Report for period ending 4/30/15 (FINAL REPORT) Work continued on our previously reported efforts to determine the level of imidacloprid in leaf tissue required to prevent ACP phloem feeding behaviors. In particular we have focused efforts at measuring the levels of neonicotinoids in different parts of the leaf tissue and how that changes with time. While this work is still underway, progress to date does show uneven distribution of neonics within a leaf that changes over time. There also appears to be some difference in movement patterns between the different neonics which may be due in part to their differences in water solubility. Further work is ongoing to better characterize these movement patterns and use that information to then determine when retreatment of plants is needed in order to ensure psyllids are not able to feed successfully on treated plants. Use of EPG techniques to determine the level of imidacloprid needed to prevent phloem feeding behaviors has continued. While still ongoing, we have narrowed down the LC50/LC90 for feeding disruption and should be able to be begin work soon for the other neonicotinoid compounds. This work will be continued in the coming year to help refine the use of soil-applied nenicotinoids for protecting young trees from HLB via disruption of psyllid phloem feeding behaviors.
Report for period ending 3/31/15 Activities of the CHMA program assistant (1/1/15-3/31/15): During this reporting period, the CHMA assistant provided support to the CHMA program by attending various local CHMA meetings as well as statewide and multi state meetings. Specifically, Mr. Page attended 12 meetings including the following: CHMA grower round table meeting in Sebring (1/7/15), Southeastern Fruit and Vegetable Conference in Savannah (1/8/15 – 1/10/15), Highlands county OJ break (1/22/15), CHMA Hardee county grower forum (1/23/15), Florida citrus show in Ft. Pierce (1/27/15 – 1/29/15), Hardee CHMA meeting (1/30/15), AFVGA (2/5/15 – 2/7/15), IRCHLB meeting (2/9/15 – 2/13/15), CHMA meeting in Ft. Pierce (2/18/15), Highland county grower forum (3/4/15), IRCHLB growers day at CREC (3/12/15), and East coast CHMA organizational meeting (3/19/15). Mr. Page also hosted visitors from Brazil and China who visited to learn more about the CHMA program and exchange information about programs being utilized in other countries. In addition to helping plan and CHMA meetings, Mr. Page continued to keep the CHMA website updated, sending out updates to the individual CHMA listserves, adding names to the listserves, and updating the ACP trend graphs and CHMA rankings. During this period, Mr. Page also worked with USDA ApHIS regarding technical glitches with the scouting data and with Tim Riley’s assistance got this resolved. He also worked with the software company that designed the CHMA mapping program to fix some problems with the program following updates. Mr. Page also continued to work with the new Citrus Economist at CREC to collect data from growers in different CHMAs and use that data to analyze citrus production in trends in areas with and without functioning CHMAs to determine the value of CHMAs in terms of increased production value.
Report for period ending 4/30/14 (FINAL REPORT) During the period of 12/31/14 to 4/30/15 this project focused on completing the season-long field trial examining the potential for movement of neonic residues into nectar to determine whether there was any potential risk of season-long repeated applications of soil-applied neonics to pollinators. These studies were conducted at the request of the EPA in order to maintain the expanded labeling of soil-applied neonics for use in citrus. This field work began in April of 2014 with neonic applications being applied to assigned blocks of 5′ tall trees every 6 weeks. The final plots were treated in October of 2014. Because our previous work suggested that neonics are likely to move into nectar at differing rates, we examined seven different schedules (treatment rotations) of neonics applied every 6 weeks. The rationale for the different rotations was to determine if there was a potential risk identified, could that risk be mitigated by changing the schedule of products applied. During the bloom period in spring of 2015, approximately 200 flowers were collected as pooled samples from ten trees in each plot. There were five replicate plots for each of the 7 rotations examined plus five untreated plots for a total of 40 plots sampled. The blooms from each plot were collected into paper bags and taken back to the lab where the nectar from each bloom was collected using capillary tubes until at least 1 ml of nectar was collected from each plot. The nectar collected was then extracted and frozen for subsequent analysis using LC/MS/MS. Analysis of the nectar showed as expected that there was difference between neonics in likelihood of movement into citrus nectar. However, for all neonics rotations tested, the average expression of residues in nectar was far below the level of concern to pollinators proposed by the EPA. Thus, no significant risk to pollinators was found in this season-long field trial where applications were ceased 2 months prior to bloom (per label directions) for trees 5′ or greater in height. These results confirm our finding in previous studies that under FLorida growing conditions and soil types, repeated use of neonics (every 6 weeks) DOES NOT have a cumulative negative effect in terms of increased neonic expression in nectar.
Report for period ending 6/30/15 (Final Report) Activities of the CHMA program assistant (4/1/15 – 6/30/15): During this reporting period, the CHMA assistant provided support to the CHMA program by attending various local CHMA meetings as well as statewide and multi state meetings. Specifically, Mr. Page attended 11 meetings including the following: Citrus grower’s Institute in Avon Park (4/7/15), citrus extension planning retreat (4/8/15 – 4/9/15), CHMA meeting in Avon Park (4/15/15), Hardee CHMA meeting (4/17/15), CHMA meeting in Immokalee (4/21/15), Volusia county CHMA meeting (4/24/15), CHMA meeting in Wauchula (4/24/15), Lake county grower round table (5/20/15), grower round table discussion in Wauchula (5/21/15), Florida Citrus Mutual Annual meeting (6/17/15), and the East coast CHMA planning meeting (6/24/15). In addition to helping plan and CHMA meetings, Mr. Page continued to keep the CHMA website updated, sending out updates to the individual CHMA listserves, adding names to the listserves, and updating the ACP trend graphs and CHMA rankings. Mr. Page also continued to work with the new Citrus Economist at CREC to collect data from growers in different CHMAs and use that data to analyze citrus production in trends in areas with and without functioning CHMAs to determine the value of CHMAs in terms of increased production value. Over the past year of funding, grower interest and participation in the CHMA program has continued to remain high. At present there are 48 CHMAs functioning across the state with additional groups functioning as CHMAs but not necessarily formalized on the CHMA website. The CHMA program has continued to keep information up-to-date on its website that has been used to document areas where the program is working well and areas where improvement is psyllid control is needed through better coordination of activities. To further document the success and benefits of the CHMA program, information has been gathered from growers from around the state to compare production costs and fruit yields. The results has shown that participation in a CHMA can have a positive effect on fruit yields. Dr. Ariel Singerman presented this information recently at the Florida Citrus Mutual Annual conference in Bonita Springs. These data will help to further increase and motivate grower interest in the CHMA program in the coming year. Plans for the coming funding cycle include further analysis of this type of data to show the continued benefit of participation in a CHMA over time.
Report for period ending 12/31/14 Activities of the CHMA program assistant (9/30/14-12/31/14): During this reporting period, the CHMA assistant provided support to the CHMA program by attending various local CHMA meetings as well as statewide and multi state meetings. Specifically, Mr. Page attended 12 meetings including the following: FFA Meeting (7/2-7/3), American Sociey for Horticultural Sciences meeting (7/25-7/29), Small farms Conference (8/1-8/2), Citrus Expo (8/12-8/14), Gulf CHMA meeting (8/28/14), Grower meeting in Sebring (9/3/14), Hardee CHMA meeting (9/5/14), meeting with county agents (9/12/14), county agent Oj break (9/24/14), Landscape tradeshow (9/25-9/26), FDACS CHMA planning meeting (9/29/14), Hardee county CHMA roundtable (9/30/14). In addition to attending meetings, Mr. Page continued to spend time working with CHMA captains and other interested growers on a daily basis identifying trouble areas, planning programs, and updating website information. Mr. Page also worte an article for Citrus Industry Magazine to update growers on the success of CHMAs statewide and also began working with the new UF/IFAS Citrus Extension Economist to gather data from the CHMA program to conduct an economic analysis of the benefits of CHMA participation.
Report for period ending 12/31/14 Work continued on our previously reported efforts to determine the level of imidacloprid in leaf tissue required to prevent ACP phloem feeding behaviors. This work involved both field studies and concurrent laboratory-based studies. In the field, trees of varying size classes were treated with the recommended rate of imidacloprid. At 2, 4, and 6 weeks post-treatment, leaf samples were collected and analyzed for imidacloprid concentration. The levels of imidacloprid detected in leaf tissue ranged from a low of 2.5 PPM (2,500 PPB) to a high of 12.3 PPM (12,300 PPB), varying with tree size and generally decreasing with increased time following application. However, in some field plots, ACP were observed feeding on treated plants 2-3 weeks following imidacloprid application. Tests of the ACP did not show any decline in susceptibility to imidacloprid where collected from trees with an average level of imidacloprid in leaves of 2-3 PPM (2,000-3,000 PPB). This was in direct contrast to previously published studies suggesting that 200-250 PPB (0.2PPM) is sufficient to control ACP. This finding was examined further in the laboratory by feeding ACP on citrus leaves with a known level of imidacloprid. Our work had previously focused on developing a system for obtaining leaves with a desired level of imidacloprid residues delivered systemically (not foliar application). Using the methods we developed, we fed adult APC on leaves containing 0, 6, 12, 60, 120, and 600 PPM imidacloprid. The results of these feeding studies yielded inconsistent results with as much as 50% survival at the 600 PPM level in some replicates. Based on these results, we suspect that the imidacloprid may be unevenly distributed in the leaf tissue over time, allowing areas of lower concentration for ACP to feed. We are now examining this hypothesis by measuring imidacloprid in different parts of the leaf (center vs leaf margin) to determine movement over time and potentially how long full leaf protection may last.
Report for period ending 9/30/14 During the period of 9/30/14 to 12/31/14 we continued work analyzing leaf tissues in order to more accurately quantify the levels of neonicotinoids present in leaf tissues. Leaf samples from more than 500 trees of different size classes treated with different rates of neonics were ground in liquid nitrogen and any potential pesticide residues within extracted, the extracts then held in the freezer for later analysis. Extracts were then analyzed using LC/MS/MS to quantify the levels of parent compounds and their metabolites in the leaf extracts. While analyses of all samples have not yet been completed, preliminary results show a consistent trend in the quantity of neonic present and tree size/rate of product applied. This work will be continued. Field trials were also continued to build upon work in previous years examining whether soil-applied neonicotinoid applications will move into the citrus flowers during bloom (in particular nectar) and pose any risk to pollinators. These studies are being conducted at the request of the EPA in order to maintain the expanded labeling of soil-applied neonics for use in citrus. Should any potential risks be identified, this project also will identify alternative timing of applications that could be recommended to mitigate any risk that might be identified. This field work began in April of 2014 with neonic applications being applied to assigned blocks of trees every 6 weeks. The purpose was to determine if the neonic applications would accumlate in the plant over time causing excessive risk to pollinators through expression in nectar. Because our previous work suggested that neonics are likely to move into nectar at differing rates, we examined seven different schedules (treatment rotations) of neonics applied every 6 weeks. The final plots were treated in October of 2014. During the next reporting cycle, the plots will be in bloom and we will collect nectar for analysis of potential neonic residues.
July 2015 The objectives of this proposal are 1) to determine if a) leaf litter biodegradation treatments reduce Guignardia spp. pseudothecia and improve control afforded by routine fungicide applications; b) if biodegradation is affected by the current fungicide application practices; and c) whether the biodegradation treatments will affect current citrus best management practices (BMP); 2) to determine the seasonal dynamics of leaf litter inoculum load in varying management regime intensities and how environment affects pseudothecia production in the leaf litter; 3) to test if the resistance to black spot in the leaves and fruit in sour orange is correlated and under simple genetic control through laboratory and field testing of progeny of sour orange crosses in both Florida and Australia. Data was collected for the large field trial and preliminary analysis was begun. The fruit incidence and severity was collected. The manuscript from the experiment to look at the effect of bagasse is still in preparation. Forty leaf litter samples were collected and assayed between January and April 2015 in a Florida grove to obtain a better understanding of the seasonal patterns of spore production in citrus leaf litter. Each sample contained 25 leaves collected from 40 randomly selected trees in a grove near Immokalee in Collier County biweekly. All leaf litter samples were evaluated under the dissecting microscope to assess degradation level. One or more pycnidium and/or pseudothecium from each leaf was examined under the microscope until positive Phyllosticta spp. fructification was identified. Leaves without Phyllosticta spp. fructification were recorded and discarded. Spores were collected by immersing leaf litter in 0.02% Tween 20, and quantification was performed by counting the spores using a haemocytometer. Preliminary weather data were obtained from the Florida Automated Weather Network (FAWN). In Australia, the proportions of the various Phyllosticta fruiting bodies (spermogonia, pycnidia and pseudothecia) were observed by microscopy. The data show a similar pattern to the 2013-14 season, with pycndia dominating at the beginning of the season, then becoming less frequent. Similar peak in pseudothecia production to the 2013-14 season can also be observed, though this peak was smaller and later in 2014-15. In the germplasm inoculation experiments the susceptible controls are starting to show symptoms. The negative controls have not. Sampling will continue for several months. In the parallel microplots for enhanced leaf litter decomposition, it was found that no one treatment over time consistently had the lowest number of fruitifications and all leaves showed similar rates of decomposition. This is similar to what we previously found when examining the rate of decomposition. The amount of water used for fungicides is very high and run-off very common. When the effect of run off was evaluated, there was no improvement for the number of fruiting bodies produced.
July 2015 The objectives of this proposal are 1) to determine if a) leaf litter biodegradation treatments reduce Guignardia spp. pseudothecia and improve control afforded by routine fungicide applications; b) if biodegradation is affected by the current fungicide application practices; and c) whether the biodegradation treatments will affect current citrus best management practices (BMP); 2) to determine the seasonal dynamics of leaf litter inoculum load in varying management regime intensities and how environment affects pseudothecia production in the leaf litter; 3) to test if the resistance to black spot in the leaves and fruit in sour orange is correlated and under simple genetic control through laboratory and field testing of progeny of sour orange crosses in both Florida and Australia. Data was collected for the large field trial and preliminary analysis was begun. The fruit incidence and severity was collected. The manuscript from the experiment to look at the effect of bagasse is still in preparation. Forty leaf litter samples were collected and assayed between January and April 2015 in a Florida grove to obtain a better understanding of the seasonal patterns of spore production in citrus leaf litter. Each sample contained 25 leaves collected from 40 randomly selected trees in a grove near Immokalee in Collier County biweekly. All leaf litter samples were evaluated under the dissecting microscope to assess degradation level. One or more pycnidium and/or pseudothecium from each leaf was examined under the microscope until positive Phyllosticta spp. fructification was identified. Leaves without Phyllosticta spp. fructification were recorded and discarded. Spores were collected by immersing leaf litter in 0.02% Tween 20, and quantification was performed by counting the spores using a haemocytometer. Preliminary weather data were obtained from the Florida Automated Weather Network (FAWN). In Australia, the proportions of the various Phyllosticta fruiting bodies (spermogonia, pycnidia and pseudothecia) were observed by microscopy. The data show a similar pattern to the 2013-14 season, with pycndia dominating at the beginning of the season, then becoming less frequent. Similar peak in pseudothecia production to the 2013-14 season can also be observed, though this peak was smaller and later in 2014-15. In the germplasm inoculation experiments the susceptible controls are starting to show symptoms. The negative controls have not. Sampling will continue for several months. In the parallel microplots for enhanced leaf litter decomposition, it was found that no one treatment over time consistently had the lowest number of fruitifications and all leaves showed similar rates of decomposition. This is similar to what we previously found when examining the rate of decomposition. The amount of water used for fungicides is very high and run-off very common. When the effect of run off was evaluated, there was no improvement for the number of fruiting bodies produced.
The second year’s applications of low concentrations (1/4 rate) of Citrus Fix (2, 4-D) and MaxCel (Cytokinin) with or without ProGibb (GA) every 45 days to Hamlin and Valencia tree canopies at two locations in central Florida for the 2015-16 growing season are underway. Trees were sampled for flowering, early fruit set and spring flush numbers and leaves per shoot. Data is being analyzed and summer shoot development is now being measured. Fruit drop for the 2014-15 season was lower than for the Controls for some treatments. In these replicated plots none of these differences were significant for individual groves except for Hamlins in Haines City, but resulted in only a 2 % reduction from the Control for the 2,4-D plus Maxcel treatment. For pooled data the 2, 4-D plus MaxCel (cytokinin) significantly reduced drop by 6 % and 7 % GA plus 2, 4-D in Hamlins. In Valencia trials the only reduction was for the pooled data for 2, 4-D plus MaxCel, but only a 3 % reduction in fruit drop occurred. A second test of applications GA to roots of nursery trees is ready to start with HLB infected trees. The trees have been infected in a hot psyllid greenhouse. GA stimulated root growth on healthy trees, now we can determine if it can stimulate root growth on infected plants.
Flowering branches were selected for data collection in 4 Hamlin, 4 Valencia, two Murcott and two Sunburst blocks in various locations from Frostproof to Auburndale, FL. Vegetative and flowering buds were tracked for leaf and flower numbers and developmental stages for 2 branch units on each side of the tree row on three trees at each site. All locations have now passed through the various stages of flowering and spring flush development. For Hamlin and Valencia blocks vegetative bud break and leaf feather stage were mostly completed by February 6th, while most flowers were pinhead or popcorn stage. This indicates there is a small window to spray new flush before more effective pesticide sprays need to be stopped. This data will allow us to now make the first restructuring of the Citrus Flower Monitoring System.
Test groves were arranged with 6 growers with 14 different tests, 4 of which were sprayed by growers. Seven tests are reported in Hamlin blocks and 1 in a white grapefruit block. Seven tests were applied in Valencia blocks. Materials across all tests included Citrus Fix (2, 4-D). Pro-Gibb (GA3), Headline, Gem, Abound and a PGR extract (Ascend) that contains an auxin, GA and cytokinin. The Hamlin and grapefruit tests were applied in September or October, and the Valencia tests in November or December. Four Hamlin tests and the grapefruit test had reductions in fruit drop from PGR treatments. Three of these had ProGibb and Citrus Fix as the significant treatments (two Hamlin and one grapefruit trial) compared to the Control and one of these had Ascend as the significant treatment. Pooled data for the Hamlin tests indicated that GA + 2, 4-D significantly reduced drop 5 % compared to the controls while 2, 4-D by itself or in combination reduced drop 3 % and GA alone or in combination reduced drop 4 %. Of 6 tests that included Headline none had a significant difference between Headline and its Control. For 8 Valencia trials no significant differences occurred between treatments within a grove, but overall for pooled data 2, 4-D reduced drop compared to the Controls by 2 % (16 versus 14 % for the treated). For 1 versus 3 decline status trees, 1 rated trees were healthier and 3 were severely declined. Hamlins had 24 versus 27 % drop, respectively, while Valencia trees had 12 versus 14 % drop, respectively, for the decline ratings. The previous year Valencia trees had drop rates averaging 28 % and in three groves 1 versus 3 rated trees averaged 35 versus 48 % drop, respectively. The lack of a better treatment response this past year in Valencia trees may have been partly due to much less drop on untreated trees, 12-14 % compared to over 28 % in the previous year. In turn this lower drop rate may have been partially due to much less fall and spring water stress on Valencia trees in 2014-15. Further work on the relationship of water stress to fruit drop in HLB trees is planned for this coming fall.
The objective of this research project is to investigate and develop a potential non-phytotoxic, environmentally friendly film-forming ACP repellent solution for preventing HLB infection. In the last reporting period, two new OSCF materials, OS-SG 11 and 12 were synthesized based on the feedback from previous OSCF ACP infection studies. These materials were synthesized using EPA approved polymers and environmentally safe inert ingredients to increase rainfastness property of the OSCF materials. In this reporting period, we have completed safety analysis of OS-SG 11 and 12 using Vinca sp and Tomato sp. Studies included temperature/ heat trapping capability of the OSCF materials and phytotoxicity. Phytotoxicity studies were conducted in a Panasonic Environmental Test Chamber (Model MLR- 352H) to control light intensity, humidity and temperature cycling to simulate summer conditions (85% RH, 34oC) Both the new OSCF materials were shown to be safe at the applied rates. The chemical composition and interaction between the raw chemical components were confirmed using Fourier transform infrared spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR), respectively. The film-forming property and elemental composition of the OS-SG 11 and 12 were supported by the Scanning Electron Microscopy and Energy Dispersive Spectroscopy (EDX) techniques. The rainfastness of OS-SG 11 and 12 is being studied using industrially-acceptable rainfall stimulation assay using tomato sp as model plant system. Plants were grouped into 4 groups (0, 1, 2 and 3 rainfalls). Using a known flow rate, each simulated rainfall was estimated to have produced 4.3 gallons of water per rainfall. Plants were allowed to fully dry between rainfalls. After all rainfalls, all leaves were collected and analyzed for OSCF presence using Atomic Absorption Spectroscopy (AAS). Both the OS-SG 11 and 12 will be delivered for ACP repellent evaluation studies within the coming month.
Abandoned citrus groves in US citrus-producing areas are potential sources for both Asian citrus psyllid (ACP), and Candidatus Liberibacter asiaticum, the bacterium that causes Huanglongbing (HLB). In Florida and Texas, no active psyllid control is being implemented in abandoned citrus. Since ACP adults are highly mobile, they can disperse from abandoned to productive citrus groves. If not controlled, these psyllids will stymie the effectiveness of area-wide management programs aimed at containing the spread of HLB in commercial citrus. Insecticide-based strategies are not feasible for ACP in abandoned citrus, but biological control of ACP could come from a combination of native or introduced arthropods and pathogens. ACP is susceptible to a native entomopathogenic fungus, Isaria fumosorosea (Ifr). A south Texas isolate (Ifr 3581) is highly pathogenic against ACP and available as a spore formulation. Autodissemination is insect dispersal of a pathogen to members of its own population. For our project, we developed an autodissemination system for inoculating ACP with Ifr 3581spores and evaluated the effects of Ifr autodisseminators (dispensers) on ACP population level and movement within and between abandoned groves and managed groves under summer, fall and winter conditions in the Rio Grande Valley of Texas. During April to December of 2013, we evaluated seven different blends of synthetic aromatic compounds (mimicking flush volatile profiles of ACP host plants) for luring adult psyllids to Ifr dispensers with sticky surfaces. We found that the numbers of psyllids captured by dispensers augmented with one of the blends (Blend # 3) was 4 times greater than non-augmented dispensers (controls) and 3 times greater than dispensers augmented with the second most attractive blend. Blend # 3 was used for our 2014 field trials with dispensers coated with Ifr spores. During January of 2014, project leader and collaborators met at the Texas A&M Citrus Center in Weslaco, TX, to finalize dispenser design and Ifr 3581 formulation for field trials. Key components of our dispensers were pre-fabricated by AlphaScent Inc., a US supplier of insect monitoring systems, to facilitate the assembly of the large numbers of dispensers required for our field trials. Sufficient quantities of components for field trials were delivered by June of 2014. During June to July of 2014, we completed summer field trials at three different sites consisting of managed grapefruit or orange groves adjacent to abandoned groves. At each site, we evaluated the impact of Ifr dispensers on ACP movement and numbers in plots of abandoned citrus trees and managed citrus trees. For treatment plots, we hung pairs of dispensers coated with Ifr spore formulation and equipped with citrus-blend lures in trees on the edge rows of the abandoned groves. For control plots, we hung dispensers with only citrus-blend lures. Under summer conditions, we found that psyllid numbers and movement among abandoned trees were reduced in treatment plots. During October to December of 2014, we repeated our trials and found that ACP movement from abandoned trees to managed trees was greatly reduced by Ifr dispensers. Prior to dispenser deployment, the numbers of ACP adults trapped on managed trees in treatment plots and control plots were not significantly different. After dispenser deployment, weekly trap counts of ACP adults were 28-50 % less for treatment plots compared to control plots. Low psyllid presence at our trial sites during March to April of 2015 prevented evaluations of Ifr dispensers under spring conditions. We propose that deployment of Ifr dispensers at the end of the active growing season for commercial citrus in Texas can reduce the numbers of ACP adults moving from abandoned groves to managed groves.
Psyllid isolines: transmission evaluations are underway for four stable genetic isolines. These isolines have demonstrated high reproductive fitness in laboratory assays, with cultures from one location reproducing significantly more than laboratory psyllid colonies. Isolines from three locations (Labelle, Homestead, and Ft. Pierce) have dropped out since previous reports, likely due to inbreeding effects. Collections have been made to reestablish laboratory isolines from these locations. To date, Wolbachia screening of isolines has identified two Wolbachia sequence types. Efforts to clear Wolbachia from these populations are ongoing. Psyllid adults were fed an artificial diet containing dsRNA targeting their endosymbionts. PCR results suggest that some endosybionts may be inhibited by this approach; however, the overall mortality of psyllids that were fed on dsRNA solutions was high, reaching 90% after 5 d in most cases. Such a high mortality rate makes this approach unfeasible for clearing Wolbachia. An alternative approach, antibiotic clearing, has had more success. An antibiotic cocktail is provided to psyllid nymphs in an artificial diet for 5 d. Insects are transferred to citrus for oviposition. Three generations of offspring are fed an an artificial diet containing the antibiotic cocktail. l One objective of this proposal is germ-line transformation of psyllids. Asian citrus psyllid (ACP) eggs were injected with several DNA/sgRNA solutions. These included the pB[pIE1-DsRed/phspBac] transformation vector system, and a new attempt to test CRISPR/Cas9 gene-editing by knock-out of the Ultrabithorax (Ubx) and Antennapedia (Antp) genes using several guide RNAs. Due to ongoing issues with relatively low numbers of eggs for collection and low frequencies of hatching and survival to adulthood for injected eggs (Table 1 and 2), we are hopeful that the broadly functional and highly efficient CRISPR/Cas9 system will overcome these limitations. The Surveyor kit method that detects genome-wide insertion/deletions in response to Cas9/sgRNA injection has verified gene-editing using sgRNAs for both genes (Fig. 1), but we have not yet detected visible phenotypes in G0s (adults from injected eggs), but screening G1 ACPs is still in progress. While the system works, visible mutations require the germline genome to be affected (and not just somatic cells). New genes with more obvious phenotypes will be targeted in the near future, in addition to testing gene cassette knock-in insertions with fluorescent protein marker genes. Nymph mortality on double-stick tape: Mortality of ACP nymphs remains high when using double sided tape to hold eggs for injection due to nymphs sticking to the tape after hatching. Various glues were tested for egg adhesion without post-hatch mortality, with the best result using Tacky glue. However, while survival of hatched nymphs was improved, the number of hatchings was somewhat lower resulting in a similar result to the use of tape. Artificial diet: To provide an alternative to tape after hatching, and improve survival of nymphs until transfer to flush, 270 uninjected eggs were placed on the Pelz-Stelinski artificial diet of which ~55% hatched, though only a few nymphs appeared to feed on diet and 3 survivied 24 hr. Potato Starch: To determine whether hatched nymphs could be prevented from sticking to tape, the exposed tape was dusted with potato starch either immediately or 1 day previous to hatch. 144 eggs on tape were immediately dusted with starch with a hatch rate of 78.1%. Some nymphs were stuck to the tape bringing the total survival to 70.5%. (Control eggs not dusted had hatch rate of 71%., but all hatched control nymphs were stuck to the tape). 57 eggs were placed on tape and dusted 1 day before hatch with a hatch rate of 89.5%. Some nymphs were stuck to the tape bringing total survival to 84.2%. Thus, dusting with starch 1 day previous to hatch yield the best results and will be re-tested on uninjected and injected eggs.
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