Since our precious report, we have completed the hiring of personnel to conduct research on this grant, including two post-doctoral associates. One objective of this project is to determine points of the Asian citrus psyllid (ACP) life cycle that may be susceptible to control programs. Previous research has highlighted that environmental perturbation such as a drop in temperature, may affect psyllid ability to transmit Candidatus Liberibacter asiaticus (Las). Work has begun to assess ACP tolerance to cold shock and how periods of cold shock may effect ACP acquisition of Las. Psyllids from laboratory colonies are subjected to 0 ‘ 1’C, 5 ‘ 1’C, 10 ‘ 1’C, 27 ‘ 1’C, 30 ‘ 1’C, 34 ‘ 1’C periods for 1, 2 or 4 weeks in controlled growth chambers. All chambers are maintained at 50 ‘ 5% RH and 14:10 (L:D) photoperiod. Psyllids are then exposed to Las infected sweet orange for 2 or 4 weeks and later tested for acquisition of Las. In further experiments we will assess the onward vector capacity of these psyllids in inoculation experiments. These data will yield clues as the effects of perturbations in temperature on ACP and their vector capacity. In addition, we are assessing the effect of Wolbachia endosymbionts on ACP, and ACP acquisition and inoculation of Las. We have begun by collecting psyllids from 10 sites in Florida between Lake county and Miami Dade county, assessing both the abundance of ACPs and the rate of Wolbachia infection within these 10 populations. During investigations we have observed low abundance of ACP in 6 of 10 sites, therefore we have concentrated on two heavily populated sites from which to raise lab colonies for further experiments; one grove in Polk County and one in Miami Dade County. Previously we have found 70-80% Wolbachia infection these counties respectively. Using single pair crossing and qRT-PCR analysis of F1, F2 and F3 generations we are currently generating one Wolbachia positive and negative colony from each county. Once fully established we will use these colonies, together with existing colonies micro-injected with non-native Wolbachia, in bioassays to assess the effect of Wolbachia on ACP fitness. Experiments will include investigations of longevity, fecundity, development and viability of nymphs, and interactions between Wolbachia and Las within ACPs. These data will yield important clues as to a potential role of Wolbachia in controlling ACP populations and Las dispersal.
The questions to be addressed by this research project are 1) how does Imidicloprid move in the sandy soils of south Florida, 2) how long does imidicloprid persist in sandy soils, and 3) how much Imidicloprid leaches below the root zone of citrus trees. Isotherms relating soil Imidicloprid concentrations in soil solutions with Imidicloprid concentrations in the solid phase of sandy soil were conducted using Immokalee fine sand. Imidicloprid was found to penetrate to a depth of 45 cm and have greatest affinity in the 0-15 cm soil depth due to higher soil organic matter at that depth. These data were supported by soil partition coefficients (Kd) of 1.68 mg of Imidicloprid. These data suggest that Imidicloprid is leached rapidly from the soil if not taken up by the target plant. Data from two field studies in the spring and summer of 2012 indicated that Imidicloprid concentrations decreased by 90% in 10 days at the 0-15 cm depth by tree uptake and recommended microsprinkler irrigation. Initial analysis of soil samples from the spring study indicate that Imidicloprid concentrations decreased rapidly in the soil and was either taken up by the plants or leached within 5 to 10 days at recommended microsprinkler irrigation rates, however reduced irrigation following applications allowed for greater soil residual time and uptake. During these initial application studies, adult psyllid populations per tree decreased from approximately Imidicloprid concentrations reached the 15-30 cm depth in 5 days after application and persisted at that depth for approximately 15 days. During this initial application study, adult psyllid populations per tree decreased from approximately 1.7 for both treated and non-treated trees to 0.1 psyllids per tree for the treated trees at 22 days after application. Psyllid populations on treated trees were below 0.1 psyllids per tree from 22 to 50 days after application. These data would indicate great persistence of the insecticide in citrus tissue and is supported by elevated leaf tissue concentrations.
Issue date for this grant was 13 July 2012. Objectives are: 1. Assess effects of abiotic factors (light quality, photoperiod, air flow, temperature fluctuations) on psyllid movement, 2. Evaluate physiological limits and biotic factors effecting of movement including feeding, egg load, infection status, and population density, 3. Evaluate techniques for tracking psyllid movement in the field for mark recapture studies, 4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field, 5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide treated trap crops (such as Bergera koenigii) to attract and kill. Objective 1. A bioassay was developed at SWFREC using a wind tunnel to determine ACP response to wind speed and direction. ACP are marked with fluorescent powders for easy observation and released into the wind tunnel. Preliminary results indicate slow movement downwind in preference to upwind in the absence of a host plant. In addition, two launch surfaces are being tested (glass petri dish and paper lined dish) to determine the best release method but results are still inconclusive. Objective 2. Lab trials at CREC indicate females avoid high densities of conspecific females, irrespective of whether direct contact is made with psyllid-infested plants. An olfactometer was used in the absence of light to demonstrate that the avoidance behavior was probably elicited volatile cues. A volatile collection system is being developed to identify this potential behavior modifying odorant. They are also in the process of establishing experiments to determine whether female egg laying or feeding may induce a systemic change in volatile release from plants that may affect subsequent settling and oviposition behavior of female and male ACP. Also in progress are bioassays to investigate a possible repellent effect of high ACP nymphal densities on ACP female movement and behavior. Preliminary results suggest little or no affect on ACP settling or movement; however, it appears that oviposition by female ACP is deterred on plants that are infested with high densities of nymphs as compared with non-infested controls. Flight mill trials to determine flight capability of ACP indicate that barometric pressure and season may be affecting ACP movement behavior with less vigorous flight being observed in winter compared to spring and summer. Objective 3. Monitoring ACP movement with sticky traps continues in a 15 acre abandoned Murcott grove in preparation for another attempt at mark-recaputre using fluorescent markers although no marked ACP were recovered during a spring trial. Objective 4. Yellow sticky traps set at three canopy heights are giving mixed results, with more ACP captures in the upper canopy of an old reset grove of ‘Valencia’ orange compared to a more even distribution at younger and more uniform grove of ‘Earlygold’. More ACP are captured at both locations on the east side of the trees than the west side, possibly indicating movement toward morning light. At both locations and a third along the Caloosahatchee river, ACP captures are greatest adjacent to a hammock or wind break indicating that these features may serve to arrest movement across the block. In ‘Earlygold’ grove, two sided traps set between insecticide sprayed and unsprayed blocks with low and high populations of ACP to evaluate the direction of movement do not indicate any predominance. Objective 5. A paper reporting positive results using UV reflective mulch to repel ACP the first 2 years after planting was submitted to and is now under revision for the refereed journal Pest Management Science. Subsequent results indicate that the 5 ft strip of metallized mulch is being shaded by the canopy and no longer effective at repelling ACP on trees more than 5 ft tall. Trials to test different ways of utilizing UV reflectance to repel ACP are being planned.
This project began 11/1/2012 and our efforts are now ongoing. We have colonies of both psyllids (Bactericerca cockerelli and Diaphorina citri) to be used in our work and are now beginning efforts to test in planta RNAi effects and discover the optimal construct development.
Dead/preserved Diaphorina citri psyllids samples collected from Florida, China, Brazil, Taiwan and Pakistan have been received from collaborators. We have performed small RNA sequencing on four separate psyllid samples so far and examined these by small RNA, next generation sequencing to find known and unknown viruses capable of infecting D. citri. We are still in the process of bioinformatic analysis by using local scripts and commercial software on our small RNA datasets. Our analyses are far along for the Taiwan D. citri RNA sample, and less so for the others. We obtained 13 million reads for the Taiwan D. citri which decreased to 11 million after adapter and sequence error removal yielding small RNAs with an average length of 21.6 nucleotides. De novo assembly was then used to create larger contigs from these small RNAs, yielding contigs with an average size of 274 nucleotides. We performed search and align Genbank queries using BLAST and have identified putative viral sequences. So far we have confirmed the identity of different genomic segments of the Diaphorina citri reovirus from the Taiwan sample. Our contigs shared a sequence identity >95% with significant E-values with this reference virus. In order to confirm our sequenced-based discovery we designed specific primer pairs based on our in silico sequences and performed RT-PCR for all of the D. citri RNA samples. RT-PCR confirmed the presence of Diaphorina citri reovirus in the Taiwan, China and Florida RNA samples, but not in those from Pakistan or Brazil. We should mention here that we have found signature sequences for other viruses in the Taiwan sample but initial E-values were not significant. We are in the process of performing more computational analysis on these and the remaining samples. These data so far validate our approach for virus discovery and give confidence for our longer term objectives for using viruses as tools for D. citri management.
The purpose of this investigation is to identify/confirm new non-neurotoxic compounds that could be useful for management of Asian citrus psyllid (ACP). Using novaluron, a chitin biosynthesis inhibitor, we are developing a high throughput assay system to more efficiently screen novel non-neurotoxic compounds for efficacy against the ACP. Novaluron is used on food crops including apples, sweet potatoes, and brassicas and has been shown to be effective against several hemipteran species including sweet potato whitefly (Bemisia tabaci), greenhouse whitefly (Trialeurodes vaporariorum), pear Psylla (Psylla piri). It is an insect growth regulator (IGR) and is effective on growing stages of the insect when molting takes place. Because it is a non-neurotoxic insecticide that targets the chitin biosynthetic pathway, a pathway specific to arthropods and fungi, it is potentially a well suited insecticide to use in a rotation scheme to reduce the use of broader spectrum, non-specific compounds in the management against insecticide resistance. Currently, novaluron is being evaluated for its efficacy against ACP egg hatch, and nymph and adult emergence. We are developing high-throughput bioassays to evaluate these parameters that can then be utilized to test other non-neurotoxic compounds, specifically other IGRs such as tebufenozie and methoprene. For testing other non-neurotoxic compounds that are most effective if ingested by the adult stage of ACP, we are developing and refining an artificial feeding assay that will enable us to introduce the compounds to the insect, including amino acids, which are known to have a range of biological effects on insects such as disruption of flight metabolics and egg production. Also, we are testing lectins, which have been shown to disrupt insect gut function. Because there is a range of sub-lethal effects expected from these compounds, appropriate bioassays to detect biological consequence of the test compounds are being developed. For example, the ingestion of L-canavanine by female insects is expected to have an effect on egg production; hypothetically, resulting in fewer eggs laid or eggs laid that are less viable. The effects in this case will be determined by dissecting the ovary of treated insects and eggs counted in addition to determining viability of laid eggs.
The objective of this investigation is to determine how enhanced nutrition of citrus plants may affect Asian citrus psyllid (ACP) biology. We have initiated this study with complementary field and laboratory experiments. Regarding the field experiment, we are still following the Keyplex’ program. The most recent treatment was applied on October 23rd. The next application (pre-bloom application) will be performed near the end of February. Starting this month we will also monitor the number of psyllids within each replicated treatment block with standard sticky traps and tap sampling. Complementary greenhouse experiments have been established and are in progress. We initiated choice test experiments with plant clippings placed in water and placed into custom-designed arenas (50 x 32 x16 cm; conditions within arenas: 23’C, 48% RH, 14:10 LD). Twenty ACP are placed within each arena and allowed to settle for 24 hours. So far, three two-choice combinations have been tested (HLB-infected versus uninfected plants, uninfected and nutrient-supplemented plants versus uninfected and non-supplemented control, and HLB-infected and nutrient-supplemented versus HLB-infected, but not supplemented control). The preliminary results indicate that ACP prefer to settle on nutritionally supplemented citrus particularly, the within a few days following the spray application. This trend seems to be more pronounced on HLB-infected than on uninfected citrus. We are also performing an experiment to determine the effect of nutrient treatment on ACP feeding. To determine whether nutritional treatments influence ACP feeding, leaf discs (60mm) are cut from the leaves of nutrient-supplemented or non-supplemented control plants and individually placed on 1.5% agar beds in 60 mm disposable petri dishes. The petri dish lids are lined with 60 mm filter paper discs, after which 10 adult ACP of mixed age and gender are released into dishes. Sealed petri dished are then inverted to collect honeydew droplets on the filter paper for 24 h. Honeydew production by ACP is an indirect measure of the amount of feeding on plants (i.e. production of honeydew is positively correlated with amount of feeding). HLB-infected and uninfected plants are tested separately. At the end of the experiment, filter papers are collected and treated with ninhydrin to stain honeydew droplets for subsequent quantification of the results. Nutrient supplemented and control (unaltered) treatments will be replicated 20 times. After 10 replications, it appears that there is more feeding taking place on HLB-infected and nutrient supplemented citrus compared with HLB-infected, nut non-supplemented controls. However, feeding is the same on uninfected plants, whether or not they were supplemented with the nutritional spray.
Our main objective for this project is to evaluate botanical compounds (plant volatiles and essential oils) as possible repellents of Asian citrus psyllid (ACP). The goal is to identify “off the shelf” treatments that are inexpensive and effective for field use. In the previous quarter, we reported on settling and field experiments with botanical oils as repellents or attractants for ACP. In this quarter, we have continued this research. We have continued to collect and analyze data from: (1) olfactometer bioassays indicating that fir oil is repellent and clove and camphor oils are attractive to ACP, (2) laboratory settling experiments with fir oil treated plants, and (3) field trapping experiments using baited yellow sticky cards. We are currently designing experiments to test fir oil as a repellent in field experiments. Based on our results to date, we are hypothesizing that fir oil may be best used to protect resets or newly planted groves that have a small canopy. Therefore, we are designing experiments to look at repellency in both of these scenarios. Furthermore, we will revisit the use of clove and camphor oils in various release devices to determine if release rate can alter the efficacy of these treatments as attractant lures. In conjunction with the field experiments outlined above, we will be conducting olfactometer assays to evaluate the repellency of fir oil to Tamarixia radiata, the main ecto-parasitoid of ACP. Next we will conduct parasitization assays in a caged arena with fir oil treated and untreated plants infested with ACP nymphs.
This project is focused on evaluating the impact of psyllid control programs on non-target pests, beneficial insects and mites. Citrus rust mites (CRM), citrus red mite, snow scale, Florida red scale, Chaff scale, citrus leafminer (CLM), citrus blackflies and whiteflies, as well as their parasitoids and predators were monitored in four commercial citrus groves in Collier, Lee and Hendry counties and an experimental orchard at Southwest Florida Research and Education Center in Collier county, linking with experiments employing insecticidal sprays to control ACP. In the commercial grove in Collier Co., 0.8 acre plots were sprayed with Delegate (5 oz/ac) and Danitol (16 oz/ac) in Nov and Dec, respectively, resulting in significant suppression of ACP and impact on non-targets. Chaff scale in insecticide-treated plots averaged 0.3 per lens field (LF) in Nov, compared to ‘ 0.09/LF in untreated plots. A similar though not significant trend was observed in December. CRM averaged 0.13 per LF and not significantly different across treatments. Predatory mites in Nov averaged between 0.04-0.09 per LF in untreated plots and less (0.01-0.03 per LF) in treated plots. It seems that predatory mites provided some suppression of CRM in the untreated plots to keep them at levels seen in the treated plots where predatory mites were reduced. In Lee Co. no differences were seen in ACP, non-target pests or beneficials in plots treated with Imidan (1 lb/ac) + 435 oil (2% v/v) in Nov compared to biweekly applications of 435 oil suggesting that both programs controlled ACP with minimum impact on beneficials. The two groves in Hendry County include plots receiving monthly sprays: Portal at 4 pts/ac in Oct and Mustang at 4.3 oz/ac in Dec. CRM averaged 0.3-0.4 per LF in untreated plots at both locations in Dec, significantly more than treated plots (‘0.006 per LF). Apparently, rust mite control afforded by Portal was still in effect with negative effects from Mustang delayed or absent. Similarly, fruit russeting in untreated and treated plots: 24% and 11%, respectively in ‘Early Gold’ and 40% and 28%, respectively, in the ‘Valencia’, indicating that the choice of ACP control products had also reduced CRM over the season. Predatory mites numbers were similar across treatments in ‘Early Gold’, averaging 0.04 per LF and surprisingly greater in the treated plots of ‘Valencia’ (0.4 per LF) compared to 0.1 per LF in the untreated plots, again indicating that the correct choice of products can maintain natural enemies. Results of four experiments conducted at SWFREC orchard comparing 39 treatments of experimental and labeled insecticides for effects on ACP, CLM, lady beetles, ants, spiders and lacewings were submitted for publication in Arthropod Management Tests. Portal 5 EC (64 oz/ac) and Apta 15 EC (14-17 oz/ac) suppressed ACP for about two months but reduced ants, spiders and lady beetles. Similarly, suppression of ACP by Dibrom 8E (16 oz/ac), Lorsban 4 E or Advance (5 pts/ac), Pyrinex 250 CS (1.2 gal/ac), Vulcan (5 pts/ac), GWN 1708 (30 oz/ac) + 435 oil (2%), GWN 1708 (24 oz/ac) + Nexter 75 WP (6.6oz/ac)+ 435 oil (2%) and Portal 5 EC (64 oz/ac) was still evident at 42 DAT, though with little effect on CLM. In another experiment, Danitol 2.4 EC (16 oz/ac), Closer 240 EC (4.25 oz/ac) Exirel (16 oz/ac), Sivanto 200 SL (10.5 oz/ac), Admire Pro 4.6 SC (7 oz/ac), Actara 25 WG (5.5 oz/ac), Voliam Flexi (5 or 7 oz/ac), Agriflex (8.5 oz/ac) and Agrimek 0.15 EC (4.25 oz/ac) all applied with 435 oil (2% v/v) were effective against ACP at 60 DAT but only Exirel, Admire Pro, Voliam Flexi, Agriflex and Agrimek showed activity against CLM. Microbial insecticides Grandevo (3lbs/ac) and MBI 206 (2 gal/ac), Delegate 25 WG (6 oz/ac) all applied with 435 Oil (2% v/v) or 435 Oil alone (2%) reduced ACP through 28 DAT and CLM through 22 DAT and were less harmful to ants, spiders and lady beetles than previously mentioned products.
Objectives of this project include: 1) scale up production of Tamarixia radiata to levels that will allow better assessment of the potential impact of augmentation on ACP populations and ultimately the spread of HLB, 2) use genetic techniques to identify parasitoids recovered from the field to demonstrate establishment and effectiveness of released strains, and 3) pass on rearing technology to both private and public sectors to encourage production and an adequate supply of these parasitoids into the future. This quarter 69,516, 70,520, 72,355 and 75,730 T. radiata were produced in colonies from South China, Pakistan, North Vietnam and Florida strain previously established from Taiwan and South Vietnam and maintained at DPI, Gainesville. The colony at SWFREC produced another 21,677 “Florida Strain” T. radiata. Wasps from these colonies were used for research, to maintain the colonies and to release in conventional and organic citrus groves in Collier, Lee, Hendry, Lake, Indian River, Hillsborough, St Lucie, Polk and Hardee counties. When available, nymphs were collected from different locations under different treatment regimes and reared in the laboratory to estimate parasitism rates. No parasitoids emerged from nymphs collected in October (n=18) at SWFREC Collier county but 20% (n=20) nymphs were parasitized in December. In an experimental block of ‘Valencia’ oranges from a neighboring conventional grove which compares (1) Nutritional (2) Insecticides (3) Insecticides + Nutritionals and (4) Untreated treatments no nymphs were parasitized in October in the first three treatments (n=243, 211 and 154, respectively) and only 1% (n=217) were parasitized in the untreated blocks. In December, parasitism averaged 82% (n=11) and 76% (n=42) in the untreated and nutritional only treatments. No parasitoids emerged from nymphs (n=20) sampled in block of ‘Valencia’ oranges in Lee county from plots either untreated plots or sprayed biweekly with 435 oil. Parasitism averaged 36% (n=26) in untreated plots of ‘Valencia’ oranges in Hendry Co. Parasitism in November averaged 65% (n=43) in a grove in Lakeland, FL. Very few nymphs were available from locations in other counties and parasitism average 16% or less. Biparental colonies of Diaphorencyrtus aligarhensis are maintained at SWFREC Immokalee and DPI Gainesville and 5,575 wasps were released in Collier county this quarter although none have yet been recovered. Experiments were conducted in the laboratory to study the effect of nutrition on survival and egg formation in T. radiata. Seven different supplements: water, honey, NuLure, psylid nymphs, honey + NuLure, honey + nymphs and NuLure + nymphs were evaluated. The result showed that, honey alone was sufficient for survival, but egg resorption took place within 5 days in the absence of psyllid hosts. NuLure alone did not support survival for more than 5 days. Survival on honey + NuLure was as good as on nymphs, but fewer eggs developed. Females with access to psyllid nymphs for host feeding were found with significantly more eggs than if fed only honey, NuLure or the honey + NuLure. However, the combinations of honey + nymphs or NuLure + nymphs were even better than nymphs alone in regard to fecundity. Nymphal hemolymph appears to be the best source of nutrition for female wasps and probably contains all essential amino acids and a balanced ratio of carbohydrates. However, the extra source of carbohydrates and amino acids provided by honey and NuLure respectively did have a positive effect on egg production, even when host nymphs were available, indicating that even hemolymph is not a perfect food.
A replicated field study was conducted in a 5.4-ha commercial block of young ‘Valencia’ orange trees through four harvests employing a factorial design to evaluate individual and compound effects of a popular foliar nutrient program and threshold-based vector management. ACP populations were maintained at contrasting levels in insecticide-treated and untreated plots despite proximity. Nevertheless, incidence of HLB, estimated by PCR at nearly 30% at the beginning of the study, rose to almost 95% early in the third year without measurable reduction from vector control. However, insecticide treatments did result in higher threshold cycle (Ct) values, indicating reduced disease intensity. Vector control significantly improved yields all but year one, while the nutrition only treatment made a significant contribution to yield only in year four. Combined foliar nutrition and vector control was the best treatment all 4years, and provided yields close to the pre-HLB regional average in the 4th year. Although the combined treatment was not profitable at current juice prices, this shortcoming could be remedied by reducing costs of the nutrient package and/or the insecticide regime. In another location where HLB incidence and tree health was monitored on grapefruit and Hamlins receiving various treatments, including initially, tree removal, S. Halbert has been conducting trapping of psyllids. Psyllids from the traps are being analyzed for HLB titer by K. Hendricks, SWFREC. Four suction traps were operated at the SW Florida Research & Extension Center from July 2009 to present. These included an 8 meter tall trap and three 2 meter traps. Of the latter, one was in managed citrus, one was in unsprayed citrus, and the other was in an open field. Samples were collected approximately weekly. The psyllids were removed and identified in Gainesville. Beginning in 2011, all Diaphorina citri Kuwayama were tested singly for presence or absence of the HLB pathogen. All three short traps collected D. citri. Both traps located in citrus collected at least occasional D. citri throughout the year, but the trap in the unsprayed citrus collected the most. The trap in the open field showed peak activity in March, coinciding with the spring flush. These collections could indicate that longer distance flights away from the crop occur at that time of the year. Overall, there were few psyllids positive for Las. There were positive samples collected from all three short traps. There was no difference in the numbers of positives by trap. This can be attributed to the fact that citrus greening disease is widespread and common in the Immokalee area. Data indicated that neither nutritional nor insecticidal sprays impacted the disease progress of HLB, because either the treatments were initiated during the long lag time between inoculation/symptom expression or another reason. Recent yield data indicates that trees in plot receiving nutritionals and insecticide are benefiting by increased yields.
Data collection and analysis of field trials with ‘Ray Ruby’ grapefruit treated with foliar sprays with copper bactericides or soil drench or trunk application with the non-insecticidal SAR-inducer acibenzolar-S-methyl (Actigard) and the neonicotinoids imidacloprid (Admire Pro) and thiamethoxam (Platinum) to evaluate their efficacy for canker control on young bearing trees has been completed. Data analysis for the Hamlin orange trial is still pending. For evaluation of contact activity, nine film forming coppers treatments sprayed every 21 days with a handgun were compared with two rates of the soluble copper formulation, Magna-Bon (copper sulfate pentahydrate) or was applied as a combination of one film forming copper with Magna-Bon at lower rates. In spring 2012, early season rains were sporadic, hence incidence of old canker lesions on fruit was 13% on the non-treated control (NTC) or half of the incidence of newer late season lesions on the NTC (27%). All the copper treatments were effective for reducing lesion incidence by more than 50% (6% old sand 10% new lesions). Magna-Bon was as effective as the film-forming copper formulations alone, mixed with film-forming copper, or applied later in the season after the filming forming copper early in the season. Comparable cankercontrol achieved with the combinations of Magna-Bon substantially reduced total seasonal application of metallic copper. Four 60-day interval soil drench and trunk applications of Actigard, Admire and Platinum reduced canker incidence on fruit from 47% to 20% compared to 16% with 21-day interval foliar sprays season-long with copper hydroxide (Kocide 3000).
Objective 1: Preliminary trials conducted with potted nursery and young field trees indicate that soil drench is an effective and consistent application method for increasing copper status of young trees over the baseline concentration in leaves. Hence, in a trial in Picos Farm at USDA-Ft. Pierce, Hamlin on Swingle citrumelo trees were root drenched in late spring with the copper bactericides EXP, Cop-R-Quik, (CQ) Magna-Bon (MB), and copper phosphite (CP) and a zinc phosphite (ZP). Spring flush leaves were collected in July to measure copper and zinc concentration in leaves to test for systemic movement of the metals. Overspray of micronutrients obscured detection of treatment differences. Leaf samples for PCR testing were also collected to track Las infection progress. So far incidence of infected trees is less than 1%. To further evaluate soil drenches of the two commercial bactericides containing copper chelate (CQ and MB), three locations were selected. Two trials are in blocks with 1 year old trees that are apparently healthy and a third trial in a block of 4-yr old Valencia trees with symptomatic and pre-symptomatic HLB infection as determined by PCR status. Hence, the preventative and curative activity of the copper bactericides are being followed in pre- and early stage HLB infected trees. In the two trials with 1 yr-old young trees, a wide range of copper concentrations in flush leaves has been established. In the 4 yr old block, no effect of MB or CQ on HLB decline rating was detected in December 2012. Samples for PCR status of the trees in all trials will be collected and analyzed this spring.
Florida growers have reported that enhanced nutritional programs (ENPs) maintain productivity of HLB-infected trees. However, efficacy and sustainability of the nutritional approach for HLB disease management remains uncertain. Complementary studies of multiple ENPs and their individual components compared to the standard nutritional program (SNP) on nursery and field trees were initiated in 2010. Two independent nursery trials were initiated with final data collection finished and data analysis currently underway. From monitoring temporal bacterial movement and replication it was found that Candidatus Liberibacter asiaticus (Las) populations are similar for ENPs and the SNP. Minor differences in Las movement have been observed. Las invaded new flush tissue faster in ENP treated trees than SNP trees. Phosphite treatments have caused Las to favor early invasion of root tissue compared to other treatments. Preliminary analysis of the final takedown results from nursery trials shows a reduction in visual symptom expression with ENPs. However, there were no differences in the HLB-associated phloem plugging or reduction in leaf number, leaf size, and fibrous root mass. The field site was chosen for its mix of healthy, presymptomatic, and HLB symptomatic trees to determine if observed differences resulted from effects on healthy or infected trees. After 3 years of nutritional treatments, fruit yield and HLB symptoms in field trees treated with ENPs have not differed significantly from the standard nutritional program. The only significant predictor of yield differences was the percent of declining trees, but there was no interaction with nutritional treatment, suggesting that nutritional programs have not affected yields of healthy or HLB affected trees in this study.
Entomopathogenic nematodes: Amending soils to increase biological control of insect pests Conducted duplicate experiments to investigate effects of soils from ACPS and conventional plots on nematophagy by fungi and subsequent entomopathogenic nematode (EPN) efficacy against D. abbreviatus larvae. Soils were oven desiccated prior to use. Trapping nematophagous fungi (NF) or zoosporous NF were added to soils with either Steinernema diaprepesi (Sd) or Heterorhabditis indica (Hi). After 10 days EPNs were extracted from half experimental units and weevil larvae were exposed to soil from the other units. ACPS soil reduced survival of both EPN species regardless of NF presence. Effect of soil very pronounced on Sd, relatively mild on Hi. Effect of NF pronounced on Sd, with additive effects of soil and trappers and significant synergism between soil and zoosporous NF. Effects of NF on Hi relatively minor with no indication of synergism. Previous study of soils under both management regimes noted increased incidence of a bacterial parasite of Sd in ACPS soils. The bacterium was not present in current experiments, thereby extending potential mechanisms for ACPS effects on EPNs to encompass biocontrol by bacteria and fungi as well as direct inhibition of ACPS to EPNs, especially Sd. Plant parasitic Nematodes: Characterizing a new nematode pest and the prevalence of resistance breaking populations of the citrus nematode. PCR primers and taqman probes designed (from published sequences) to distinguish Xiphinema citricolum from X. laevistriatum (two dagger nematodes that infest citrus orchards) conflicted with our morphological characterization of several populations of dagger nematodes. The results indicate the need to re-evaluate the molecular and morphological characterization of dagger nematodes from type localities. This work has been initiated using specimens isolated from localities given in the original report (2 localities for each species). Continued survey of sites identified by DuPont soil sampling program as positive for dagger and/or citrus nematode coincident with tree decline.
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