Two 3-year field experiments are being conducted in two commercial orange blocks in Hendry County (southwest Florida). One of the groves is planted with ‘Earlygold’ oranges and the other with ‘Valencia’ oranges. Average HLB incidence estimated in both groves at the beginning of the experiment based on PCR analysis of a random sample of 160 trees was 98% in ‘Earlygold’ and 42% in ‘Valencia’. Experimental design is randomized complete block with 4 replicates and 4 treatments: (1) No insecticide, (2) Calendar applications, in order to drive vector populations close to 0, (3) nominal threshold of 0.2 psyllids per tap, and (4) nominal threshold of 0.7 psyllids per tap. Calendar applications are being applied every month. The insecticides used for these applications in the last three months were fenopropathrin (Danitol 2.4 EC) at 16 fl oz/ac in September, spirotetramat (Movento MPC) at 16 fl oz/ac plus horticultural mineral oil 2% in October and carbaryl (Sevin XLR plus) in November at 0.65 gallons/acre. In December, treatments (2) and (3) received the first dormant spray with phosmet (Imidan 70-W) at 1 lb per acre. In the third week of November the “Earlygold” block was harvested and yields, yield increments with respect to the 2010 harvest as well as several fruit quality parameters were measured for each treatment. Values obtained were correlated to cumulative number of ACP adults obtained by tap sampling during the last season. A significant effect of treatments on yields was found (F = 4.03; df = 3, 15; P = 0.045). Treatment (2) had significantly higher yields (266.9 ‘ 10.9 boxes per acre) than the rest of the treatments (208.9 ‘ 11.7, 213.4 ‘ 22.5 and 220.7 ‘ 14.6 boxes per acre for treatments (1), (3) and (4) respectively). Similarly, yields with treatment (2) significantly increased (45.25 ‘ 15.73 boxes per acre) compared to 2010, in contrast to the rest of the treatments (-7.00 ‘ 15.59, 14.60 ‘ 13.34 and 10.14 ‘ 15.19 boxes per acre from 20110 to 2011 for treatments (1), (3) and (4) respectively). There was also a significant treatment effect on fruit diameter (F = 4.27; df = 3, 639; P = 0.0053), fruit weight (F = 5.23; df = 3, 639; P = 0.0014) and the fruit weight/volume ratio (F = 6.07 df = 3, 639; P = 0.0004) with higher values for the three parameters in treatment (2). Despite the fact that we also found significantly ACP treatment (2) compared to the rest of the treatments (F = 6.13; df = 3, 15; P = 0.0148), no significant correlation was found between cumulative ACP numbers and yield losses (P = 0.8353). Future harvests are expected to increase differences among treatments. In a preliminary cost-benefits economic analysis the estimated additional cost of the calendar insecticide program was $555.6 per acre and the estimated yield increment from 2010 to 2011 due to the insecticide calendar applications was 39.3 boxes per acre. According to this, the juice price that would return the insecticide investment would be $2.28 per pound solids. Currently, markets price are under this number, around $2.00 per pound solids. Therefore, after the first year of applying the ACP insecticide calendar program, increase profits due to this program in last harvest would not cover the extra costs of the program.
Objectives of this project include 1) refinement of sampling methods, 2) testing the influence of adult density and shoot infestation on precision of estimated means and distribution of population within blocks, and 3) evaluation and integration of methods for assessing psyllid density, shoot density, and infestation rates into a user friendly system accessible to consultants and managers. Counts of psyllid adults by stem tap and sticky traps were compared for two years in a 12-acre commercial block planted with Valencia x Carrizo oranges subjected to four treatments (1) Nutritional (2) Insecticides (3) Insecticides + Nutritionals and (4) Untreated. This block was divided into 16 plots, equally distributed among four treatments. Plots under treatment 2 and 3 were treated with insecticides when psyllid adults exceeded 0.2 per tap sample. The remaining eight plots were also continuously monitored, but were not treated with insecticides although four received nutritional sprays. In each plot six trees were selected and in each selected tree, one ‘sticky trap’ was deployed inside the canopy. Traps were replaced biweekly, at which time two tap samples were conducted in each tree. A ‘tap sample’ count consisted of adult psyllids falling on a white clipboard placed under randomly chosen branches which were then struck 3 times with the PVC pipe. Sticky traps were examined under the stereoscopic microscope in the laboratory and adult psyllids counted. Biweekly captures of adults on sticky traps averaged 2, 3, 13 and 15 in treatments 3, 2, 4 and 1, respectively. Adults per tap sample conducted only at trap replacement averaged 0.1, 0.2, 0.7 and 0.7 in treatments 3, 2, 4 and 1, respectively. The relationship between the two sampling methods was analyzed using Pearson Correlation Coefficient. Analysis of cumulative data showed that two methods were highly correlated in treatments 1 (r = 0.66, P = < 0.0001) and 4 (r = 0.69, P = < 0.0001) not treated with insecticides but weak in treatments 2 (r = 0.21, P = 0.12) and 3 (r = 0.18, P = 0.20) that were treated. Analysis of data on individual dates showed that out of 37 comparisons 10, 16, 17 and 23 were statistically significant in treatments 3, 2, 4 and 1, respectively. Examination of data plots showed that although both methods were detecting psyllids at low and high densities, either one or the other was detecting more psyllids at low density at different times, particularly in treatment 3 averaging lowest population. These findings once again indicate that tap sampling is an effective and efficient method which provides reliable and instant information for management decisions. Detailed comparisons of different sampling methods are provided in the EDIS document listed below. We are now comparing stem tap sampling with vacuum sampling in commercial citrus. The vacuum sampler is made from a leaf blower and collects many psyllids even at low populations. A single strike captured an average of about twice what a single tap sample would catch and the two samples were significantly correlated. Use and effectiveness of the tap sampling method was promoted through several meetings and workshops including the one arranged by FAO on "Taller subregional 'Control Biol'gico en HLB': Recomendaciones para Centroam'rica". It was encouraging to see that stem tap method is already being used in some concerned countries such as Costa Rica and Belize. Arevalo, A. H, J. A. Qureshi and P. A. Stansly. 2011. Sampling Asian citrus psyllid (ACP) in Florida citrus groves. EDIS, http://edis.ifas.ufl.edu/in867.
We continue to conduct replicated experiments and measure the impact of psyllid control programs on non target pests and beneficial insects. During this quarter, experimental blocks at commercial and SWFREC groves were sampled for psyllids, citrus rust mites, citrus red mite, predatory mites, snow scale, chaff scale, Glover scale, purple scale, citrus blackflies, whiteflies, mealy bugs, as well as their natural enemies. Only more common species are reported. The experiment in Collier county compares 4 treatments (1) Nutritional (2) Insecticides (3) Insecticides + Nutritionals and (4) Untreated control. Insecticides currently recommended for psyllid control are used when adults exceed 0.2 per tap sample and nutritional products and their application timing follow a program developed by McKinnon Corporation averaging three sprays per year. CRM were high on fruits in the treatments using insecticides (0.2-0.3/lens field) compared to nutritionals and control (0.02-0.03/lens field). However, chaff scale was not observed in the samples conducted in insecticide treatments compared to 0.1/lens field in the nutritional and control treatments. Shoots infested with woolly whitefly averaged 9-12% in insecticide treatments compared to 19-27% in nutritional and control treatments. Threshold levels of psyllid population were not reached this quarter in two experiments in Hendry county, one in a 35 acre block of ‘Early Gold’ and the other in a 16 acre block of ‘Valencia’. However plots designated for calendar sprays received of a rotation of insecticides recommended for ACP control applied every month: abamectine (Agri-Mek SC) at 3.5 fl oz/ac + horticultural mineral oil 2% in July, malathion (Gowan Malathion 8F) at 2.5 pt/ac in August and fenpropathrin (Danitol 2.4 EC) at 16 fl oz/ac in September. Psyllid adults per tap sample averaged 0-0.01 in the treated plots and 0-0.08 and 0-0.2 in the control of Early Gold and Valencia, respectively. Citrus rust mites and chaff scale did not differ between treated and untreated plots. However, only 2% shoots were infested with woolly whitefly in treated plots compared to 34% in control plots of ‘Valencia’. Shoot infestation with woolly whitefly averaged 36% and did not differ between plots treated with grower standard sprays, biweekly spray of 435 oil or untreated check in a10.9 acre block of ‘Valencia’ in Lee county. Citrus rust mite and chaff scale averaged 1.9 and 0.35 per lens field, respectively, and did not differ between the three treatments. Psyllids in the grower standard, oil and control treatments averaged 0.03, 0.24 and 0.35 per tap sample, respectively. A study conducted at the SWFREC used 16 yr old Valencia orange trees and tested 13 treatments of Danitol 2.4 EC, Actara 25 WG, Agri-flex, Voliam Flexi, Dibrom 8 E, Portal 0.4 EC and MBI203 DF1. All treatments provided significant reduction in psyllid populations for one month. However, treatment effects against citrus leafminer were very short lived. Significant reduction was observed at 3 days after treatment (DAT) with Danitol 2.4 EC, Actara 25 WG, Agri-flex, and Dibrom 8 E all applied with 435 oil and Voliam Flexi and Portal 0.4 EC applied alone. Only Agri-flex + 435 oil and Voliam Flexi were providing significant reduction at 10 DAT. It seems that calendar or grower standard treatments and experimental treatments suppressed psyllids but did not reduce non target pests in most situations, creating need for additional applications and negative impacts on predators and parasitoids. Psyllid populations were very low during the reported period and fewer or no sprays were warranted based on thresholds.
The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apple psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. The production of greenhouse-grown citrus trees with nutrient deficiency symptoms for both zinc and boron continues. Deficiency symptoms continue to develop for boron and zinc deficiencies have started to be apparent. The high mobility of zinc within the plant allows redistribution of existing zinc to new flush, delaying the development of deficiency and the associated symptoms. HLB symptom development continues in our plants with the different HLB strains. As soon as strong deficiency symptoms develop, then psyllid testing for objective 1 and 2 will commence and is expected to move rapidly. We are also interested to determine if strains of Las will have any effect on the attractiveness of trees to D. citri. It has been reported that Las strains have varying levels of virulence and symptomatology (Tsai et al. 2008). We have analyzed DNA samples from HLB positive trees from Polk and Highlands counties as well as the ‘Smoak Grove’ CREC greenhouse strain by PCR and sequencing. Three putative strains of Ca. Liberibacter asiaticus (Las) were found with 5 (CREC greenhouse isolate), 13, and 15 tandem repeats of DNA in the LAPGP locus described by Chen et al. 2010 and have identified sources of budwood. Cloning and sequencing of loci including the b-operon, OMP (outer membrane protein) gene and phage DNA polymerase to support the differentiation of the three strains is complete (Bastianel et al. 2005; Lin et al. 2008; Okuda et al. 2005; Tomimura et al. 2009). Results from sequence analysis clearly defines two strains based on conserved mutations in the b-operon sequence, matching strains from Japan and Vietnam and a strain from Vietnam for the northern and southern Florida strains, respectively.
One of the most recent challenges with deploying DMDS in the ISCA release device called SPLAT (specialized pheromone lure application technology) has been the phytotoxicity of the treatment to leaves and branches. A substantial amount of the DMDS active ingredient is needed in order to affect psyllid population densities. Unfortunately, we have found the DMDS active ingredient burns tree foliage and can even kill entire small tree branches, if applied directly to the wood surface. Therefore, we have been developing alternative release devices that would allow deploying the DMDS active ingredient within trees without touching tree surfaces. Our most recent prototype is a sachet that is hung in trees with a wire hanger. The sachet contains the SPLAT-DMDS, which is allowed to evaporate through a porous membrane. Therefore, the DMDS dispenser is deployed in trees; however, the active ingredient does not come in direct contact with tree branches or leaves. We are currently investigating whether these devices will be effective. The experiment was initiated in September and we are still analyzing data. The results of an earlier experiment conducted in August with a newer formulation of SPLAT-DMDS have been analyzed. The results of this test did not replicate the success we observed with the same formulation last fall. Psyllid populations were not significantly reduced by deployment of this formulation in this mid-summer test (as compared with control plots)as was observed last fall. We are unsure why we were unable to replicate the earlier success. We are trying to determine if the inconsistent results are because of differences in psyllid population densities between these tests or differences in environmental conditions when the different tests were conducted. It is possible that the formulation is not holding up to the intense temperatures and rainfall experienced in the summer as compared with cooler and dryer conditions in the fall. Finally, we have initiated testing of three new formulations of SPLAT that contain repellents other than DMDS that have proven effective against psyllids in laboratory tests. These experiments have only recently been initiated and we should have initial results in 3-4 months.
The purpose this investigation has been to evaluate and improve low volume spray technology for psyllid control. We have been investigating how low volume treatments (2-10 gallons per acre) compare with standard applications using airblast sprayers at 200 gallons of finished spray per acre. We have been investigating longevity of residual activity of the sprays against Asian citrus psyllid (ACP). Also, we have been investigating alternative application practices, such as border row treatments with low volume technology. 1) Leaf residue toxicity studies that compare standard high volume with low volume spray applications in the field. In these experiments, we investigated the longevity of residual activity of low volume spray residues against adult ACP by comparing low and high volume treated leaves. Trees were treated in the field with commercial airblast or low volume sprayers. Leaves were removed from the plots where fenopropathrin (16 oz/acre), phosmet (1.5 lb/acre) or imidacloprid (15 oz/acre) were applied by low (12 gpa) or high volume (200 gpa) applications. Leaves were removed at four and eight days post-application. A bioassay was then performed on leaf discs held in Petri dishes. Approximately 15 adult Asian citrus psyllids were placed per assay dish with leaf discs of either the treated leaves (low volume versus standard) or untreated control leaves. Adult mortality was assessed at 24 h and 48 h post exposure. The results showed that mortality of adult psyllids on 4-day old insecticide residues was between 60 and 100% for high volume application, but <10% for low volume treatments. For residues on day eight, only the high volume fenpropathrin treatment caused mortality (80%). The other treatments caused <10% mortality. A residue trial of the adjuvant, Induce, was conducted with the insecticides fenpropathrin and chlorpyrifos at both low and high volume applications. At day three, discs from all treatments caused 100% mortality. On day seven; however, fenpropathrin + Induce low volume and high volume were the only treatments to cause adult mortality: 65 and 80% respectively. 2) Effectiveness of the border row treatment tactic for ACP control. The objective of this experiment is to investigate the reduction in numbers of ACP adults caused by treating the two border rows of grove plots as compared with blocks in which every row was treated. An untreated control of three plots was included for comparison. Numbers in both fully and border only treated blocks were significantly reduced by applications three and seven days after applications were made. On day 14, the effects were not significant in the border row-treated plots as compared with the controls. The three scheduled low volume applications of 16 oz/acre of fenpropathrin with with 2% V/V 435 crop oil added as a LV application of 12 gpa have now been made and spatial analysis of the data is underway. Leaf disk bioassays were also performed on leaves taken from the upper canopy (~2.5 m) of mature trees and and compared to leaves collected from 1 m. At three days after treatment, the 1 m leaf disks caused 100% mortality versus 0% for the upper canopy leaves. Results from day seven indicate that neither low (1m) or high (2.5m) leaves were significantly toxic to the adults after low volume treatment. Overall, these results indicate that low volume treatment applications have very short residual activity and most of the ACP kill occurs during or very shortly after treatment.
One of the main objectives of this research is to identify attractants for Asian citrus psyllid (ACP) that could be used for monitoring, as well as, developing attract-and-kill technologies for ACP. Although we have focused on identification of ACP pheromones considerably, we have also made substantial progress recently with respect to identifying citrus-based volatiles as ACP attractants. Recently, we examined host plant volatiles from uninfested flush and young leaves of citrus genotypes that were reported to vary in susceptibility as measured by colonization by ACP in the field (Westbrook et al. 2011). Five species of Rutaceae were selected for plant volatile analysis from a list of genotypes assayed for colonization by Westbrook et al. (2011). In that study, 87 genotypes were examined in the field for presence of eggs, nymphs and/or adults. We collected and analyzed volatiles from three genotypes on which adults were abundant, including Bergera koenigii L. (curry leaf tree), Murraya paniculata (L.) (orange jasmine) and Citrus macrophylla Wester. Volatiles were also analyzed from Citrus jambhiri Lush. (rough lemon), a genotype on which the ACP population was nearly midway between the highest and lowest recorded, and Poncirus trifoliata L. (trifoliate orange), whose population of adults was near the lowest recorded in the study. C. sinensis (L.) Osbeck ‘Valencia’ was also included because of its widespread commercial use and susceptibility to ACP. Plant volatiles were collected by placing freshly excised 15 g bouquets consisting of shoots of terminal leaves and flush of each genotype into separate 50 ml beakers containing distilled water. Bouquets from B. koenigii were collected from field-grown plants whereas bouquets from the other five genotypes were collected from potted greenhouse-grown plants. The beaker was then placed into a closed ca. 1-liter glass container. Charcoal-filtered humidified air was pushed through the container at a rate of 500 ml/min for 24 h. Plant volatiles were trapped in glass tubes packed with Porapak Q. The collected volatiles analyzed by GC-MS using a ThermoElectron Trace GC coupled to a DSQII quadrupole MS, all controlled by Xcalibur software. Limonene and (E)- .-ocimene were the only volatiles that occurred in the headspace over all six genotypes. Whereas limonene was present at high percentages in headspace collected from C. jambhiri and C. macrophylla collections (55.4% and 60.7%, respectively), it constituted 5.0% of volatiles from C. sinensis ‘Valencia’ and <1% from B. koenigii, M. paniculata and P. trifoliata. Similarly, (E)- .-ocimene was found at levels of 10.1% and 14.0% in C. jambhiri and C. sinensis 'Valencia' headspace, respectively, but only at 2.4% of volatiles collected from B. koenigii; <1% of this compound was found in extracts of C. macrophylla, M. paniculata and P. trifoliata headspace. The collection from B. koenigii, the genotype with the second highest susceptibility to adult ACP in the Westbrook et al. (2011) study, contained 48.3% '-phellandrene. Our most recent data report the proportions of various compounds found in the headspace volatiles among six species in the family Rutaceae differing in their susceptibility to ACP. Some compounds occurred exclusively while others were common to several species. In genotypes such as P. trifoliata, it will be important to assess whether it is a lack of key attractive components that renders it unsuitable for ACP or whether it is the presence of repellent compounds. Behavioral analysis of individual compounds as well as blends of compounds are underway and should produce useful information for psyllid management and plant breeding strategies.
We have been investigating levels of insecticide resistance in populations of Asian citrus psyllid (ACP) in Florida. There are three distinct color morphs of ACP. In other words, ACP adults in the field are either gray/brown, blue/green or red/orange, based on their abdominal color. Over the course of an annual season, the proportions of ACP representing a particular color morph change. For example, gray/brown psyllids may be more abundant in the summer than winter, while blue/green ACP may be more abundant in the winter than summer. In insects, variation in body color among individuals of a species is a common phenomenon. These color variations play an important role in mate selection, keeping predators away, body temperature regulation, and habitat selection. Color variations in individuals of a species are possibly the result of phenotypic plasticity, nutritional status, bacterial symbionts, infection status, genetic factors or combinations of two or more factors. We noticed that certain color morphs may be more suspectable to insecticides than others in experiments in which resistance levels of FL ACP populations were being evaluated. Our hypothesis is that factors such as nutritional status and bacterial symbionts dictate the abdominal color of ACP, which in turn influences levels of insecticide resistance in corresponding color morphs of ACP. Previous studies with other insect species have shown that nutritional status and bacterial symbionts influence insecticide susceptibility. Insecticide susceptibility for three insecticides was compared among gray/brown, blue/green and red/orange color morphs of field-collected ACP. ACP from the red/orange morph were significantly more susceptible to fenpropathrin than ACP of the blue/green morph. Imidacloprid caused higher mortality of red/orange than blue/green and gray/brown morphs. Likewise, chlorpyriphos caused significantly higher mortality in red/orange than in blue/green and gray/brown morphs. To confirm the genetic basis of variable susceptibility levels among the three color morphs, relative expression of five CYP4 genes (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1 and CYP4G70) was compared among the color morphs. These genes regulate expression of the specific enzymes that degrade the insecticide imidacloprid (and certain other insecticides/toxins). Expression of all five CYP4 genes was significantly different among the various color morphs tested. CYP4C67 was expressed at significantly higher levels in blue/green than in red/orange and gray/brown morphs. CYP4DA1, CYP4C68, CYP4G70, and CYP4DB1 were expressed at significantly higher levels in blue/green and gray/brown than red/orange morphs. These results suggest that expression levels of five CYP4 genes are highly specific to the color morph of ACP tested. Red/orange ACP color morphs are characterized by lower levels of CYP4 genes, indicating lower levels of cytochrome P450 monooxygenases. Lower levels of CYP4 genes in red/orange morphs suggest that decreased expression of such genes might be responsible for higher susceptibility to the three insecticides tested in ACP. These CYP4 genes may serve as DNA-based screening markers for cytochrome P450-mediated insecticide resistance in field populations of ACP. In addition, these results will be useful for insecticide resistance monitoring, since we know that certain morphs are more susceptible than others. This will allow us to standardize our tests during various points in the season. Finally, during the times when red/orange ACP are most prevalent, ACP populations may be most susceptible to several currently used insecticides.
The goal of this project is to evaluate and find effective botanical repellents for Asian Citrus Psyllid (ACP) management that will be safe to the environment and not harm beneficial insects. The objectives include examination of repellent and toxicological properties of selected botanical chemicals against ACP that are known to be active against other insect species. For behavioral bioassays we are using a customized T-Maze olfactometer. The olfactometer consists of a 30 cm glass tube that is bifurcated into two equal halves with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the ACP to make a choice between two potential odor fields. The olfactometer arms are connected to odor sources placed in solid-phase micro-extraction chambers (ARS, Gainesville, FL) through Teflon’-glass tube connectors. The samples are diluted in appropriate solvent and subsequently pipetted onto a 5 cm Richmond cotton wick, which will serve as the release device. Each chemical is evaluated individually on at least 4 dilutions on a log scale to determine optimum release rate. The control treatment consists of a cotton wick impregnated with solvent only. To evaluate insecticide contact activity of compounds we have secured an automatic microapplicator. Seven serial dilutions for each test chemical are prepared by dissolving the test chemical in acetone or absolute alcohol. Thereafter, 0.4 ‘l of each serial dilution of test chemical or a solvent only (control) treatment is applied from to the dorsal mesothorax of adult psyllids. Treated psyllids are transferred to Petri dishes containing citrus leaf discs placed on a solidified agar beds. Mortality of ACP is scored 48 hr after exposure to treatments. Data are analyzed using standard probit analysis. Toxicity bioassays using treated glass jar techniques will also be carried out using higher dosage for the selected botanicals. In this project we are evaluating 30 plant-produced chemicals for their attractant, repellent, and insecticidal activities against ACP with an intent to identify effective tools for ACP. Preliminary bioassays with some of the botanicals indicated that a ketone compound, gernyl acetone from cardamom attracted psyllids at a 1 ul dosage in a customized T-maze laboratory olfactometer. Geranyl acetone is also a component of general citrus volatiles. Similarly, another ketone, alpha damascene, from rose attracted psyllids at a 10 ul dosage. A sesquiterpenoid, citronellol from lemon grass oil repelled psyllids. However, topical applications of a 0.8 ug dosage per insect of these two chemicals did not show toxic activity against psyllids. Beta-damascone, 9-decen-ol, and geraniol did not influence psyllid behavior up to a 100ul dosage. Behavioral and toxicity bioassays with more botanical repellents/insecticides are in progress.
As the number of Citrus growers interested in providing certified organic products increases in the Lower Rio Grande Valley, there is a need to evaluate the efficacy of biopesticides products available for farm use. Many of these biopesticides have previously worked with other horticultural crops throughout the US but remain unproven on South Texas citrus pests and citrus varieties. Previous studies performed at TAMU-Kingsville Citrus Center over the past two years focused on the impacts of various compost and bio-pesticide sources at reducing Asian Citrus Psyllid (ACP) populations. These studies yielded encouraging results on the potential of biopesticides to provide ACP management in organic farms. Objectives: The purpose of this project is to determine the effect of using organic practices such as the use of bio-pesticides and different compost sources (i.e., green waste compost, Sugarcane mill waste compost, and seafood based compost) on the growth parameters of young grapefruit trees and their pest load and compare it to conventionally managed citrus trees in South Texas Data Analysis: A randomized split plot design was used and a ‘Mixed model analysis’ evaluated the effects of the main factors (treatments , controls and pest evaluation) and their interactions on the biopesticides used against pest and or fungal diseases. Correlation analysis will establish relationships between active ingredient levels and densities of each target. Pest survey in the field, pre-treatment sampling and weekly post-treatment sampling and surveys was made to assess pest densities and bio-pesticide control efficacy. Pest population densities and fluctuations will be evaluated throughout the experimental period. Greenhouse and laboratory chamber studies will utilize 1 year-old trees or leaf tissue samples to more accurately determine the mechanism involved in compost or biopesticide source impacts on citrus pest control. Results: Application of bio-pesticides reduced ACP egg, nymph, and adult populations immediately following spray application. Although, the reduction in each case was only temporary, suggesting that routine and continuous spray of bio-pesticides would be essential for ACP management in organic citrus production. Kaolin+Oil treatment had a better trend of reducing ACP populations than the other two programs, but the Kaolin negatively impacted plant growth and photosynthesis and is recommended for only periodic use and not as part of a continual routine spray program. Interestingly, the major finding from this study came from the sugarcane mill waste compost source applied to young citrus trees. A negative trend correlation was found between decreasing ACP populations and increasing calcium (Ca) in citrus leaves. This Ca increase arose from the sugarcane compost source, and was not particularly significant for the other two compost sources. Benefits to Citrus Industry: With an increase in citrus organic farming, growers will benefit from the data as findings may provide safe mechanisms such as appropriate compost source or foliar inorganic sprays containing available Ca to reduce ACP population without the use of bio-pesticides.
Florida citrus production is facing increasing acreage losses due to disease threats arising from pests like the Asian Citrus Psyllid and Citrus Greening Disease (Huanglongbing, HLB). Some citrus growers in Florida have experienced positive results at combating the physiological damage and yield loss commonly associated with HLB by following a good nutrition plan, even in fields with up to 95% of trees confirmed with HLB disease. A foliar nutrient spray solution is one such nutrient solution that has promise at boosting citrus tree vigor to assist in the combating HLB disease. Some growers in Florida have observed varying results when mimicking the Maury Boyd nutrient cocktail mixture in orchards with confirmed HLB present. However, one nutrient currently not listed in the Maury Boyd cocktail provided to many of these growers is the macronutrient Calcium. Initial data from field trials after 12 weeks from these treatments supports finding from preliminary studies, with lower ACP populations ontrees with foliar Ca treatment applied , as compared to other treatments. Objectives: The purpose of this project was to determine the effect of using various micro- and macro-nutrient foliar sprays, in combination with nitrogen and oil spray treatment, on the physiological growth parameters of young grapefruit trees, their pest load, and subsequent impacts on fruit yield and quality. Data Analysis: A randomized split plot design was developed and a ‘Mixed model analysis’ will evaluate the effects of the main factors (treatments , controls and pest evaluation) and their interactions ofthe foliar nutrient sprays used against pests. Correlation analysis will establish relationships between nutrients on mites, leafminer, scale, ACP pest densities and leaf nutrient status. Time is still needed to evaluate findings throughout an entire growing season on all pests, plus to adequately evaluate the impact of the foliar treatments on fruit yield and quality during the harvest season. Funds for this study were only approved in April 2011. Due to the short time span for evaluation, we are only 4 months into a one year assessment to satisfy minimum objectives for this study, thus a request has been made for a continuance of financial support by the Texas Citrus Producers Board to finish the study during the upcoming 2011-2012 year. We have requested and have received continued funding to support this project and pay for the soil and leaf sample analytical procedures with final accomplishments to be finalized by August 2012. Benefits to Citrus Industry: With an increased threat of HLB disease and other insect related diseases facing LRGV citrus growers, we anticipate that this data may allow us to find another aspect of decreasing this threat to the industry without the use of bio-pesticides. Furthermore, yield and fruit quality assessments will provide economic data as the financial incentives for foliar applied nutrient plans for South Texas citrus production.
Research targeting methods to block Asian citrus psyllid feeding as a means to prevent the spread of HLB in citrus was initiated. A technical person was hired to maintain psyllid colonies and these colonies were initiated. A postdoctoral research associate with hired and initiated research. Early work was in developing new methods to study salivary sheath biosynthesis in a way that is compatible with our screening protocol. This work resulted in the identification that the sheaths are primarily of carbohydrate composition with few other substances. Specific proteins were identified and are now being characterized, but abundance of these proteins is so low that they are most-likely not structural components of the sheaths. Our earlier finding of EDTA inhibition of salivary sheath synthesis was incorporated into research showing that EDTA changes secreted protein profile during psyllid feeding. We are currently characterizing the proteins to determine their function. Torrey Pines Institute for Molecular Studies has initiated the synthesis of peptide libraries that will be tested in our psyllid feeding bioassay looking for compounds that will block the feeding process or otherwise affect psyllid behavior.
Screening of a peptide combinatorial library was initiated using structured combinations. Separate peptide combinations that induce either increased psyllid mortality of improved psyllid survival were identified in the first round of screening. Discovery of peptides that improve psyllid survival on an artificial diet may prove to improve laboratory experiments designed to develop new psyllid control strategies. Discovery of a peptide that will block the ability of psyllids to feed on citrus is being pursued in research aimed at developing novel insect control strategies based on either application of the peptides to plants or production of plants that produce this peptide as a resistance mechanism. Furthermore, continued analysis of salivary sheath composition has led to a new hypothesis on how salivary sheaths are synthesized and how their building blocks are recruited for polymerization to form the sheath.
The objective of this project was to evaluate methyl salicylate dispensers to determine whether their deployment in citrus can enhance biological control of Asian citrus psyllid (ACP). Locations selected for the field trials with the commercially available methyl salicylate (MeSA) lure, Predalure (AgBio Inc.; Denver, CO) were unmanaged grove plots (‘Valencia’ oranges, Clermont, FL), minimally managed grove plots (mixed citrus cultivars, Lake Alfred, FL) and heavily managed grove plots (‘Hamlin’ oranges, Auburndale, FL). The plots were 0.4 ha in area. Treated plots had 1 MeSA lure attached per tree and control plots were untreated. Lures were replaced at 8 week intervals and data were collected season-long. Adult ACP and natural enemies were monitored every 2 weeks with Pherocon AM yellow sticky traps and by sweep net samples. Collection of field data for the methyl salicylate (MeSA) study was concluded on 10/5/2010. In all groves, psyllid populations remained low throughout the season. The overall average number of psyllids per trap was <1 for the intensively managed grove, ~1/trap for the minimally managed grove and up to 1.5/trap for the unmanaged grove. No significant differences were recorded between psyllid numbers or in the number of beneficial insects captured between plots treated with MeSA and control plots. These results were different from our initial investigation in 2009, which indicated that natural enemies were higher in MeSA-treated plots. However, in that year, psyllid populations in that grove were much higher than in the groves studied in 2010. In 2009 an average of > 10 psyllid adults were captured per week in control plots whereas ~2 psyllids were captured/week in MeSA-treated plots. Also in 2009 we saw an increase in natural enemies, particularly coccinellid beetles, in MeSA-treated plots as compared with control plots. We hypothesize that the low psyllid numbers overall in 2010 could account for the lack of effect of MeSA treatment. If natural enemies were initially attracted to the plots, the lack of psyllid prey may have caused them to leave the plots in search of resources elsewhere. Annual variation in attraction of natural enemies to MeSA has been observed in other crops (Rodriquez-Saona et al. 2011. Biological Control. In Press. doi:10.1016/j.biocontrol.2011.06.017). It is also possible that insecticide use or the unusually cold winter affected our findings in 2010. In laboratory bioassays, MeSA was repellent at a high concentration (100 ug) but attractive at a low concentration (0.01 ug) to adult ACP in an olfactometer. In settling experiments on plants, however, more ACP were recorded on MeSA-treated plants than on control plants for the 2 concentrations tested (0.001 and 0.01 M), perhaps indicating that the sprayed concentration was not high enough to observe repellency. In conclusion, MeSA treatment in citrus had mixed results with respect to attraction of natural enemies and reducing psyllid populations in citrus in Florida. MeSA may have an effect in situations when psyllid populations are high at attracting natural enemies and thereby increasing the effect of biological control by earlier and enhanced recruitment. MeSA appears to be a psyllid attractant at low dosages and a repellent at high dosages.
We proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. To date, we have cloned sequences at least 400 bp in length from nine homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 (GSTS1) and constitutively expressed S4e ribosomal protein from M. persicae, as well as a control derived from green fluorescent protein (GFP) sequence. In a side project, we have cloned and characterized two novel IP-free SUS1 promoter alleles (CsSUS1-1 and 2) from Citrus sinensis cv. valencia and have found them to drive phloem-specific expression in both Arabidopsis and tobacco when fused to a reporter gene. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, we evaluated the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). In this objective, Gateway-based vectors were used to express the selected insect dsRNA either constitutively (35S promoter) or in a phloem-specific manner. We successfully generated Gateway vectors that will result in constitutive (35S promoter) or phloem-specific (CsSUS1 promoter) expression, respectively, of M. persicae-specific Coo2, GSTS1 and S4e dsRNA, as well as a control derived from GFP. Our results suggest that the M. persicae-specific dsRNA expressed in planta has a negative effect on both the lifespan of the insects and the number of offspring generated. In the fall of 2010, we began working on objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Previously, we conducted 3′ rapid amplification of cDNA from vacuolar ATP synthase subunit G, S4e, and .-tubulin transcripts from D. citri. We have now inserted sequences of the aforementioned transcripts into Gateway-based vectors downstream of both the constitutive 35S and our novel phloem-specific citrus CsSUS1 promoters. To date, we are in the process of transforming and regenerating citrus with the D. citri-specific gateway vectors for evaluation and use by the Florida citrus industry. Initial attempts to transform and regenerate Citrus sinensis ‘Valencia’ and ‘Hamlin’ containing reporter gene constructs were successful. Currently we have completed transformation of citrus callous tissue using gateway vectors with the vacuolar ATP synthase subunit G or S4e transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter. To date, we have generated several lines, and we are in the process of growing them up for testing in Florida. At the same time, we are preparing to generate additional lines of the aforementioned constructs and with the other transcript/promoter combinations from the second objective. In summary, we have cloned a number of transcripts from both D. citri and our model organism, M. persicae, and have analyzed a subset of derived dsRNAs to test their effect on M. persicae using in vitro assays (objective 1). We have also cloned and characterized several novel phloem-specific promoters from C. sinensis, and have evaluated their expression patterns. We have also created new Gateway-derived vectors bearing a native citrus phloem-specific promoter, for use in RNAi in our M. persicae model and evaluated them in planta (objective 2). Finally, we’ve now generated similar vectors specifically designed against D. citri (objective 3), and are generating transgenic lines expressing D. citri dsRNA for evaluation in Florida.
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