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.
This research seeks to determine whether young trees infected with CLas and displaying typical HLB symptoms can be brought to maturity and produce an economically viable yield. This will be achieved by managing a 58 acre grove of 3-year-old Valencia / Kuharske Carrizo trees using a combination of three different foliar and three different ground applied nutritional programs. Factorial AxB treatments consist of A) ground-applied: 1) Liquid/dry+Ca (BHG standard), 2) Liquid+Ca, 3) Liquid/dry-Ca B) foliar-applied: 1) BHG standard-Ca, 2) BHG standard+Ca, 3) “Prescription”(+Ca). The prescription treatment was designed to be dynamic, customized for optimization, with feedback based on frequent leaf tissue analyses, visual symptoms, and the growth of the tree canopies and yield. There are six replications of treatments, with two being pure replications. This report updates the fruit yield and quality measurements of the 2014/15 season. Measurements and analyses include fruit yield (boxes/acre), and fruit quality as brix, acid%, ratio, pounds solids per box, fruit weight/size, no. of fruit per box, percentage juice, juice yield per acre, and pounds solids yield per acre. The trial average yield was 171.6 boxes/acre, which translates to 1.14 boxes/tree. This is still quite a low yield for 6-year old Valencia trees, which should be producing more than 2 boxes/tree at that age. As mentioned before, the economic productivity of HLB-affected young groves like this could be greatly enhanced if higher planting densities were utilized. Theoretically had this grove been planted at 8×18 feet (303 trees/acre), the average yield could be 344 boxes/acre. The trees have grown about half their normal rate due to HLB and therefore in 6 years have not properly utilized the amount of space allowed by a low planting density of 151 trees/acre. The only analyzed yield variate that showed a statistical treatment difference was the ratio, for response to foliar nutrition, shown below: FOLFERT* STD-Ca STD+Ca Prescrip 17.70 19.13 18.49 The ratio measured from fruit grown with foliar nutrient sprays including added calcium as calcium nitrate was significantly higher than the standard formula without calcium. The average pounds solids yield per acre was 781 lb/acre, which is considerably lower than 6-year old Valencia trees would be yielding in the absence of HLB. Again, a higher planting density (double) could have helped offset the losses incurred due to greening.
The overall goal of this 3-year research project is to efficiently deliver antimicrobial molecules into citrus phloem against HLB bacteria. In the past three years, we developed two nano-delivery systems (W/O or O/W) for foliar spray or bark-application and applied them in a field trial. Based on various physiochemical characteristics of oils, surfactants, and organic solvents, a novel oil in water (O/W) nanoemulsion was optimized to combat the citrus huanglongbing disease. This nanoemulsion was produced using a spontaneous emulsification method for the efficient delivery of ampicillin into citrus phloem by bark application. The nanoemulsion prepared from Cremophor EL (viscous oil), acetone (water miscibility organic solvent) and Span 80/Tween 80 (surfactant) exhibited small droplet size (13.68 0.26 nm). This nanoemulsion had an improved absorption rate (time of peak concentration (tmax) = 2 d, peak of maximum concentration (Cmax) = 71.9 ng/g) and bioavailability of ampicillin (Relative Bioavailability= 274.63%) in HLB-affected citrus, as compared with Amp solution alone (tmax=6 d, Cmax=56.4 ng/g and RBA=100%). The same nanoemulsion was used to deliver six antimicrobials to control citrus HLB by bark application. The results showed that Candidatus Liberibacter asiaticus in HLB-affected citrus was reduced to undetectable levels 6 months after the initial treatments of two agro-antibiotics (ACT and VA) and two beta lactam antibiotics (AMP and PEN), while SDX and ZS had less therapeutic efficiency. Based on the physiological and chemical characteristics of leaf cuticle, a transcuticular nanoemulsion formulation was developed to enhance the permeation of an effective antimicrobial compound against HLB disease through the citrus cuticle into the phloem via a foliar spray. The results demonstrated that efficiency of cuticle penetration using an enzymatic method (pectinase and cellulase) was dependent on the citrus cultivar and whether the tree was infected with Las, and it was more difficult to penetrate cuticles from valencia orange (Citrus sinensis) and HLB-symptomatic leaves. Of eight adjuvants tested, Brij 35 provided the greatest increase in permeability of the HLB-affected cuticle with a 3.33-fold enhancement of cuticular permeability over water control. An in vitro assay using Bacillus subtilis showed that nanoemulsion formulations containing Amp (droplets size = 5.26 0.04 nm and 94 1.48 nm) coupled with Brij 35 resulted in greater inhibitory zone diameters (5.75 mm and 6.66 mm) compared to those of Brij 35 (4.34 mm) and Amp solution (2.83 mm) alone. Furthermore, the nanoemulsion formulations inhibited Las bacteria in HLB-affected citrus in planta more efficiently than controls. Our study shows that our oil in water (O/W) or water in oil (W/O) nanoemulsion formulations may provide a useful model for the effective delivery of chemical compounds into citrus phloem via bark application or foliar spray for controlling citrus HLB. Two papers have been or will be published PLOS (2015) and Crop Protection (2015).
This overall 3 year project was focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB. In this quarter (April 2015 to June 2015), we continue to evaluate 1) the effect of Pen and SD on control of HLB disease by gravity bag infusion in the field; 2) the efficiency of effective chemical compounds (Pen, SDX, Pcy and Carv) against HLB disease by gravity bag infusion; 3) the effectiveness of a combination of chemotherapy, thermotherapy and nutrient therapy against HLB in field trials. The preliminary results showed that Pen and SD reduced the disease index of citrus HLB, but Las bacterium still could be detected in the citrus. The HLB disease index and Las bacterium titer in citrus treated with Pen was decreased significantly. However, the citrus treated with SDX, Pcy and Carv displayed HLB disease indexes and Las bacterial titers not significantly different than controls. The HLB-affected citrus trees at the USDA Farm were treated by combination of thermotherapy, chemotherapy and nutrient treatment in this quarter. The citrus trees were treated by steam at 125~128 F for 120 seconds or 180 seconds, respectively. Some foliar drop was observed with both heat treatments. Two weeks after the heat treatment, a lot of new flush appeared. The chemical compounds (Pen and EBI-602) and additional nutrients were applied to the heat-treated citrus by foliar spray, using our optimized nano-delivery system. In the next quarter, we will continue to monitor the HLB disease index and Las bacterium titers in the citrus treated with Pen, SD, SDX, Pcy and Carv by gravity infusion, and investigate field performance of these treatments. And the data on therapeutic efficiency and agronomic traits of the citrus treated by combination of thermotherapy, chemotherapy, nutrient therapy will also be collected.
Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are valuable for monitoring CLM but need further evaluation to optimize use for management. The objectives of the proposed project include: (1) assess species composition of trap catches, (2) optimize spatial and temporal distribution of pheromone traps. Sampling from the Okaloacoochee Slough State Forest and three commercial groves was completed. Samples, sequenced for COI barcoding to target the 658 bp region of COI gene, were merged with the previously published genetic dataset (Kawahara et al. 2013). The OK Slough had the highest number of samples sequenced, and within these samples, P. citrella was the most common (56.0%, 83.5% and 78.4% for samples from 2011, 2012 and 2013, respectively), followed by a relatively large proportion of P. vitegenella (28.8%, 8.4% and 15.5% for samples from 2011, 2012 and 2013, respectively). P. vitegenella was captured in only one of the commercial groves sampled and represented 13.3% and 12.5 % of the captures from 2011 and 2012, respectively. Phyllocnistis new sp. 1 and new sp. 2 were also represented in OK slough trap by-catch at 6.0%, 2.1%, and 2.6% and 1.6%, 4.0%, and 3.4%, for samples from 2011, 2012 and 2013, respectively. Phyllocnistis new sp. 1 and new sp. 2 were not captured in commercial groves. These results indicated that males of multiple species of Phyllocnistis, such as the two new Phyllocnistis species, are attracted to P. citrella lures. These results are consistent with Kawahara et al. (2013). It is not yet clear if trap captures are an accurate representation of by-catch species population densities or are influenced by differential attraction to the pheromone lure. These results suggest interesting avenues for future research. A new ML tree with additional samples analyzed on UF’s High Performance Computing Cluster (HPC) indicated that the majority of samples obtained grouped with already-known specimens of P. citrella. Bootstrap values for clades were generally low for most groups (<50%), which is comparable to prior results based on a single marker (e.g., DePrins and Kawahara [2012]). However, there were some notable results, such as the strong sister group relationship of P. citrella (North American) to P. citrella (Asia). Further, there is strong support for the monophyly of P. citrella (NA and Asia), P. insignis, and Phyllocnistis new sp. 1. Other taxa that were included in the dataset, but not generated from this particular project, also show strong bootstrap support for their monophyly, which will be relevant to future studies on non - P. citrella related species in the genus (e.g., P. hyperpersea (100% BP), P. longipalpus (100% BP), P. perseafolia (100% BP), P. populiella (93% BP), P. saligna (100% BP)). We also see relatively strong genetic distance between each group (>2-4% in many cases), supporting the hypothesis that these clades should be treated as separate species. Sequences (N = 212) of the 658 bp COI barcode region were submitted to GenBank (www.ncbi.org). Mark-release-recapture work using fluorescent powders on eight dates indicated that male moths are capable of moving 280 m in 24 h. However, captures at 280 m were very infrequent. No captures were made at greater distances. These results are being prepared for publication as a research note. We have received and addressed a review, and are nearing re-submission of “Placement Density and Longevity of Pheromone Traps for Monitoring of Citrus Leafminer, Phyllocnistis citrella (Lepidoptera: Gracillariidae)” by P. Vanaclocha, M. M. Jones, C. Monz’ and P. A. Stansly.
Combinations of frass (insect excrement) collected from Diaprepes abbreviatus males containing the putative pheromone methyl (E)-3-(2-hydroxyethyl)-4-methyl-2-pentanoate combined with plant volatile compounds previously identified as potential kairomones, failed to attract weevils to baited traps in the field. We also tried manipulating natural populations of adult weevils in the field to test the hypothesis that male and/or female distribution in a regular grid of trees could be manipulated by creating artificial aggregations of males, females or both sexes. Weevils were collected from all trees within plots of small trees of Pongamia pinnata known to be fed upon and infested with Diaprepes. Weevils were sexed, marked and placed in sleeve cages on single trees in the center of plots. Treatments consisted of center trees with males only, females only, or males and females. The hypothesis was that weevils freely moving in the area would be attracted to baited trees. All trees in the experimental plots were examined for weevils daily. No aggregation of weevils were found to occur on baited trees regardless of the sex of weevils used to created the artificial aggregations. These experiments suggest that orientation to pheromones is not a primary behavior of this weevil. As a final effort to identify an active volatile component, we are re-visiting the question of a female-produced pheromone. The previously identified pheromone is only found in male frass and we have confirmed that finding with new collections of volatiles from the headspace of males. We have collected volatiles from previously isolated males and females weekly for 3 months. Male pheromone only appeared in measurable amounts at ten weeks after adult emergence from the pupal stage. Volatiles have been collected on Super Q filters, eluted with dichloromethane and stored at -20’C. Collections from males and females will now be analyzed by gas chromatograph-coupled electroantennography (GC-EAD) for additional peaks we may have missed in previous collections of volatiles.
This is a project to develop novel approaches to controlling psyllids . Effective techniques to reduce the rate of Huanglongbing (HLB) spread are key to slowing its incidence, especially for new citrus plantings. RNA-interference (RNAi) is a natural regulatory and anti-viral response in eukaryotes and can be manipulated to target mRNAs/gene expression, including to control insects. Our on-going collaboration has found that RNAi inducers, expressed in citrus trees using the Citrus tristeza virus (CTV) vector, reduce the survival of adult Diaphorina citri moving onto the trees, and greatly reduce their reproduction and acquisition of Candidatus Liberibacter asiaticus by progeny. Our goal is to further improve RNAi activity such that it will help to manage D. citri and HLB, allow reduction in pesticide use and lower grower costs for U.S. citrus. Sequences of specific psyllid genes that are thought to be needed for the survival and reproduction of psyllids are cloned into the CTV vector. As the virus replicates in phloem cells, it produces large amounts of dsRNA intermediates that now also produces dsRNAs containing psyllid sequences. The normal plants RNAi defense mechanism processes the dsRNAs into small 21 nt siRNAs that target mRNA degradation. These siRNAs migrate from virus infected cells into the sieve element. As the psyllid feeds, it sucks up these siRNAs that now target the psyllid mRNAs and prevents the psyllid from making this protein. The lack of this protein has detrimental effects on the survival, reproduction, and CLas acquisition of psyllids. So far, we have seen reduction in survival of adult psyllids placed on RNAi expressing plants, but the effects on reproduction of the new generation of psyllids has been much greater. This is likely because the nymphs are rapidly growing an need lots of new protein synthesis and because they uptake large amounts of phloem sap. We had a technical glitch that slowed us down for a couple of months, but we are testing about twenty different anti-psyllid sequences to identify the most efficacious sequence. We recently have found that the RNAi target sequences that are expressed from near the 3′ terminus of the CTV vector appear to not be stable enough to be useful for controlling HLB spread in the field. We are recloning all of the target sequences between p13 and p20 or between CPm and CP looking for an optimal compromise between efficacy and stability.
The three growers are applying the 1/4 rates of Citrus Fix (2, 4-D), MaxCel (cytokinin) and now also ProGibb every 45 days on approximately 1 acre each of Hamlin and Valencia orange trees. Treated and Control trees are categorized by three levels of tree health and are monitored for fruit drop, flushing, yield, etc. Two locations are receiving similar treatments in a replicated trial. For Hamlin tree trials, 2, 4-D plus MaxCel (cytokinin) had a 6 % reduction in fruit drop, while 2, 4-D plus GA resulted in a 7 % reduction in fruit drop. For Valencia blocks, 2 had a 2 or 3 % significant reduction in fruit drop and overall the 2, 4-D plus MaxCel treatment reduced drop from 14 down to 11 %. Fruit set and flush development are now being measured.
Recent efforts to disrupt mating of the leafminer Phyllocnistis citrella have focused on the use of SPLAT’, a flowable wax emulsion intended to serve as a slow-release matrix for pheromones. Early success with this approach was overshadowed by the expense and difficulty of application, and variation in wax component chemistry that contributed to reduced longevity of pheromone emission in the field. Solid elastomer dispensers (DCEPT CLM’, ISCA Technologies, Inc.) loaded with a 3:1 blend of (Z,Z,E)-7,11,13-hexadecatrienal and (Z,Z)-7,11-hexadecadienal, the major components of P. citrella sex pheromone, provided disruption of trap catch in commercial citrus orchards for periods exceeding 30 weeks. The triene component alone worked as well or better than the 3:1 blend. The height of dispensers placed by hand in the tree canopy had a significant effect on trap shutdown. Dispensers placed low (0.6 m) in the canopy resulted in a reduction of trap shutdown in the upper third (>3 m) of the canopy suggesting that the net movement of pheromone molecules was downward during the period of active moth flight. Although moth flight appeared equivalent among the heights tested, placement of dispensers higher in the canopy appears more effective given this downward movement of pheromone plumes. These studies suggest that season-long trap catch disruption can be attained in citrus with a single application of a hand-applied dispenser. Efficacy of trap catch disruption was excellent in the 3 field trials and compares favorably with longevity of disruption previously reported for SPLAT CLM. The ability of the pheromone blend or triene-only formulation to disrupt trap catch of males in traps baited with the ‘natural’ 3:1 blend agrees closely with what we have observed and reported previously for SPLAT-based formulations. The longevity of disruption provided by DCEPT far exceeded results obtained with SPLAT and raises the possibility that a single application of emitters may provide year-long disruption of mating for P. citrella. Dispensers loaded with the triene component performed equally well regardless of the presence of (Z,Z)-7,11-hexadecadienal. The two formulations tested contained the same amount of triene; the addition of the diene component did not increase disruption. We conclude that inclusion of the diene in mating disruption products for this species has no benefit that would justify the additional expense of its synthesis and formulation. Also, the fact that highly effective trap catch disruption was obtained with an off-ratio blend supports the conclusion that disruption in this species falls into the category of non-competitive mechanisms because the triene alone is not attractive to males. The height of traps did not affect the number of males caught suggesting that males were active throughout the canopy. But the location of pheromone dispensers in the tree canopy did have a significant effect on trap catch disruption. Dispensers placed low in the canopy (<0.6 m) resulted in decreased disruption in the center and tops of the canopy compared with disruption achieved low in the canopy. Similarly, dispensers located in the canopy center (2m) were more effective at disruption of traps located low or in the center of the canopy; less disruption was achieved in the canopy top. These results suggest that net displacement of pheromone was downward perhaps due to the fact that the triene (molecular weight 234) is heavier than air and would tend to settle under calmer conditions that prevail at the time of greatest flight activity by males.
Laboratory and field trials failed to demonstrate attraction of males or females to artificial aggregations of males, females or male-female pairs. During the period, we focussed our efforts on collecting headspace volatiles over groups of adult males and females of known age. The adult weevils were collected directly from diet cups and maintained in cages segregated by sex. Headspace volatiles are being collected weekly from cohorts for analysis by GC-MS and for electroantennography. We have been able to confirm the presence of the pheromone methyl (.)-3-(2-hydroxyethyl)-4 methyl-2-pentanoate in headspace (by SPME) and in jar washings from 8-week-old males. Headspace collections from females are being stored (-20’C) for later analysis by GC-coupled electroantennography (GC-EAD) to look for female-produced compounds with antennal activity. However, at this juncture, the most parsimonious explanation of our results to date is that the Diaprepes root weevil does not rely upon a sex pheromone for locating conspecifics in the field.
Canker disease on fruit and foliage is under evaluation in the following 2015 trials: 1) Canker management in grapefruit: Foliar applications of novel bactericidal formulations will be compared with standard film-forming copper and zinc formulations: 1) Nordox 75WG, 2) Nordox 30/30 with 0.5 lb of metallic copper and an equivalent amount of metallic Zn, 3) Nordox 45/15 WG, 4) 2 Zinkicide formulations 5) 6 T-sol formulations; 6) CuSiNG-Quat-P (Cu+Quat), 7) Fixed-Quat DDAC, 8) CS-CuSiNP, 9) SG0025 (treatments 4-9 are experimental formulations from Dr. Swadesh Santra, a Nano-chemist at Univ. Central Florida), 10) Untreated check 2) Canker management in Hamlin: Foliar applications of standard film-forming copper formulations and soluble copper formulations will be compared in 4 yr old trees 3) Greenhouse assay to evaluate local systemic and residual activity of selected UCF formulations and FireWall will be against Xanthomonas citri subsp. citri on potted Pineapple orange trees inoculated by injection infiltration 4) Assessment of Cu bioavailability and residual activity on grapefruit for copper oxide (Nordox 75 WG) and the copper-zinc formulation Nordox 30-30 during two periods of summer season (July and August) to test whether zinc may be enhancing the bioavailability of Cu from the copper oxide in the 30/30 and 45/15 formulations compared to 75 WG copper oxide alone 5) Selected results from 2014 trials were presented at the Florida Citrus Show in January and Citrus Institute in April and published in the April 2015 issue of Citrus Industry magazine
The canker field trial of Zinkicide compared to industry standard management on grapefruit showed better control of canker on fruit than all other treatments. We also observed control of some fungal pathogens. A second year of this field trial has begun to confirm the results and test the consistency of control. Yield and juice quality data from the HLB trial in young Valencia trees has been collected. The timing of first treatment and the trees flush cycle make it difficult to determine efficacy in the first year of treatment. An unexpected problem occurred with a very strong gradient of Las titer in the small tree field trial that was not apparent based on an even distribution of symptoms in the plot layout. This gradient is confounding titer analysis and we are trying to determine the cause. While HLB field trials will continue, they will be performed at a larger scale in a project funded by the SCRI and are no longer a part of this project. HLB symptom ratings were also collected for infected trees in the canker trial because treatment began earlier. The data is promising, however, it was not the main goal of this trial, so initial HLB disease assessment was not done. Additionally, the data may be biased because the reduction in canker could reduce leaf drop and cause the trees to look healthier. Dr. Santra’s group has improved synthesis of Zinkicide to reduce the amount of some ingredients needed for synthesis while maintaining efficacy. This improvement is aimed at reducing cost of production.
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