No additional treatments or data collection occurred during this period. Data will be summarized in the final report.
There were no additional tests or measurements during this period. Results will be summarized in the Final Report for the project.
Test groves were arranged with 5 growers with 11 different tests 4 of which will be sprayed by growers. Seven tests are in Hamlin blocks and 5 are in Valencia blocks. Materials included in tests are Citrus Fix (2, 4-D). Pro-Gibb (GA3), Headline, Gem, Abound and a PGR extract that contains an auxin, GA and cytokinin. Hamlin tests were applied in September or October and Valencia tests in November or December. Data collection will include preharvest fruit drop, total yield and juice analysis on some tests particularly if Headline is included.
Low concentrations (1/4 rate) of 2, 4-D and Max-Cel were applied every 45 days to Hamlin and Valencia tree canopies at two locations in central Florida starting in Spring 2014. Concentrations were 12.6 ml Citrus Fix and 480 ml Max-Cel. Every other 45 day period, GA3 (0.04 g ai/tree) was applied in 3 gal of water per each microjet irrigation zone. Treatments were applied from Spring through October. Trees were sampled in late spring for phloem development at four locations in the scaffold system (root flare, trunk, small scoffolds and leaf main veins). Comparable samples will be taken at the end of the growing season to compare phloem development with and without the PGR treatment. Comprehensive PGR profiles on the treatments were run by Dr. Killiny. Almost all of the treatment trees were positive for HLB. Fruit drop and fruit per tree data are now being collected. Root densities and canopy condition will also be evaluated.
The person to redo the Internet model for flowering evaluation was identified and hired to start work in December. Data collection and rewriting code will be done from December through January. Off season blooms related to HLB stresses are a problem that needs to be dealt with. We will try to program them if their induction relates to mild or moderate water stress periods.
This project, in general, seeks to determine if there are any observable influences of foliar fertilizers on HLB-affected grapefruit in the Indian River marketing district. To-date, this project has established 3 separate research trials in commercial grapefruit groves and university research farms in St. Lucie county, Florida. Trial #1 is ~25 years old of ‘Flame’ grapefruit on Swingle rootstock. Trial #2 utilizes ~6 year old ‘Ruby Red’ on Sour orange trees. The first 2 trials are located in commercial groves. Trial #3 seeks to determine the effect(s) that foliar fertilizers have on young tree growth and their ability to protract HLB disease symptoms in 1 year old ‘Ray Ruby’ grapefruit on Kuharske rootstock. For trial #1, there are a total of 30 replicated experimental units; for trial #2, there are a total of 50 replicated experimental units; and, for the trial #3, there are a total of 24 replicated experimental units. Foliar fertilizer applications have been started since February 2014. Tree growth data, leaf nutrition data, and CLas titer measurements have been made. Fruit drop monitoring will begin in September 2014 in trials #1 and #2. In prosecution of these efforts, 2-part-time OPS employees have been hired to do our foliar sprays and collect data observations. We also completed the refurbishment of a mobile fruit grading line. Fruit quality and quantity assessments will begin in late fall/early winter of 2014.
The insectary at the Florida Department of Agriculture and Consumer Services, Division of Plant Industry (DPI) was a longstanding source of Diaprepes root weevil larvae for researchers in Florida and elsewhere. Reduced demand for these larvae caused this agency to discontinue their production in September 2014. In anticipation we met several times with the agency to learn their methodology. We purchased cages and supplies and constructed an insectary, acquired adult weevils and began rearing larvae on diet in axenic conditions. To date we have produced 4 ‘production runs’. Contamination of cultures in which the larvae grow has been a problem, but fewer units have become contaminated over time. We will have enough 3-5 instar larvae going into the winter season to conduct the upcoming field trials at sites in Lake and Polk Counties. Repeated experiment using H. floridensis, validating results reported in previous report. We shall include H. floridensis in upcoming field trials.
Entomopathogenic nematodes: Amending soils to increase biological control of insect pests We repeated experiments to test whether EPN community structure can be engineered by managing soil water potential (see June 2014 report for results). Mixtures of Sd, Sx, Hi, and Hz were maintained with weevil larvae in soil low and high water potential. EPNs that recycle in weevils were periodically added to fresh columns with weevil larvae for several ‘generations’ (21 days each). During the first three ‘generations,’ Sd gradually, but consistently increased its presence in the EPN population from 45% to 55% in the dry (6% moisture) soil and remained steady at 22-30% in the wetter (18% moisture) soil. Steinernema sp. (Sx) constituted between 27-41% of EPNs in dry soil and 55-63% in moist soil, whereas Hz represented between 6-23% of EPNs without an effect of soil moisture. It is interesting that Hi did not persist in any treatment, given that Hi is the dominant EPN species in all of the ecoregions where citrus is grown. We reported that Hi is little affected by Catenaria spp., a fungal predator of nematodes, whereas the fungus is lethal to most other EPN species in controlled studies. Similarly, during monthly sampling for two years in 4 citrus groves, detection of Catenaria sp. was found to be inversely related to Sd abundance and positively related to Hi abundance (unpublished). Results such as these suggest the need to increase the complexity of the biota in these experiments to include natural enemies of EPNs in order to recreate selection pressures in microcosms that produce EPN communities comparable to those in the field. A future microcosm experiment will test this hypothesis. We continued comparing the biology of two closely related native species, Steinernema diaprepesi (Sd) and Steinernema sp. (Sx), with respect to survival mechanisms. When subjected to a range of relative humidity (50%-100%), Sx did not survive RH ‘90% whereas, Sd persisted on filter paper disks at 90% and greater RH. Moreover, when the two species were maintained 48 h in aqueous solutions of 30% glycerol, both lost volume with the loss of water but, upon rehydration for 24 h, most Sd became motile whereas all Sx ruptured. Thus, Sd (an inhabitant of well drained, central ridge soils) has superior capacity for osmoregulation and desiccation survival compared to Sx (an inhabitant of poorly drained, flawoods soils). An inferior capacity for osmoregulation may also affect the spatial pattern of Sx which has not been detected in coastal orchards where hypertonic conditions tend to be greater than in the inland flatwoods orchards. Plant parasitic Nematodes: Characterizing a new nematode pest and the prevalence of resistance breaking populations of the citrus nematode. Treated plots two nematicide trials conducted in east coast grapefruit on swingle. We identified a second site on the central ridge with 18-month-old trees heavily infested by dagger nematode (Xiphinema vulgare). Installed irrigation lines adjacent to exiting lines to initiate nematicide trials (oxamyl and an experimental nematicide/fungicide) that will continue for three years.
The project began on May first 2014 with two citrus groves selected for the project. A mature grove owned by English Bothers consists of 20 year-old Hamlin sweet orange trees predominately on Swingle rootstock. Sixteen full row main plots of 40 trees each were selected for irrigation water acidification at one of four target water pH (7.5, 6.0, 5.0, and 4.0). The second experimental site was established in a block of two year old Hamlin sweet orange trees on Swingle rootstock at Orange-Co’s Joshua block. Rows of trees with similar treatment plots were selected randomly with one row split into two main plots of 20 trees each. Initial soil measurements, indicated that soils at both sites were alkaline with average soil pH of 7.43 for English Bothers and 7.05 for Orange-Co. After irrigation water acidification treatments were initiated, soil pH at both sites decreased by October, 2014 to a range of 6.24 to 6.55 with no significant differences for treatment at either site. Soil ph in control blocks with no acidification remained at 7.3 for English Brothers and declined to 6.55 at Orange-Co. These treatments were applied during the rainy season with little irrigation applied at both sites. A total of approximately 39 minutes of irrigation at was recorded for a four month period (July through September) at Orange-Co. Irrigation during these four months were primarily to apply acidifying fertigation. Likewise, no significant differences in tree trunk measurements (17.0to 21.1 Mm), Phytophthora propagules (0.008 to 0.02 propagules/mg soil) ir root density (0.07 to 0.31 mm root/mm soil at 0-15 cm and 0.01 to 0.03 mm/mm at 15 to 30 cm) were found in June, indicating no difference in tree size due or root pathogen density for the acidification treatments. These results indicate that little soil acidification occurred during the summer rainy season because of low irrigation inputs.
Feb 2014 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: All preliminary experiments have been completed for propiconazole, difenoconazole, and tetraconazole. Preliminary experiments with imazalil and fenbuconazole are on-going Objective 2: We’ve taken our first rating in the fungicide trial and will continue until fruit harvest. Too early to say how treatments are going to shake out. We are planning for next season’s trial. CBS lesions are now apparent on fruit at most sites so we’re planning to start some of the survey data next week. Objective 3: Suitable sites to conduct the spatial studies have been located. This study will begin in earnest close to fruit maturity when symptoms become apparent. Objective 4: Since the last progress report, work has continued evaluating the efficacy in vitro of the postharvest citrus fungicides imazalil (Freshgard 700, Active Ingredient: 44.6%) and pyrimethanil (Penbotec 400, Active Ingredient: 37.14%) at concentrations of 0.001, 0.01, 0.1, 1, and 10 mg/l active ingredient in the PDA medium on mycelial growth inhibition of four Guignardia citricarpa isolates. After 14 day, there was a near log-linear increase in mycelial growth inhibition increasing with 100% inhibition at the highest concentration. To evaluate heat effects on G. citricarpa, balls of mycelial masses cultured in liquid media were placed in solution at 55C for 0.5, 1, 2, 3, 4, 5, or 6 minutes, the flasks cooled by placing in an ice-water bath, the mycelia plated on . strength PDA media, and growth measured over 10 days. The control was exposed only to a solution at 25C. No treatment duration killed the G. citricarpa, but 55C exposure for 1 min reduce mycelial growth by half, and inhibition gradually increasing to about 75% after 6 minutes exposure. Neither chitosan at low concentrations (0.5 ‘ 10 mg/L), nor 50, 100, 200, or 300 mg/L salicylic acid (SA) inhibited G. citricarpa mycelia growth, but instead appeared to stimulate its growth slightly (SA) to moderately (chitosan) in the mid to higher concentrations tested. These experiments will be repeated.
We have compared the qualitative and quantitative expression in plants of amiRNAs engineered to target psyllids by directly expressing them from Agrobacterium tumefaciens, by using Tobacco mosaic virus (TMV) and by using a modified Begomovirus expression system (Tomato mottle virus, A component, TAV). Based on the results of Hi-seq illumina deep sequencing, TAV is the best candidate to express artificial microRNAs in plants. We are currently evaluating more samples by deep sequencing and engineering additional constructs to target psyllids. Objective 2 is to evaluate and optimize in planta expression of anti-psyllid interfering RNAs. We have started the leaf-disc feeding assays using our transgenic plants expressing double-stranded RNAs expressed from two different promoters (the 35S promoter for general tissue expression and the AtSuc2 promoter for phloem-specific expression) and the in planta transient expression of specific artificial microRNAs. So far the transient delivery/expression system has not worked for citrus. Thus, we are evaluating additional candidate interfering RNAs by in vitro feeding assays, and evaluating specific interfering RNAs in citrus plants infected with recombinant Citrus tristeza virus (CTV). The latter is in collaboration with Dr. W. O. Dawson. We have also been optimizing the conditions of our on plant feeding tests to have them close to natural feeding environment.
This proposal aimed to continue improving a novel psyllid trap and to use the trap to gather new information on the behavior, biology, population dynamics and biological control of ACP/Candidatus Liberibacter asiaticus. Lab and field testing was conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps and behaviorally active components. Obj. 1: We continued to conduct field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulations of visual cues as well as trap physical components and component orientation. During this final quarter we have successfully tested, winnowed out inferior traps, retested old and modified trap prototype configurations and developed new traps. Results indicate that the cylinder trap is the most efficient trap but other shapes have merit for other functions with ACP. For example, a cone shaped trap was found to attract ACP and also enhanced the ability to capture ACP in ways that indicate the trap could be readily used to protect and dispense fungal spores in the field. No increased responses by ACP with trap additions related to odor cues as reported to be potential attractants in the literature were observed. A sampling regime was initiated to try to delineate the northern most established populations of ACP in Florida. Sampling locations toward this end have been Gainesville, Live Oak, Quincy and Marianna, FL. To date ACP have not been detected at any sampling location north of Gainesville. A subsample of captured ACP (n=150) from Gainesville were analyzed monthly (W. Hunter) for the causal CLas bacteria of greening. Interestingly, to date none of the psyllids analyzed have been infected with CLas. An outbreak of another invasive psyllid species, Acizzi jamatonica, on the mimosa tree, Albizia julibrissin, was found in north Florida and this opportunity was exploited in several ways. ACP traps were tested for sampling of the adult psyllids, psyllid parasitoids were sought via observation and rearing, but none were detected. Adults of the psyllids were also evaluated for pathogen associates (A. Rooney, W. Hunter). The ACP trap worked equally well for monitoring of A. jamatonica. A similar effort was also deployed for the persimmon psyllid, Trioza diospyri. The ACP cylinder trap did not capture this psyllid species, but during sampling, a high percentage of the T. diospyri nymphs were found to be parasitized. Obj. 2: The areawide psyllid sampling to detect and develop new biological controls for use against ACP was pursued around the state. Personnel collected psyllids in “out-of-the -way” places that may hold relic citrus trees and relatively isolated ACP populations. So far several new pathogens have been identified. We have obtained cooperation with other USDA-ARS research personnel with expertise in the identification and rearing of entomopathogenic fungi and they have evaluated samples toward this end from Florida and Puerto Rico. Project Enhancement Grant to develop sentinel plants. A methodology to improve the management and use of sentinel citrus plants that are flushing was tested and developed. Flushing citrus plants in portable containers were highly attractive to ACP and appear to have merit for developing a sentinel plant platform that will provide a number of useful functions in the continued suppression of ACP, especially with initiating fungal epizootics, release of parasitoids, as a monitoring method, etc. This approach appears to have excellent potential as a system to deliver bio-based suppression tactics for ACP in non-commercial citrus venues such as abandoned and organic groves, as well as for urban areas.
The Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is naturally infected with Wolbachia (wDi). Recently,we calculated the within-host density of Wolbachia in Florida D. citri populations using a novel quantitative PCR for detection of the Wolbachia outer surface protein gene, wsp. Gene quantities were normalized to the D. citri wingless gene (Wg) to estimate Wolbachia abundance in individual D. citri. Using this method, significant geographic differences in Wolbachia densities were detected among Florida D. citri populations, with higher infection levels occurring in male versus female hosts. More recently, we have determined that Wolbachia densities are low during larval development and increase throughout adulthood. These data were recently published in Environmental Entomology. Three sequence types have been of Wolbachia have been identified in D. citri populations in Florida. These putative Wolbachia strains were characterized by identification of accumulated nucleotide differences in 5 conserved genes using multi locus sequences typing (MLST). Differences between ACP Wolbachia genes were determined by comparing consensus sequences to an MLST database. One sequence type in particular in found in only geographic region of Florida, and as such is a promising candidate for investigating interactions with Las transmission. Consequently, we are working to establish additional isofemale ACP lines from populations of psyllids predominately infected with the low frequency sequence type of Wolbachia through field collections. We are currently evaluating the phenotypic effects of strains (including cytoplasmic incompatibility) in established isofemale psyllid lines. Recent data suggests competition among Wolbachia strains. Evaluations of co-infection with native Wolbachia strains are currently underway to evaluate insect and symbiont fitness. In addition, we have identified an inverse relationship between Wolbachia and the ACP endosymbionts, Ca. Carsonella ruddii and Ca. Profftella armatura. Evidently, densities of these endosymbionts differ geographically, and in relation to Wolbachia strain infection.
Kaolin clays have been shown to be effective in reducing Diaphorina citri feeding on citrus leaves by inhibiting normal behavior. The overall objective of this project is to investigate the feeding deterrence provide by reflective colored kaolin clays on citrus plants from ACP. In the first quarter of this project kaolin clays have been procured from different sources including Surround kaolin clays product. We have also selected dyes, including FDA certified colorants, those approved for agricultural use by the EPA (list ‘180.920), covering a range of visible spectrum for coating clays. Particles size and zeta potential measurements of selected clay materials were performed to characterize the clay particles. Since the selected dyes have different structures and chemical properties, surface modifications of the clay are required for doping with dyes. Surface modification experiments were performed for kaolin clay products from different suppliers. It was observed that some of the clays changed the optical reflectance of the absorbed dyes and while others leached out earthly material during modification treatments which interfered with doping process. Clay that retained the dyes reflectance property as well as remained suspended in solution (making it easier for spraying) were selected for further studies. In the trials conducted, several dyes were successfully were sorbed to kaolin clays. Some examples of dyes used are FD&C Blue 2, FD&C Red 40, Basic Blue 54, Crystal Violet, and Basic Yellow 29, D&C Violet 2 etc. Experiments are being conducted to characterize and scale up the lab procedure to produce color clays for ACP greenhouse bioassays to determine reflective wavelength deterrence efficiency. Tests are also being conducted to further modified kaolin formulations for increasing rain-fastness.
Canker disease on fruit and foliage is under evaluation this month (October) for the following trials. Final results will be reported in the December progress report: 1) Soil-applied Actigard in an EPA-approved Experimental Use Program (EUP) in two east coast grapefruit groves conducted in collaboration with Syngenta. 2) Foliar applications of soluble copper and novel bactericidal formulations in comparison with film-forming copper formulations. Experimental formulations are tested at much lower metallic rates in grapefruit and Hamlin. Evaluation of fruit drop due to canker in Hamlin will be completed in November/December. 3) Evaluation of performance of copper sprays in grapefruit groves with windbreaks: 6 and 7 yr-old red grapefruit blocks surrounded by a 20-30 ft tall Corymbia torelliana windbreaks. 4) Performance of two applications of Firewall (streptomycin) in a grapefruit trial with and without the penetrant Nanocanopy that is purported to increase uptake and systemic activity of the active ingredient. Efficacy of Firewall + Nanocanopy will be compared to Firewall alone. Also evaluating Cu bioavailability and residual activity on grapefruit for copper oxide (Nordox 75G) and copper-zinc formulations (Nordox 30-30) during two periods of summer season (July and August) when rainfall and fruit expansion rates differ.
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