Leaf residue toxicity studies. The objective of this experiment has been to determine the residual efficacy of low volume and high volume insecticide applications on citrus leaves. Fenopropathrin (16 oz/acre), phosmet (1.5 lb/acre) and imidacloprid (15 oz/acre) were applied to citrus either as a low volume (LV) at 12 gpa or a high volume (HV at 200 gpa) application. For the fenpropathrin and imidicloprid treatments, 2% V/V of 435 crop oil was added and for the phosmet treatment LI-700 was added to adjust the pH of the solution to the recommended range. At 4 and 8 days post-application, leaves were removed from the plots and a leaf disc mortality bioassay was performed in Petri dishes. Approximately 15 adult Asian citrus psyllids were placed in the dishes with leaf discs of either the treated leaves or untreated control leaves. Adult mortality was assessed at 24 h and 48 h post exposure. Four days after treatments were applied, the 24 h exposure mortality for HV treatments was 100% for fenpropathrin, 84% for phosmet and 61% for imidicloprid. The only treatment with any mortality for LV applications was phosmet at 2.2%. After 48 hrs of exposure, all the HV treatments had >90% mortality. Mortality in all the LV treatments was <10%. Leaves removed from the field at day 8 caused much lower mortality in HV applications than at day 4. After 24 h of exposure exposure the mortality for fenpropathrin was 79%, phosmet was 14% and imidicloprid was 16%. Interestingly, the LV mortality at day 8 was somewhat higher than at day 4 for phosmet (14%) and imidicloprid (18%). After 48 h of exposure, mortality was higher for both HV and LV treatments. Border row treatment. In these experiments our goal is to compare the reduction in psyllid populations when treating all rows of citrus grove plots versus border row only treatment. All treatments are made to plots where psyllid populations were low initially because of a previous comprehensive treatment. In a 44 acre commercial grove, plots have been sprayed with 16 oz/acre of fenpropathrin with 2% V/V 435 crop oil added as a LV application of 12 gpa. In 3 of the plots, every tree was sprayed and in another 3 plots, only the outer 2 rows of trees were sprayed. Three plots were left untreated as control plots. We have completed the first trial of this experiment and are analyzing the results. A second replication is planned for early July, a third in August. Canopy investigation. This experiment is designed to investigate the distribution of adult psyllids within the tree canopy with the goal of optimizing insecticide spray applications. Sampling of the mid and upper parts of the canopy of mature trees for psyllid adults has been initiated. To date, three fold more adult psyllid have been found in the upper canopy than at mid-canopy level.
The goal of the present project is to establish economic thresholds under different juice price scenarios that optimize returns on investment when a nutrient/SAR package is being applied in groves with moderate to high incidence of HLB. Two 3-year field experiments were initiated 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 based on PCR analysis of a random sample of 160 trees is 98% in ‘Earlygold’ and 76% 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 approximately every 1 month and consist of a rotation of insecticides recommended for managing this pest. Treatment (2) has been sprayed in both blocks with carbaryl (Sevin XLR Plus ) at 0.75 gal/ac in April, spinetoram (Delegate WG) at 4.5 oz/ac in May and imidacloprid (Admire Pro) in June at 4.5 oz/acre. No sprays have been applied for treatments (3) and (4), during the last 3 months because nominal threshold populations have not been reached. Adult psyllid populations are being monitored every two weeks by tap sampling. Flushing patterns and flush infestation are being estimated by assessing the number of new shoots per tree and evaluating the proportion infested with psyllids. Effects of treatments on natural enemy populations are being evaluated by counting beneficials in tap samples, suction samples, flush observations. Additionally, exclusion studies on ACP infested flushes are being conducted in treatments (1) and (2) to evaluate the potential negative incidence of the sprays on the ACP beneficials. A branch with emerging flush is caged with a small number of adult psyllids for 3 days. Resulting eggs and 1st instar nymphs are counted and the cages replaced on half the branches. When the nymphs have reached 5th instar or are ready to emerge as adults the brances are removed and all surviving nymphs are counted. The difference in survival between caged and uncaged branches is taken as the effect of predation on the psyllid cohort. The first such study run in June indicated ACP mortality of 82.8 ‘ 4.9 % in treatment (1) and 69.1 ‘ 9.0 % in treatment (2). The greater reduction in treatment (1) may indicate greater activity of natural enemies in the absence of insecticide applications, and provide a better picture of the faunal changes that have occurred in response to treatments. Secondary pests and their natural enemies are also being studied during the growing season in the four threshold treatments. Citrus leafminer (CLM) densities are being monitored using pheromone traps. A trend toward lower CLM activity in treatment (2) has been observed although differences are not yet significant (P = 0.1024; df = 3,15; F = 2.577 for the ‘Earlygold’ block and P = 0.1018; df = 3,15; F = 2.638 for the ‘Valencia’ block). In contrast, citrus red mites showed significantly higher populations in treatment (2) compared to other treatments in the ‘Valencia’ block (P = 0.0003; df = 3,1599; F = 6.424), in concert with significantly lower populations of predatory mites (phytoseids) (P = 0.0009; df = 3,1599; F = 5.600). These results would indicate that the calendar applications had a negative effect on phytoseids, and therefore their prey, citrus red mites, increased their numbers. Further studies in the following months on these and other secondary pests will help us to elucidate the costs incurred by spraying due to disruption of biological control.
Ultra low-volume and Aerial Application of Insecticides and Horticultural Mineral to Control Asian Citrus Psyllid in Commercial Orchards. Low volume (LV) aerial and ground sprays have become an important method of application during the dormant season and at other times in SW Florida. We continue our evaluations of LV application of 435 horticultural mineral oil (HMO) which has shown promising results the last 3 years. This report documents updated results from the fifth trial begun on February 2011 in a 10.9 acre plot of ‘Valencia’ orange in Lee County that compares the efficacy of low volume spray spray of 435 horticultural spray oil (HMO) with the grower standard and an untreated control. Seven sprays of 435 oil (23Mar, 12Apr, 21Apr, 28Apr, 5May, 19May, 2June, 14June) have been completed since the last report. A grower standard of 6 oz of Actara/ac + 2gal HMO/ac was applied in 10 gpa spray mix on 2 May. On 14 June, 10fl oz/ac of abamectin 0.15 (Agri-mek) plus 2 gal HMO oil was applied in a 10-gal mix. Thus far mean psyllid populations have been below the 0.2 threshold for grower standard between 12 Apr and 20 June, whereas the oil treatment has been above threshold for 26 Apr, 9 May, 24 May, 6 June, and 20 June. There were significant differences between grower standard and oil treatments on 9 May (p = 0.06), 24 May (p = 0.04), with grower standard having less psyllids present (0.08 +/- 0.06SEM, 9 May; 0.07 +/- 0.06SEM 24May) than oil (0.25 +/- 0.06SEM 9 May; 0.25 +/- 0.06SEM). However, during June tap sampling, these significant differences disappeared (p > 0.05), and the mean psyllid counts for grower standard were 0.12 +/- 0.08SEM, 0.10 +/- 0.10SEM, and for oil (0.23 +/- 0/8SEM, 0.33 +/- 0.10SEM) on 6 June, 20 June, respectively. We added citrus leafminer assessments to this grove to determine if treatments are affective at reducing leafminer numbers. We placed pheromone traps in the grove on 24 May and have conducted one damage assessment of leaves using a modified Horsfall Barratt scale. There were significant differences between the grower standard and oil (p = 0.03) with the grower standard having less leafmining damage (0.50 +/- 0.05SEM) than the oil treated plot (0.68 +/- 0.06). All the spraying with broad-spectrum insecticides appears to be increasing incidence of various secondary pests including scale insects, mites and citrus leafminer (CLM). Furthermore, canker is an increasing problem throughout the area which has increased concern about CLM. CLM control requires precise application timing on emerging flush to be effective so there is considerable interest in aerial application. Therefore, we are evaluating aerial application of two products widely used for leafminer control, the insect growth regulator methoxyfenozide, and spinetoram, an analog of spinosyn A which is derived from actinomycete bacteria. Both products are selective with low vertebrate toxicity. The replicated trial in being conducted on an 80 acre block divided into 12 large plots in a replicated complete block design with 4 treatments and 3 replications. Pheromone traps are being used to monitor CLM populations within all plots before and after the applications which are planned for 7 July when summer flush will be optimal for maximum control.
In California, citrus nursery stock must be treated with both a systemic and foliar pesticide in order to move it within areas quarantined for the Asian citrus psyllid (ACP). The purpose of these treatments is to prevent ACP from establishing in nurseries and being moved around California when the plants are sold. Imidacloprid is one of the systemic insecticide options available to nurserymen and is the focus of our research at UC Riverside. However, there are other systemic neonicotinoids recommended by the CDFA that growers are permitted to use on their citrus before shipments leave the nursery. These insecticides include Safari (dinotefuran) and Platinum (thiamethoxam), and this report summarizes our data on these compounds. We are evaluating Platinum and Safari uptake and persistence in 2 citrus varieties, ‘Kinnow’ mandarins (1 year old) and ‘Tango’ mandarins (3 years old). Admire Pro (imidacloprid) was also included in the trial. Both varieties are budded on ‘Carrizo’ rootstock. Trees were treated with the current label rate for Platinum and Safari, and the 0.33 ml/cu ft potting media rate of Admire Pro. Within 1 week of treatment with Admire Pro, imidacloprid levels were above the 200 ppb threshold established by Mamoudou Setamou as the dose needed to kill ACP nymphs. We use this concentration to evaluate the efficacy of our treatments. Mean concentrations in both the Kinnows and Tangos were at 1800 ppb. At week 2, the concentrations had increased further. The levels in the Kinnows and Tangos were 6,000 ppb and 6,550 ppb, respectively. Clearly, the trees were well protected within 1 week of treatment. As yet, there are no data available to guide us on how much thiamethoxam (Platinum) or dinotefuran (Safari) we need to have in the trees to protect them. However, at the label rates we tested, the concentrations were in the ppm range within 1 week of treatment, suggesting that the trees would also be protected from ACP. Thiamethoxam concentrations in the Kinnows and Tangos were 1,600 ppb and 2,400 ppb, respectively, at one week, while concentrations of dinotefuran in the Kinnows and Tangos were 12,000 ppb and 8,100 ppb, respectively, at one week. The substantially higher concentrations of thiamethoxam and dinotefuran compared to imidacloprid are not surprising given their higher water solubilities. Thiamethoxam (4 g/l) is about 8-fold more water soluble than imidacloprid (0.5 g/l) and dinotefuran (40 g/l) is 80-fold more water soluble than imidacloprid. The higher water solubility will permit a more rapid uptake of insecticide into the trees; however, it could also result in a less persistent product due to more rapid depletion of soil reservoirs of insecticide. We will monitor the concentrations in these trees for 6 months to determine the longevity of residues within the trees. This information will guide future uses of these products on nursery citrus. We are continuing to monitor imidacloprid applications in a commercial nursery. By doing this, it allows us to evaluate the efficacy of treatments in citrus growing under normal production conditions, as opposed to the more controlled environment of a laboratory or greenhouse trial. We generally measure lower levels of imidacloprid in commercial citrus, although the treatments do reach the 200 ppb threshold concentration. The more frequent appearance of flush at the commercail sites results in lower titers of imidacloprid because the new tissue grows at a rate that is faster than the rate of movement of imidacloprid. This result reaffirms the importance of the foliar treatments for nursery stock that is due to leave the faciity. The foliar treatments will protect the citrus until the imidacloprid establishes at threshold concentrations.
The agreement between CRDF and ARS is now in place for this project. This initial report covers the period from the agreement start date of 1 May through 30 June 2011. No expenditures have been charged to this project to date. Field work has continued uninterrupted this year with 3 applications of SPLAT-CLM made at two locations: 2 applications to 90 acres in a grapefruit grove in St. Lucie county and 1 application to 82 acres in an orange grove in Charlotte county. A second application will be made in Charlotte county in early July. Complementary experiments were installed at the two sites to determine the potential for reducing the cost of SPLAT-CLM applications by leaving untreated beds (gaps). End of year analysis of results of various gap treatments will attempt to identify coverage patterns that optimize mating disruption and amount of product applied. The prototype applicator has been improved by the addition of two cylindrical hoppers capable of holding sufficient SPLAT-CLM to treat 50 acres at a time. GPS telemetry and software-driven pump speed contributed to a very high degree of precision in terms of the amount of product applied per acre. Precise application is particularly important given the relatively high cost of SPLAT-CLM. The longevity of a single application continues to be studied to determine the frequency of application required to maintain effective control throughout the year. We will continue to focus on reducing the cost of mating disruption to control CLM and associated citrus canker disease by identifying the most cost-effective coverage pattern and frequency of application. Craig Keathley has been selected and has accepted a position as postdoctoral research entomologist at USHRL, Ft. Pierce. He will defend his thesis in late August and begin his duties related to this project in early September. Craig will receive his PhD from the University of Kentucky’s Entomology Department where he studied under Dr. Daniel Potter. In addition to his academic qualifications, the soon-to-be Dr. Keathley worked as a vegetable crop pest control adviser in California prior to his doctoral studies at UK. We are excited to have Craig join our team and look forward to his contributions to this effort.
We have completed a confirmation test to demonstrate control of bean thrips by fumigation with Vapormate. More than 30,000 adult bean thrips were placed into the navels of navel oranges (10-15 per orange) and stored at 41F overnight to drive the thrips deep into the navel. The next day, the oranges were packed into boxes and loaded into a marine container with filler fruit to make 8 full pallets of packed orange boxes. Following the 1 hour fumigation, the infested oranges were retrieved and thrips mortality was determined to be 100%. Fruit quality was also assessed on export quality fruit following fumigation with Vapormate. There were no significant effects on fruit quality. We have also developed a dose mortality curve through small scale tests in the laboratory and determine the concentration of Vapormate required to achieve Probit 9 mortality at 41, 50 and 59F. We are preparing a manuscript on this work for publication in the Journal of Economic Entomology.
This project was designed to examine the potential disease control of citrus huanglongbing (HLB) by interplanting citrus with guava. In Vietnam guava has been shown to be an effective deterrent to HLB, slowing the disease and keeping plantings alive for up to 15 years that normally succumb in 2-3 year. For all plots and experiments, Guava trees, (Vietnamese white cultivar) were propagated and grown to appropriate size requiring ~1 year. Both nursery and field citrus trees are assayed for HLB every 60 days, and have been assayed multiple times. Psyllid populations are monitored continuously every 2 weeks to document repulsion of the vector. Results: Guava vs no guava nurseries: Two nursery sites, a guava protected citrus nursery versus an unprotected nursery, have were established with disease free, PCR-negative citrus trees (2 sweet orange and 1 grapefruit cultivars) in June 2009 and were located in the protected and unprotected plots. The guava trees were grown to appropriate size as indicated in Vietnam prior to outplanintg. To date HLB appears to be progressing more slowly in nursery plots interplanted with guava than in non interplanted plots. The freezes discussed below did not adversely affect these nursery plots. Citrus/guava interplantings: 2 commercial plantings with multiple replications were established but due to freezes and property sales these plantings are no longer viable. A third trial planting was established at the USHRL Picos Farm in Fort Pierce. The Picos plot was interplanted with citrus in August 2009. Severe frosts during 2008/2009 and again during 2009/2010 winters affected the USHRL plots and caused a delay in the experiment. A final hard freeze during the 2010/2011 season killed most of the guava trees. Data analysis to date indicates no differences were observed among treatments, i.e., guava interplanted vs. non-interplanted plots prior to the final demise of the plots. Our interpretation is that Florida is actually a subtropical environment, prone to intermittent freezes and cool or cold temperatures. Whereas, Vietnam and Indonesia, where the guava effect seems to work, are truly tropical without such broad temperature swings. After freezes it takes a considerable time to either replant guava or for the freeze damage guava to recover. Even during cool weather guava trees are very sensitive and do not continue to flourish and grow. It is the new flush of guava which appears to be the best at producing ACP repellent volatiles. Cool or freezing temperatures inhibits volatile production and thus the citrus crop is left unprotected from ACP. While guava not be a viable deterrent as an intercrop, it still may be possible to identify individual volatiles from guava that might be useful under field applications as chemical applications. Moving forward: We have switched direction slightly to investigate other more temperate Myrtaceous plant species that are more cold tolerant and might be useful as intercrops. Research continues using a Y-tube olfactometer to continue to investigate guava volatiles as repellents of the psyllid as well as to investigate the feasibility of other Myrtaceous plants. To date we have not bee able to determine any differences in y-tube olfactometer experiments
We completed a trial in Ventura County where we monitored the uptake of imidacloprid in lemons applied at 3 timings during the season. The uptake of imidacloprid (determined by whether the 200 ppb target threshold for ACP control was reached) was not good at this site. The reason for the poor uptake was likely due to a 3 week interval between irrigations, and heavy soil conditions. The soil texture was determined at this site to be 25% clay and an organic matter content of 6%. On May 11, 2011, we retreated trees that had been treated in 2010 with a further application of imidacloprid. Thus far, we are not detecting imidacloprid in trees that received 2 successive years of imidacloprid treatments. We have started a new trial on mandarins in Kern County. We plan to put on 4 imidacloprid treatments leading up to the Fall flush to determine how effective imidacloprid will be at protecting the young leaf tissue. The trial is being conducted in 2 blocks that are under different irrigation systems (drip v fanjet). The first treatment timing was administered on June 9 following the removal of the nets from the trees, and we have begun our residue analyis. We are working with Terry Nelson of Oxnard Pest Control to establish additional sites in Ventura County to evaluate imidacloprid uptake. Our goal is to select sites with lighter soils where the irrigation is more frequent. In conjunction with the imidacloprid treatments at the Ventura site, we are also evaluating thiamethoxam uptake. We have not detected thiamethoxam at 2 weeks after treatments were applied. In August, we plan to apply further thiamethoxam treatments to the grapefruits at Hemet, in an effort to determine what is happening to the insecticide. We have applied clothianidin as a soil treatment and trunk spray to navel oranges at Lindcove Research and Extension Center. Working with Pat Clay of Valent, we were able to get Belay 2.13 SC and an adjuvant for the trunk spray work. The method of application appears to be relatively easy and would be a good option for some growers. We have no data for clothianidin on citrus so this is an important study for the citrus industry. The first samples were taken on June 15 (1 week after treatments) and no clothianidin was detected in either set of treated trees. The results of the Spring 2010 nectar collections have been summarized in a report that has been submitted to the CA-DPR and US-EPA. Imidacloprid did not accumulate in the nectar sampled from trees treated in successive years with the 1X rate of application. Residues were higher in trees treated in the Fall versus those treated in the Spring, but the levels were still well below the No Effect Level of 20 ppb set by Bayer CropScience. In Spring 2011, further nectar samples were collected from field sites where imidacloprid treatments had been applied. In all, 105 samples were collected from sites that differed in soil type and in the number of successive years that imidacloprid had been used. The samples will be analyzed by Dr Bill Leimkuehler of Bayer CropScience.
Two trials were conducted in May and June, 2011 to determine the levels of horizontal transfer (psyllid-to-psyllid spore transfer) of Ifr spores achievable with the autodisseminator (dispenser) in greenhouse tests. The results demonstrated that adult ACP transferred Ifr spores from the dispensers to nymphs developing on potted orange jasmine in a greenhouse. In the first trial, 44% of the adults and 34% of the nymphs became infected with Ifr while in the second trial 35% of the adults and 27% of the nymphs became infected. Interestingly, Ifr mycelia grew extensively as a saprophyte on molted skins, honeydew, and waxy excrement, resulting in fungal coverage of entire nymph clusters. We are currently incubating Ifr inoculated waxy excrement and molted skins to determine whether Ifr mycelium grown on this material will develop conidiospores and whether these spores, in turn, will infect nearby psyllids. A second set of tests is currently underway to compare the level of adult ACP emergence from nymph colonies that were exposed to Ifr-infected adults to that of unexposed nymph colonies. Initial testing indicates that blastospores on the dispenser surface remain viable at least 10 days after deployment in the greenhouse. Tests have just been initiated to monitor spore viability, infectiveness, and phenology for 21 days following deployment of the dispenser. These tests will determine the phenology of blastospores and condiospores on the trap coating and their relative importance in primary infection. Laboratory tests using novel behavioral assay methods to measure ACP response to combinations of visual, scent, and taste cues have showed that: 1) ACP response to particular scents is strongly influenced by scent concentration. For some scent mixtures, stronger and weaker concentrations are less stimulatory than mid-range concentrations while for other scent mixtures, response is strong to low concentrations and tapers off as concentration increases. 2) ACP can differentiate among scents composed of only three ingredients, with a synthetic mixture of terpenes from Mexican lime and orange jasmine being the most stimulatory. We will determine whether the addition of targeted compounds will enhance the attractiveness of the mixtures 3) ACP can differentiate between different shades of green and are more strongly attracted to medium-dark green objects than to pale green objects.
Native California non-target psyllid species have been collected from California’s natural habitats for host specificity tests with Tamarixia radiata. Five native psyllid colonies, Diclidophlebia fremontiae (host plant Fremontodendron californicum), Calophya nigrella (host plant Rhus ovata), Heteropsylla texana (host plant Prosopis glandulosa), Heteropsylla sp. (Host plant Acacia farnesiana) and Bactericerca cockerelli, a native pest of potatoes, tomatoes and peppers are being continuously cultured and maintained on their natural host plants in the I & Q facility at UCR. We have been unable to establish and maintain colonies of Neophyllura arctistophyli (host plant Manzanita Arctistaphylos glauca) in Quarantine. This psyllid has been dropped from the test list because of extreme difficulty in locating wild populations, an inability to maintain lab colonies and problems sourcing adequate numbers of host plants. We have been unable to produce A. glauca from seeds or cuttings (a specialist native CA plant nursery also tried and failed to produce this plant), and no California native plant nurseries produce adequate numbers of plants for this research project. Consequently, N. arctistophyli has been replaced by olive psyllid as this species is phylogenetically closely related to N. arctistophyli and ACP. A self-introduced weed biocontrol agent, the Scotch broom psyllid, Arytainilla spartiophylla, is univoltine and because of winter chilling requirements to break diapause it has not been possible to maintain a colony at UCR. These psyllids were collected from the field in northern California and shipped to Quarantine for testing. A continuous supply of healthy seedlings of host plants is very important for rearing test psyllids. Seedlings of eggplant/sweet pepper Prosopis glandulosa and Acacia farnesiana, Rhus ovata, Fremontodendron, and olive are being acquired from local nurseries or grown by CDFA at the Mt. Rubidoux facility. We have completed safety tests for three native psyllid species that occur naturally on native California host plants; Heteropsylla sp. on Acacia farnesiana, Heteropsylla texana on Prosopis glandulosa, and Calophya californica on Rhus ovata. None of these native psyllids on their respective host plants were parasitized by T. radiata. Testing of potato, scotch broom, and olive psyllids has been completed also. Scotch broom and olive psyllids were not attacked by T. radiata. A few nymphs of the pestiferous potato psyllid were attacked. We are currently running additional replications of Fremontia psyllids and Rhus psyllids for their suitability as hosts for T. radiata. Upon completion of this last round of tests the host testing of T. radiata should be concluded and the Environment Assessment report will be prepared and presented to USDA-APHIS and NAPPO for review.
Spatial and Temporal Incidence of Ca. Liberibacter in Citrus and Psyllids Detected Using Real Time PCR, April 2011. In 2010, psyllid adults from our HLB negative colony on orange jasmine were caged on new shoots (10-12 adults/shoot/ per tree) that had been caged immediately after trimming and were not exposed to feral psyllid population. On the same tree an additional cage was placed on a previously uncaged shoot that was infested with feral psyllid nymphs. These adults and nymphs remained caged on the shoots for three weeks. Some adults from laboratory reproduced in the cages so when collected there were some nymphs available. Similarly cages with feral populations also had some nymphs and adults at the time of collection. Using PCR, 40 shoots tested HLB negative with average Ct value of 39.8 ‘ 0.07 and 15 shoots tested HLB positive with average Ct value of 27.4 ‘ 0.7. From lab reared adults caged on HLB negative shoots 69 were tested and all were negative. From their nymphal progeny 58 were tested and 1.7% were positive with Ct value 29.8. From the ones caged on HLB positive shoots 51 adults were tested and 12% were positive with average Ct value of 29.6 ‘ 0.7. Among 31 nymphs none were positive. Out of 88 adults that emerged from feral nymphs caged on HLB negative shoots 10% were positive with average Ct value of 28.9 ‘ 0.9. Only 5 nymphs were tested and all were negative. Among 53 adults that developed from feral nymphs caged on HLB positive shoots 9% were positive with average Ct value of 29.6 ‘ 0.9. No nymphs were tested. These colonies were re-treated on April 8, 2011 and titer values and CT values are being calculated to correlate with data from 2010. This data was also reported in April 2011 however the quantification of HLB within the psyllids is still being analyzed therefore results are pending. In April, we completed the third year of a replicated experiment in a 12-acre experiment commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test effects of micro-nutrients + systemic acquired resistance inducers, and Asian citrus psyllid (ACP) chemical control on ACP populations on Can. Libericacter asiaticus (CLas) titer, and plant yield. Psyllid populations in the insecticide and insecticide+nutritional plots reached the 0.20 threshold on 22Apr, 6May, and the insecticide+nutritional plot reached threshold on 4June. Insecticide and Insecticide+nutritional had significantly (P< 0.05) less psyllids than control or nutritional only on dates 8Apr, 22Apr, and 7May. All three treatments were significantly (P < 0.05) less than control on 21May, but no differences were found between treatments on 4Jun. Since our last report we have sprayed three applications on the treated plots: Danitol 4EC at 16oz/acre on 15 Mar11, Dibrom 8 at 16oz/acre on 28Apr, and Delegate WG at a rate of 30oz + 2gal/ac 435 oil on 12May. Three years data of sticky card sampling of adult psyllids are now being examined to analyze movement of adult psyllids between treatments. Tap and flush sampling has been modified to include bi-monthly counts of nymphs and eggs on flush to determine any correlation to tap sampling counts. In January, we collected plant samples for HLB detection, and these are still in process. Following the third harvest, fruit samples were sent to the CREC fruit quality laboratory (Apr 11) for analysis of lbs. juice per box, acid, total brix, and ratio. An initial citrus leafminer damage assessment using a modifield Horsfall Barratt scale will be done following summer flush to determine if CLM should be monitored. The objectives of this grant are ahead of schedule since grant was initiated prior to release of grant funds the first year, therefore we have three years of data as of the 2011 harvest.
The objective of this project was to investigate three questions: 1) what is the seasonal pattern of Ca. Liberibacter asiaticus (Las) prevalence in leaf tissue on a grove scale; 2) what are the flushing patterns of citrus and whether these flushing patterns affect the prevalence of Las in Diaphorina citri or citrus leaves; and 3) what is the prevalence of Diaphorina citri carrying Las on a grove scale and how does it compare the results from the citrus trees in the same grove. In 2008 and 2009 Ebert and Rogers demonstrated that the prevalence of Las in the Asian citrus psyllid (ACP) varied seasonally but the pattern between seasons was not consistent. It was suggested that perhaps the reason for the differences between the years related to the flushing patterns of citrus and the prevalence of the bacterium in the leaves where ACPs are feeding. This project aims to determine if there is a relationship between the frequency of disease on branches and ACPs. The backlog of plant samples has been cleared and we continue to collect additional samples from 3 groves. Two groves are in Polk County near Lake Alfred (high HLB levels) and Lake Wales (low HLB levels) and the third is in Lake County (moderate HLB levels). The Polk County groves are Hamlin and the 3rd is Early gold. We are restricted in site selection by the presence of significant populations of ACP. In each grove, two hundred trees were selected from 10 consecutive rows. One leaf/tree was randomly picked every fortnight. For qPCR detection of Las, the midribs of five random leaves were pooled to obtain 40 samples/date. An estimated Las prevalence in the branches was generated from the pools with PooledInfRate v3. Simultaneously, ACPs were collected from the same location to compare the seasonal prevalence of Las in citrus branches and ACPs. There were 2 peaks of Las prevalence in the moderately infected grove at approx. 0.50 mid-October 2010 and April 2011, dipping to 0.20 in February 2011. Las prevalence in ACP had a similar pattern but the main peak reached 0.75 in December. In the highly infected grove, there has been a steady increase of Las prevalence in branches since July 2010 until March 2011 where a plateau of 0.75 was reached. There was the same general trend observed with ACP although the prevalence levels were 0.15 to 0.20 higher, reaching 0.90 in March 2011, but there was a large decline in ACP prevalence to 0.60 in May 2011. In the low HLB intensity grove, no Las has been detected in since the initiation of sampling in July 2010. However, very low prevalence has been detected in branches. Prevalence has hovered below 0.02 until April 2011 when it reached 0.06. The phenology data was collected at all sites since November 2010. Flush was highly synchronous in 2011 likely because of the extended cold temperatures this winter and the drought conditions. As would be expected with the very dry conditions, there is currently little new flush on the trees.
A combinatorial peptide library was synthesized by Torrey Pines Institute for Molecular Studies (TPIMS) and tested ‘in-bulk’ by addition to an artificial diet fed to the Asian citrus psyllid. This represented a collection of millions of 10-amino acid peptides (decamers). When the peptides were present in the psyllid diet, there was a clear inability to form normal salivary sheaths by the psyllids. This research is now being expanded to identify which of the peptides is responsible for the salivary sheath inhibition through a combinatorial screening protocol developed by TPIMS. 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.
We have completed the second year of our project. Our longterm goal is to identify and then use specific psyllid (B. cockerelli) RNA sequences to induce RNA interference (RNAi) activity in recipient psyllids. As we reported previously, we have cloned midgut sequences from B . cockerelli, and are continuing to evaluate these sequences for their ability to induce detrimental RNAi effects in psyllids. We have evaluated effector RNA sequences by intrathoracic injection (as a positive control), and by two feeding approaches. All methods have shown some potential, depending on the sequences evaluated. In vitro-generated double-stranded dsRNAs were evaluated via micro-manipulator-driven intra-thoracic injection (200nL/psyllid). This is a tedious process but is used routinely to induce whole insect RNAi effects for many different insect types. Increased mortality was observed for actin dsRNA in our experiments when compared with the control GFP dsRNA. Injection of an ATPase specific dsRNA also consistently suppressed the endogenous ATPase mRNA expression by 30% in independent experiments. We also identified specific siRNAs, hallmarks of RNAi activity in recipient insect. Taken together, these data demonstrated that psyllids are susceptible to RNAi. We developed a robust and effective in vitro feeding system for B. cockerelli. We have used this artificial diet system for screening 18 candidate sequences (as dsRNAs and siRNAs) for B. cockerelli. Four out of 18 sequences, including the homologues of actin, ATPase, Hsp70 and CLIC, caused substantial psyllid mortality as compared with GFP dsRNA controls. Consistent and specific down-regulation of endogenous mRNAs also was revealed by qRT-PCR, and corresponding siRNAs were detected by Northern blot analysis. These latter two analyses are important and demonstrate that the orally-induced mortality is not an artifact, but due to RNAi. We also constructed an artificial chimeric sequence for actin, ATPase, Hsp70 and CLIC. Oral feeding of dsRNAs for this chimera sequence resulted in significantly higher mortality compared with the dsRNA of any of the individual sequence, demonstrating possible synergistic effects of these sequences on psyllid survival. We are also using means for more rapid evaluation of RNAi effects in plants by using plant virus-based expression systems. We have used a Tobacco mosaic virus (TMV)-based plant expression system to express B. cockerelli sequences in tomatoes. However, TMV systemic expression of insert sequences requires approximately 3-4 weeks and resulting infections are non-uniform within tomato plants. We have now evaluated different virus vectors and host plants, including TMV, Tobacco rattle virus (TRV) and Potato virus X (PVX) in different host species including tomatoes, tobacco, Datura, tomatillo and Nicotiana clevelandii, all of which are good host plants for B. cockerelli. TMV systemic infections in tomatillo develop very rapidly, within one week and the tomato psyllid readily feeds on tomatillo. TRV and PVX both also offer opportunities in other plant species. The combination of viruses and plants will allow us to rapidly test RNAi activities in plants.In summary, we illustrated the efficacy of RNAi silencing in the tomato psyllid and selected candidate sequences for RNA interference using two feeding systems, and efforts for plant virus-based and whole plant transgenic expression systems are underway.
In the previous study, imidacloprid, 0.35 g AI/plant, and thiamethoxam, 0.25 g AI/plant, applied in the nursery tree bags, before planting, were efficient to control Asian Citrus Psyllid (ACP), Diaphorina citri, until 60 days after application (DAA). The time to cause 100% of ACP mortality was between 5 to 7 days after the confinement of adults in treated plants. The first experiment was performed to determine if the systemic insecticides are effective until 90 days after application in nursery trees and if this application is effective to prevent transmission of the bacteria. In this experiment, the time to reach 100% of the adult ACP mortality ranged from 3 to 7 days for both systemic insecticides tested (imidacloprid and thiamethoxam), in the same doses of the previous experiment. The insecticides were effective up to 90 DAA. The results of PCR carried out for ACP, 15 and 30 days after application, were positive for 100% of the samples, consisting of 10 insects tested, but in the confinement held at 46 DAA, in any sample was detected the presence of the bacteria. No acquisition in this period. In bioassays performed at 75 and 90 DAA, the percentage of positive samples was from 50 to 70% and from 10 to 40%, respectively. Plants treated with insecticide, the proportion of insects reaching the phloem was similar between plants treated with imidacloprid (0.35 g AI/tree), thiamethoxam (0.25 g AI/tree) and control (untreated plants), being respectively 74, 72 and 76%. The time to perform the first ACP salivation was also similar between treatments, 118.4, 103.2, and 112.6 minutes, respectively. However, the time of phloem ingestion was drastically reduced compared to untreated plants. Apparently, ACP can only distinguish between plants with and without treatment from the moment that start ingesting the phloem sap. In this case, it was observed that after ingestion of sap with insecticide, the ACP removes the stylet from the plant and rarely returns to start a new probe on the same plant. With systemic insecticides, the main interference on probing behavior occurred during the phloem phase; phloem sap ingestion, as measured by duration of waveform E2, is significantly reduced (approximately 91%). The second experiment was conducted varying the doses of the systemic insecticides and confinement of the ACP in plants treated only 7 DAA. For thiamethoxam, the doses tested were: 1.0, 0.5, 0.1 and 0.05 g/nursery tree nursery and for imidacloprid were: 1.75, 0.9, 0.2 and 0.08 ml/nursery tree. For both insecticides, the control was more efficient in the two highest doses (mortality over 85%). The percentage of ACP positive samples ranged from 60 to 100%. As in the first experiment, there were no transmission results. In the third experiment, were tested different foliar spray insecticide to determine if they are effective to prevent the transmission and for how long. Except for etofenprox, all other foliar applied insecticides were effective in controlling adults of ACP, with variable control period. The last experiment was performed to evaluate the effect of mineral oil (MO) on the feeding behavior of ACP and its effect on the vector repellency. The results showed that up to 21 days after application, mineral oil, 1.5% and 1.0%, shows repellent effect on adults of D. citri, which prefer oil-free leave flush for oviposition. Using electrical penetration graphs (EPG) techniques showed that plants sprayed with insecticides and MO affects mainly the initial phase of probing (pathway). In the case of foliar insecticides, ACP was able to perform probes 1 DAA, but it was able to reach the phloem only in the evaluation of 15 DAA. However, on plants treated with MO around 32% of insects were able to reach the phloem 1 DAA. We still do not consider that the project is finished. To finish the project we need an extension of one year to obtain such data.
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