of PGRs in citrus has been limited to influencing fruit development and for maintaining post-harvest quality. PGRs not only control vegetative growth, but also offer the potential to reduce insect pest populations either by reducing pest-required vegetative growth or by altering host plant metabolites or nutrition. A greenhouse trial of six different plant growth regulators and their effects on citrus tree growth and psyllid oviposition has recently been completed. While data analysis on this trial is not yet complete, early indications are that four of the six PGRs successfully reduced the growth of citrus trees and the products had varying effects on psyllid behavior, from dramatically reducing oviposition to increasing oviposition. The results of this trial are very promising and future trials are being designed to build on these findings. In addition, a wide range of PGRs are being screened in ongoing greenhouse trials for toxicity and other adverse effects. These data will be used in designing field trials during spring 2010.
This is a continuing project for which funding was released this year on 13 April. Tamarixia radiata colonies from south China, North Vietnam and Pakistan established in quarantine have now been approved by USDA-APHIS and DPI for release into the environment. Additionally, we obtained 3 more shipments of mummified nymphs of D. citri from Pakistan this quarter to refresh the Pakastani colony of T. radiata and establish an additional colony of the parasitoid Diaphorencyrtus aligarhensis at DPI ,Gainesville. For our second objective, a collaborative study with Dr. Norman Barr, USDA-ARS Mission TX, on the genetic characterization of T. radiata strains from USA and some other regions of the world was completed and is in press for publication. Using the genetic markers being developed by Dr. Barr, we hope to track the establishment and performance of different T. radiata in the field. We are consistently increasing the production of parasitoids and continuing to release and evaluate in the field (Objectives 1,3). Between March and September 2009 we produced 42,000 T. radiata wasps that were used to maintain our colonies, initiate the colony at OrangeCo, conduct experiments here and at Lake Alfred, and release in experimental, conventional, and organic groves. Twenty three percent nymphs were parasitized by T. radiata in August-September in a block of 1 m tall citrus plants at the SWFREC, Immokalee. Nymphs recovered on sentinel plants in the conventional grove were 52% parasitized by T. radiata in April but none in August and September. Diaphorencyrtus aligarhensis, an endoparasitoid of D. citri from southern China is also being reared and released throughout the state. A total of 2400, 1325, and 5050 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. In August-September 2009, 1500 wasps were released in dooryard Murraya and citrus and in organic groves, however, none were recovered. We are also monitoring the populations of psyllids, predatory mites, parasitoids, and other natural enemies in an organic grove in Lake Wales, FL., where we released over 0.5 million predatory mites (Amblyseius swirskii) on mature orange during bloom. Predatory mites, mostly Typhlodromalus and Euseius species averaged 3 and 5 per two tap samples per tree in March and April, respectively, declined to 1 per sample during May-June and < 1 during July-August. Psyllids per two tap samples averaged 1 during March-April, 3 in May, 1 in June, and < 1 in July and August. Incidence of parasitism is low throughout the state, presumably due to widespread use of insecticides for psyllid control; thus the need to augment populations of T. radiata. For objective 4, we assisted Orange Co. mass rear T. radiata, made several presentations and published our findings to reach the target clientele. An International Tamarixia Workshop is being organized with APHIS for Feb 2010 in McAllen TX. 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 (in press). 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.
This is a 3-year project with 3 main focal points: 1. To build a foundation of understanding of the host- Candidatus Liberibacter asiaticus (LAS) interactions that involves testing multiple genetic variants of citrus, examination of environmental and seasonal effects on symptoms and pathogen replication and movement, and understanding effects of genetic variation in the pathogen system, so that better methods of controlling the vector, detecting and monitoring the disease, and growing trees will allow better production of citrus; and, 2. To develop an understanding of how Las interacts with citrus genotypes to cause disease in sensitive varieties and to not cause disease in tolerant varieties in the hope that this information will lead to the development of approaches to produce citrus economically in the present situation with citrus greening in Florida; and, 3. To provide knowledge and resources to support and foster research in other laboratories. A substantial number of projects to be supported are based on our research and reagents (propagated healthy and HLB-infected plants, HLB inocula, nucleic acid extracts, etc.) supplied by our laboratory. We view this as one of our most important goals. We have developed a containment plant growth room to allow natural infection of citrus trees by psyllid inoculation. We are testing a series of citrus relatives for preference and inoculation by aphids. This test includes a series of different sources of poncirus. This test is at the 4 month stage. Preliminary observations suggest that psyllids are able to acquire Las from exposed plants months before PCR detection and before development of symptoms. Preliminary observations suggest that psyllids prefer to feed on older infected plants compared to younger plants with new flush, although reproduction occurs on the new flush. We also have a series of experiments testing the susceptibility of poncirus and latipes in the greenhouse. Most plants remain PCR negative after graft inoculation although the living inoculum is definitely positive. Yet an occasional plant becomes PCR positive. We continue to investigate whether this is a genetic response that can be used to produce resistant or tolerant trees. Similar experiments are being done by psyllid inoculation. A wide range of citrus relatives have been inoculated with Las and are being evalutated in the greenhouse. We are also screening a range of elite lines for the citrus improvement group and a series of transgenic trees. All of these experiments are underway. We also continue to be be a resource to supply infected trees and infected psyllids to several other research laboratories.
This is a continuing project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. With the loss of trees due to canker eradication and development and the continuing losses to greening, there is a major concern whether sufficient fruit will be produced to keep processing plants open. We are approaching this problem in two ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen to focus on antibacterial peptides because they represent one of the few choices available for this time frame. Secondly, we are developing virus vectors based on CTV to express the antibacterial peptides in trees in the field as an interim measure until transgenic trees are available. We think that this approach could be used beginning 2-3 years from now and until probably 15 years from now when resistant trees should be available. The milestones for this year’s research are: 1) Continue to screen peptides for activity against Las in citrus trees using the CTV vector. We have completed one large screening of antibacterial peptides against Las in sweet orange trees. Eighteen different peptides were tested in this experiment. The trees were infected with CTV expressing the different peptides and two months later were inoculated with HLB by grafting. We found three peptides that allow much better growth of infected trees. Some trees had no symptoms and no detectable Las, some trees had no symptoms and low levels of Las, and other trees had leaf symptoms but continued growth of the trees with normal levels of Las. This experiment also demonstrated that leader peptides for the export of the peptide from the cell from which it was produced is not needed for HLB but is needed for citrus canker. We have developed systems to test the peptides under more natural conditions. We are concerned that graft inoculation of HLB into the trunks of small trees is a too severe challenge that might cause peptides that could work in the field to be missed. We have established a containment plant growth room in which psyllids inoculate the plants expressing the peptides. An advantage is that the psyllids feed on the areas of the plants with the highest levels of expressed peptides. This experiment is at the 4 month stage. This experiment also is evaluating a series of transgenic plants for resistance or tolerance to HLB. 2) Begin high-throughput screening in tobacco against solanaceous liberbacter. (Falk, UC Davis) The subcontract has just been completed to UC Davis and the work is beginning. 3) Improve CTV-based vector to produce 2-5 peptides and to overcome cross-protection. We have built several new versions of CTV vector and have shown that two foreign genes can be expressed efficiently at the same time. We are making progress in understanding the process of cross protection to allow development of a CTV vector that will allow subsequent application of antibacterial gene products. 4) Examine survival of peptides in fruit and juice. We have developed the plants for these assays, but this project is just beginning. 5) Prepare trees for year 2.
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 metagenomics approach, and published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin in USDA-ARS, Parlier, California, we have obtained approximate 1.2Mb, a nearly complete genome of Ca. L. psyllaurous with less than 20 contigs, which has ca. 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. Preliminary comparison revealed significant difference between Ca. L. asiaticus and Ca. L. americanus. 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. We have characterized the ATP/ADP translocase of Las, and proved its function in the heterologous E. coli system. We are currently developing a antibody-based “drug” to target this protein, aiming to disrupt the life cycle of the Las bacterium. 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.
To demonstrate that Candidatus Liberibacter bacterium is the causative agent of Huanglongbing disease (HLB), or greening, KochÕs postulates need to be performed with a pure culture of Liberibacter and is still challenging various research laboratories. We want to obtain a primoculture of Candidatus Liberibacter by co-culturing the bacteria with insect cells to study Candidatus Liberibacter physiology, metabolism, virulence and its interactions with the insect vector. Indeed Liberibacter is vectored by psyllid insects and is able to proliferate inside the insect. We therefore hypothesize that insect cells could act like feeder cells, providing nutrient complements in a continuous way but also a favorable environment to Liberibacter development. We tested various insect cell lines to in vitro culture Candidatus Liberibacter asiaticus (LAS), the Asian form of HLB disease, also found in Florida. We tested Lepidoptera cell lines (Mamestra brassicae and Spodoptera frugiperda) and Diptera cell lines (Drosophila melanogaster). We inoculated this cell cultures with different inoculums (from infected Macrophylla, Lime or Periwinkle plants). We checked for the presence of LAS in inoculated cell cultures by direct and Nested PCR. Current results: -Inoculums from LAS infected citrus (Lime from Vietnam) were the best of the inoculums tested. -To this day we didnÕt detect any Las in Mamestra (hemocyte and ovarian cells) or Spodoptera (hemocyte cells) cell lines. -We detected LAS in two drosophila cell cultures by direct PCR and the signal was maintained after 6 transfers of the culture, over more than 6 weeks (and still running). It means the bacterium is multiplying. -In order to reach higher bacterial concentrations, we are currently testing complementation of the Drosophila cell culture medium with various sugars, vitamins or trace elements that could be found in citrus/periwinkle phloem. We analyzed metabolic pathways potentially encoded by the released Liberibacter genome sequences to improve growth conditions and to define limiting factors and/or growth inhibitors. We so far selected 6 complements and got positive results with 3 of them. Current work: -The detection of LAS varies over time and transfers in drosophila cell cultures; it seems to be linked to drosophila cells concentration. In order to define this link, we are setting up multiplex qPCR assays to monitor the ratio of LAS vs. drosophila DNA over culture time. -We are combining complements to the insect cell culture media and looking for new ones to improve the bacterial concentration in the insect cell cultures. -We are analyzing sugar and amino-acids variations (in particular depletion) in insect cell culture media over culture time to identify potential LAS growth limiting factors.
Funds for this study were received March 18, 2009. Since these experiments take place early in the season when fruit require degreening to meet market requirements, the bulk of the funds will be used in October and November when late summer/fall rains result in small lesions on green fruit and washing the fruit may inhibit degreening. As part of another project, we recently built a research grading line that will be used for this study and allow evaluation of a number of washing treatments and other variables on the ability to effectively grade the fruit to find canker lesions and other blemishes. Modifications to our research citrus washing line are complete to evaluate different washing techniques and we’ve arranged to run fruit over a local commercial packinghouse wash line. Preliminary results from earlier experiments were presented at the FSHS meetings June 2009 and a manuscript submitted and accepted as a peer reviewed publication in the next 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 (HPW) compared to longer (1 to 2 min) brush washing only (no HPW). On October 6th, 2009 we began our first set of experiments using Fallglo tangerines that were either washed on a commercial line (the entire brush and HPW, brush washer only, HPW only, or . or . time on the HPW), washed or washed and waxed (carnauba) on our research line, or were not washed at all but only passed over rollers on a grading table. Fruit from all these treatments were degreened under simulated commercial conditions and color development and weight loss measured almost daily. The fruit continues to be evaluated. The week of October 19 we are scheduled to conduct another, larger study using navel oranges and Ruby Red grapefruit. In all, we plan to evaluate different methods of washing on fruit coloration during and after degreening, total required degreening time to reach acceptable color, and the affect of fruit cleanliness, presence of other peel markings (i.e., windscar, melanose, etc.), speed of fruit travel, lighting conditions, roller color, grading area width, and the number of graders per unit area on the ability to detect fruit with canker lesions and other surface defects.
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