Based on results from the previous Contest Project (CRDF#400), eleven compounds have been selected by the contest committee as candidates for further research to determine their efficacy for control of HLB based on their ability to substantially reduce the titers of the bacterium Candidatus Liberibacter in our grafted citrus assay, lack of phytotoxicity to citrus and potential for registration. After first evaluating combinations of these molecules using the graft-based chemotherapy method under a separate agreement, this project extension will evaluate these 11 compounds as control agents against the HLB bacterium individually and in combination using infected, container-grown citrus and HLB-affected scions. The objective of this project is to determine an optimum chemical formulation that may be registered for field control of HLB. In this quarter (July to Oct, 2013), 18 treatments from 11 compounds or combinations have been applied to HLB-affected potted-plants coupled with three heat treatments (40, 42 and 45 degree). All these treatments will be analyzed every 2 months for Liberibacter. The preliminary results showed that heat treatment promoted the growth of new flush from the seriously HLB-affected citrus. No bacterium was detected in the new leaves but was present in the old leaves two months after treatment with compounds coupled with the heat treatments. Four months post treatment, several treatments reduced the Las bacterium to undetectable levels both in the new and the old leaves of the containerized citrus. The research is ongoing.
We have initiated this investigation with two laboratory experiments. The aim of the first experiment was to understand how communication between citrus plants may affect psyllid host acceptance as influenced by HLB infection. We now have strong evidence that ACP are more attracted to uninfected plants exposed to volatiles from another HLB-infected citrus plant than to uninfected plants exposed to volatiles from other uninfected plants. The second experiment aims to test the effect of drought stress on the recruitment of ACP, natural enemies, and the expression of Las-inducted plant volatiles. We demonstrated that control plants infested with 50 ACP were more attractive to the parasitoid, Tamarixia radiata, than control plants without psyllids. However, under drought stress, plants infested with 50 ACP were no more attractive than uninfested, drought stressed plants. These findings were confirmed by GC-MS analysis that showed a strong decrease of volatiles emitted by drought-stressed plants even when infected by ACP. Our next objective is to test if submitting citrus plants to drought stress will lower the attractiveness of HLB-infected plants to psyllids as compared with non-stressed controls. To complement these two laboratory experiments, we are now working to identify the gene(s) involved in methyl-salyclate production in citrus. Methyl salicylate is a known attractant of ACP and our objective is to determine if this gene is up regulated under: 1) pysllid infestation 2) HLB infection and/or 3) when submitted to volatiles from infected citrus plants. Regarding the field portion of this project, we have identified groves comprised of only reset trees, as well as, groves where some resets were replanted among mature trees within a mature grove. We have monitored psyllid population densities within these groves. We have strong evidence from this year that reset trees planted in a habitat consisting of other reset trees harbored more ACP than within groves comprised of mature trees only or on citrus resets located among mature trees. In other words, psyllids were most abundant in areas containing only resets. Reset trees likely produce more flush to support higher population densities of psyllids. To better understand this observation, we plan to collect data on ambient and canopy temperatures, leaf nutrient status, and volatile production between treatments.
Our objective for this project has been to evaluate botanical compounds as repellents for Asian citrus psyllid (ACP) with the purpose of developing possible repellent formulations for use in the field. In the past quarter, we have completed a study of a new dispenser of a potential botanical insecticide. This dispenser was a hand applied product. It is based on some of our initial research with guava-based chemicals as psyllid repellents. The product is proprietary and was provided to us by ISCA Technologies as part of prototype product development. The experiment was conducted at the Water Conserv II Research and Extension site in Orange County, FL in a planting of ‘Hamlin’ oranges on Swingle rootstock. Trees were 3-4 ft in height (approximately 2.5 years old) and were planted at 12 x 25 ft. The purpose was to test the technology in young trees. All treatments were assigned randomly to 15 tree plots in a randomized complete block design replicated 5 times. There were two treatments: 1) control and 2) plots treated with SPLAT-ACP Repel Hand applied dispensers. A pre-count of all plots prior to treatment was conducted on the day prior to treatment application. Thereafter, all treatments were sampled for five weeks on a weekly basis. On each sampling date, ten terminal flush samples were collected from the interior portions of each replicate plot. Flush samples from each plot were placed in paper bags and immediately transferred to the laboratory for inspection. Flush samples from each plot were carefully examined using a stereomicroscope for pysllid nymphs. Data were collected according to the following rating scale: 0 psyllids per flush ‘ 0 1-5 psyllids per flush’1 6-10 psyllids per flush’2 11 or more psyllids per flush’3 In addition, on each sampling date, adult psyllids were counted by conducting 10 tap samples per replicate plot. For each tap sample, a tree branch was vigorously tapped with a PVC pipe directly over a horizontally placed 210 . 297 mm plastic white sheet. All Asian citrus psyllid adults found on the sheet following branch agitation were counted and recorded. Unfortunately, the results of this experiment did not result in reduced psyllid populations in the treated plots as compared with the controls. We are currently analyzing the release rates from these dispensers to determine why there was no effect on ACP population densities before we conduct a subsequent field experiment with this emerging technology. Also, we are planning experiments in more mature citrus trees.
The objective of this research is to investigate potential non-neurotoxic insecticides against Asian citrus psyllid (ACP) that could be incorporated into ACP management programs in Florida. We are especially targeting those that have shown promise against insect pests similar to ACP. Such additional tools may not only prove effective against ACP, but also could assist in ACP resistance management programs as needed tools for effective rotation of insecticides. We are specifically investigating those non-neurotoxic insecticides that have shown promise in controlling other insects pests. In the past quarter, we have evaluated methoprene in the laboratory. Methoprene is a juvenile hormone (JH) analog that acts as a growth regulator. We evaluated this compound for its ability to inhibit ACP egg hatch of eggs of various ages. We treated 0-48 hrs and 49-96 hrs aged eggs with six different concentrations of methoprene ranging from 0-320 ‘g/ml. For 0-48 hrs aged eggs, we observed egg hatch inhibitions of 14, 24, 31, 41, 65 and 84 percent for concentrations of 0, 10, 20, 40, 80 and 160 ‘g/ml, respectively. Similarly, the concentrations of 0, 10, 20, 40, 80, 160 and 320 ‘g/ml led to 13, 23, 29, 34, 39, 62 and 95 percent egg hatch inhibitions of 49-96 hrs aged eggs. These results show that treatment with methoprene successfully inhibited ACP egg hatch. These data are currently being analyzed to ascertain LD 50 and LD90 doses. We are also investigating the effect of methoprene on nymphal development. We will also investigate the effect of methoprene on adult survival, fertility and fecundity. We have also started investigations on the effectiveness of the juvenile hormone (JH) analog, Diofenolan, to inhibit the development of immature stages of ACP. The results of our research suggest that JH-analog, non-neurotoxic insecticides are effective in disrupting growth of immature stages of Asian citrus psyllid, specifically, and likely useful tools.
The objective of this study 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 trial, we are still following the Keyplex’ program. The two latest spray applications occurred in July and August. We followed ACP populations in the replicate sprayed and non-sprayed control plots from March to September. As mentioned in the previous report, from March to May we consistently observed more ACP on HLB-infected trees compared to uninfected trees. However, since June, when the ACP population increased significantly, we did not observe this difference between populations on infected and uninfected trees. Regarding the effect of Keyplex’ on infected versus non-infected plants, we observed the reverse trend on psyllid behavior: from March to May we did not observe a difference in ACP population between trees with the foliar nutrient supplement applications versus the control trees. However since June, we observed consistently more ACP on the nutrient supplemented trees than on the untreated control trees. Samples from these trees are being analyzed to see if the difference observed are statistically significant, but these results corroborate what we observed in previous laboratory experiments, where psyllids were more attracted to infected plants that were treated with the nutritional program compared to untreated, but infected control plants. Currently, we are performing an experiment to investigate the effect of nutrient sprays on HLB acquisition by ACP. Earlier, we observed that fewer ACP acquired the HLB bacterium from trees that were infected and supplemented with nutrients than from infected trees that were not supplemented after 10 days of psyllid exposure to trees in laboratory experiments. However, the results from field experiments did not corroborate these laboratory data. We did not find a significant difference between HLB acquisitions by psyllids when they were exposed to HLB-infected trees during the nymph stage on supplemented versus non-supplemented trees. We are currently analyzing the results of Candidatus Liberibacter (HLB pathogen) acquisition by ACP from field experiments after 10 and 28 days of psyllid exposure to trees. In similar laboratory experiments, we are analyzing psyllids after 28 days of exposure to trees. These experiments have compared psyllid acquisition of the HLB pathogen from infected trees that were either supplemented or not supplemented with nutritional sprays.
We have completed our annual survey of insecticide resistance in populations of Asian citrus psyllid across various growing locations throughout Florida. Five sites were evaluated in Lake Alfred, Winter Garden, Ft. Pierce, and La Belle across which 18,000 psyllids were collected and used in resistance screening assays. Insecticides at the LD50 value evaluated were carbaryl, chlorpyrifos, fenpropathrin, flupyradifurone, imidacloprid, and thiamethoxam, representing a range of chemistries available for psyllid population management. A focus on neonicotinoids was conducted to make comparisons of currently used chemistries, imidacloprid and thiamethoxam, first and second-generation, respectively, against, flupyradifurone, a new neonicotinoid, to determine base-line susceptibilities and to evaluate potential cross-resistance. An overall decrease in resistance levels was observed during this year’s survey for all insecticides tested as compared to previous surveys in 2011 and 2012. This drop may be due to better rotational practices between insecticides with different modes of action by growers, movement of psyllids from non-treated areas, or collection of psyllids from different sites than from previous years. The bottom line is that in the spring/summer of 2013, we did not observe that resistance appeared to be a significant problem in psyllid populations throughout Florida. Resistance found to be significantly higher in the field as compared with the laboratory susceptible control culture occurred for imidacloprid (1.8 fold ratio) and fenpropathrin (2.2 fold ratio) at Ft. Pierce and Lake Alfred, respectively. No other levels of resistance of statistical significance were observed in the field as compared with the susceptible laboratory population. A concomitant resistance against flupyradifurone for psyllids tested from Ft. Pierce was not observed; however, there was a trend suggesting cross-resistance. It is possible that resistance levels to neonicotinoids were too low at this site for a conclusive determination of cross-resistance between imidacloprid and the fourth-generation neonicotinoid, flupyradifurone. Additional sites are being screened for higher levels of imidacloprid resistance to identify populations to better evaluate potential cross-resistance between these insecticides. In addition, we have been developing insecticide resistant psyllid colonies against imidacloprid and flupyradifurone (and other modes of action) to address the possibility of cross-resistance directly in the laboratory.
Florida growers have reported that enhanced nutritional programs (ENPs) maintain productivity of HLB-affected 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. ENPs did slow the development of nutrient deficiency in HLB-affected trees, but it did not prevent the development of deficiencies. The slowed nutrient deficiency was not reflected in a slowed yield decline. In both field and greenhouse trees, treatment of HLB-affected trees with ENPs resulted in a reduced fibrous root density compared to trees under the SNP. Healthy trees treated with ENPs had a slight increase in fibrous root density compared to SNP. It appears that ENP treatment of HLB-affected trees may accelerate fibrous root loss, increasing the susceptibility of the trees to stress conditions such as drought. In nursery trial, ENPs reduced the intensity of visual foliar symptoms, but not the percent of canopy with symptoms. Only on HLB affected trees, ENPs caused a reduction in total number of leaves in the canopy, although they did not change the number of new leaves produced. This suggests that ENPs induce increased leaf drop. Based on symptom observations, it is likely that the leaf drop is of leaves that would have expressed strong symptoms. Combined, these results suggest that ENPs are masking HLB damage to the tree by decreasing the percentage of canopy with easily visible yellowing, but not improving the biomass or health of the tree. A manuscript describing the results of this project has been submitted to a peer-reviewed journal and is currently under review for publication.
The objectives of this project were to screen insecticidal peptides for efficacy against the Asian citrus psyllid (ACP) citrus leafminer, brown citrus aphid (BCA), and T. radiata and to determine the lethal and sublethal effects of antimicrobial peptides on ACP and their associated microorganisms, including Candidatus Liberibacter asiaticus (CLas). A citrus tristeza virus (CTV) vector system was used to express candidate insecticidal peptides in plants tissue. After screening a range of insecticidal peptides against ACP in artificial feeding bioassays, four insecticidal peptides (A-D) were selected and transformed into citrus tristeza virus (CTV) vectors for further evaluation against citrus pests. For each insect species, we evaluated life history parameters (fecundity, nymph development, and survival) and feeding behavior. The effect of CTV-expressed peptides on ACP host preference and CLas titer were also evaluated in laboratory bioassays. We have identified one peptide that, when expressed in Valencia in a citrus tristeza virus-based expression vector system, elicits negative responses from two phloem-feeding citrus pests. Additionally, we have demonstrated that all peptides evaluated disrupt various aspects of ACP biology and behavior, including feeding and settling. Reductions in feeding and ACP CLas titer in response to peptide presence suggests that use of the CTV-peptide expression system may provide an effective tool for management of CLas transmission as part of an integrated pest management program.
Reducing metallic copper for canker control: Control on grapefruit – Nordox at 33% of the full rate (0.66 lb/acre) was mixed with soluble Magna-Bon (100 ppm rate ) and compared with Nordox at the full rate (1.33 lb/acre) and at a reduced rate (1.0 lb/acre). Nordox at the full rate (1.33 lb/acre) was applied in the first 5 applications and Magna-Bon (100 ppm rate; Nordox 1.33/lbA in the frst 5 applications followed by 5 applications of MB was compared with 10 apps of Nordox the full rate (1.33 lb/acre). Fruit disease control for the mixture of Nordox with Magna-Bon or the program with Nordox applied early and Magna-Bon applied later in the season provided comparable control to the full rate of Nordox. Each program reduced the metallic Cu applied per season by 50% Control on Hamlin oranges – Kocide at 33% of the full rate (1.0 lb/acre) was mixed with soluble Magna-Bon (100 ppm rate ; 37 oz.) and compared with Kocide at the full rate (3.0 lb/acre ) and at a reduced rate (2.5 lb/acre). Reduction of canker-=induce fruit drop for the mixture of Kocide plus Magna-Bon was comparable to the full rate of Kocide at half the metallic rate. The Kocide plus Magna-Bon mixture achieved canker control with 50% of the applied metallic Cu. SAR for canker control – Currently, we are collaborating with Syngenta in an EPA-approved Experimental Use Program (EUP) in two east coast grapefruit groves to support the labeling of Actigard (ASM) integrated with copper sprays for control of canker on bearing grapefruit.
Objective 1: Assays of non-bearing trees indicate that soil drench of chelated copper formulations is a consistently effective application method for increasing copper status of young leaves well above the baseline concentration. Therefore, preventative and curative activity of chelated copper bactericides are being followed in pre- and early stage HLB infected trees. Tree health ratings and samples for PCR status of the trees in all trials will be collected and analyzed later this fall. In the meantime, grower trials of Magna-Bon (MB) and Copper Quik (CQ) are underway based on promising visual observations of improved tree health after 18 months of soil applications of rates varying from 1-2 gal product per application and 2-3 applications per season. One grower location will provide the opportunity to compare fruit drop due to HLB in matched 3 acre sub-blocks with and without soil applications of CQ for the last two seasons.
Abandoned citrus groves in US citrus-producing regions are potential sources for both Asian citrus psyllid (ACP), and Candidatus Liberibacter asiaticus (CLas), the bacterium that causes Huanglongbing (HLB). In Florida and Texas where grower-driven area-wide management programs are underway in commercial groves, no psyllid control is being implemented in abandoned citrus. Since ACP adults are highly mobile, they can disperse from abandoned to productive citrus groves. If not controlled, these psyllids will stymie the effectiveness of area-wide management programs aimed at containing the spread of HLB in commercial citrus. Insecticide-based strategies are not feasible for ACP in abandoned citrus, but biological control of ACP could come from a combination of native or introduced arthropods and pathogens. ACP is susceptible to a native entomopathogenic fungus, Isaria fumosorosea (Ifr). Two Ifr strains highly pathogenic against ACP and available as spore formulations in the US are the commercial strain PFR97 WDG’ (Certis Inc.,USA) and a south Texas isolate (Ifr 3581). We will develop a novel autodissemination system for inoculating ACP with Ifr and use these infected psyllids to instigate epizootics and rapidly reduce ACP populations in abandoned groves. Our ultimate goal is to provide optimized strategies for integration of Ifr autodissemination into current management practices. Suppression of ACP in abandoned citrus by Ifr autodissemination will significantly lower the risk of immigrating adults spreading HLB to commercial groves. Our proposed project objectives for the first and second quarters of Year 1 (April 2013 to September 2013) were to evaluate: (1) Impact of Ifr dispensers on ACP populations in abandoned and managed trees, and (2) Effect of time and exposure on Ifr dispensers. For Objective 1, we will mark adult psyllids in abandoned grove plots in South Texas and compare the numbers of marked psyllids that move from abandoned into managed grove plots in the absence of Ifr dispensers (control) and when Ifr 3581 dispensers are hung in trees on the edge rows of the abandoned groves. ACP movement, infestation, densities, and Ifr infection will be monitored weekly on edge row trees of the abandoned and managed plots. For Objective 2, we will expose dispensers to outside weathering on citrus trees from groves also used for Objective 1 and evaluate the efficacy of Ifr formulation recovered at different time intervals. To accomplish Objective 1 and 2, we have located five suitable pairs of managed groves and abandoned groves in the citrus producing regions of Hidalgo County and Cameron County in the Rio Grande Valley. These pairs of managed and abandoned groves consist of orange or grapefruit trees and the abandoned groves have not been managed for at least 2 years. The efficacy of our management tactic will largely depend on the ability of our Ifr dispensers to attract adult psyllids to the dispensers’ spore-coated surfaces. The use of effective attractants for our dispensers is essential for the completion of Objectives 1 and 2. During the spring and summer of 2013, we conducted field trials to evaluate the relative efficacy of six different blends of synthetic aromatic compounds mimicking the flush volatile profiles of orange jasmine and Valley lemon for luring adult psyllids onto yellow sticky traps. Based on the results of these trials, we have selected two proprietary blends for dispensers that will be evaluated in field trials.
We conducted 3 assays to compare the effectiveness of a commercially available entomopathogenic nematode (EPN) species Heterorhabditis bacteriophora to that of Steinernema riobrave which is documented to be the most effective EPN pathogen of Diaprepes abbreviatus, but is not currently available commercially. In the basic assay, weevil larvae are preconditioned at 10 C or 27 C for 48 h. Then weevil larvae are exposed to very low rates (2 nematodes/cm2 soil surface) of EPNs at 18 C or 27 C for 6 days, during which mortality is evaluated at 2 day intervals. Thus, temperature regimes of 10C+18C simulate a wintertime cold event and 27C+27C simulates summertime conditions. Results of these trials revealed that: 1. H. bacteriphora was 50% as effective as S. riobrave in summer. 2. S. riobrave was 10% more effective in winter than it was with nonstressed weevils during summer 3. H. bacteriophora was 60% more effective in winter than it was with nonstressed weevils during summer 4. H. bacteriophora was 80% as effective in winter as Sr was with nonstressed weevils during summer We subsequently obtained an isolate of H. bacteriophora from upstate New York. In a single trial, not yet repeated, the New York isolate was somewhat more effective than the commercial isolate of H. bacteriophora during simulated winter cold events. We most recently obtained a commercial product containing Steinernema krauseii a temperate species capable of infecting hosts at temperatures as low as 8 C. In a single trial, not yet repeated, S. krauseii was ineffective against weevils under simulated summertime conditions. It was much more effective in the cold temperature regime, but inferior to H. bacteriophora and S. riobrave in its ability to kill weevil larvae.
Entomopathogenic nematodes: Amending soils to increase biological control of insect pests Because we found increased numbers of Steinernema diaprepesi in low pH treatments (as measured by qPCR of nematodes from soil samples) at the Bartow experiment (May 2013 report), we buried caged weevil larvae in all plots and evaluated the mortality rate after 7 days in the soil. In contrast to our predictions, weevil mortality and infection of weevils by S. diaprepesi were only 15% (74% vs 64%, ns) and 32% (33% vs 25%, ns) higher, respectively, in sulfur amended than non-amended treatments. Heterorhabditis indica infection of weevils was 8-fold higher (18% vs 2%, P<0.05) in the non-amended plots which conforms to the relationship measured in the Schumann ACPS trial in which H. indica were significantly more abundant in the high pH ACPS plots than in the low pH conventional citriculture plots. We Initiated a second round of buried sentinel weevils to confirm the results, to be reported in September 2013. In June 2013 the average fibrous root weight and numbers free living nematodes from sulfur-amended trees at Bartow were 60% (ns) and 56% (P<0.05), respectively, of those from non-amended trees. The abundance of Tylenchulus semipenetrans was 5-fold greater in non-amended than in sulfur amended plots (P<0.05). These results continue trends noted previously and suggest that sulfur is reducing fibrous root density somewhat (perhaps because roots are more efficient at lower pH), but is also affecting the citrus nematode independently of its affect on root availabilty. This is consistent with previous reports of the effects of pH on the citrus nematode and demonstrates that the potential for managing this nematode by pH adjustment is greater than previously thought. Completed a second manuscript (Cultural practices to manage a bacterial disease alter entomopathogenic nematode communities and increase the severity of a pest-disease complex) describing effects of ACPS and fabric mulch on citrus tree growth and yield, Diaprepes abbreviatus, Phytophthora nicotianae, EPNs and natural enemies of EPNs. First manuscript in this series (Campos-Herrera, R., et al. 2013. New citriculture system suppresses native and augmented entomopathogenic nematodes. Biological Control 66: 183-194.) published in June. Plant parasitic Nematodes: Characterizing a new nematode pest and the prevalence of resistance breaking populations of the citrus nematode. Evaluated nematicidal efficacy in the east coast, dagger nematode nematicide trial in June. No chemical treatment (oxamyl, and three experimental nematicides) reduced dagger nematode populations significantly. Plots will receive additional treatments at the end of the rainy season in early autumn and treatment effects measured in November. Dagger nematode spatial pattern at site on the central ridge was characterized and a second trial will be initiated this autumn.
Our long term goal is to develop a system for inoculating Asian citrus psyllid (ACP) with a native pathogenic fungus, Isaria fumosorosea (Ifr), and use these infected psyllids to rapidly ‘autodisseminate’ the pathogen to ACP populations in residential citrus. Specific research objectives for 2012-2013 were as follows: (1) Initiate Ifr-epizootics among ACP populations on dooryard citrus trees, (2) Evaluate seasonal and environmental effects on infection levels, (3) Determine the impact of pathogen on arthropod biological control agents. For objectives 1 and 2, we secured the permission of trailer park residents in the Rio Grande Valley to use their citrus trees for our field studies. To minimize the risk of spreading HLB, we agreed to: (1) inoculate and release only ACP adults collected from the study sites, and (2) use only trees infested by wild ACP. Since our last report submitted in April of 2013, the following trials were completed: “Quantify infection of Ifr-inoculated adults under field conditions and the effects of incubation treatments prior to field release”. To better assess what release rates of inoculated psyllids would be required to initiate epizootics within target trees, we conducted trials to quantify infection of Ifr-inoculated adults under field conditions and the effects of incubation treatments prior to field release. The rationale behind the incubation treatments were to develop a means of maximizing infection of inoculated psyllids after release on target trees. During June to September of 2013, we conducted field studies using orange trees at the ‘Victoria Palms’ trailer park in Donna, TX. Adult psyllids collected from orange trees at ‘Victoria Palms’ were inoculated with Ifr formulation and held (incubated) in humid vials with orange leaf disks for: 0 h, 24 h, and 48 h . Another set of adults were not inoculated and set aside as controls. Each psyllid was released into a ‘clip cage’ attached to a leaf on an orange tree at the trailer park. Groups of four clip cages (one cage for a control psyllid and one cage for each of the three types of inoculated psyllids) were attached to four apical leaves on the same stem. Ten groups of clip cages were setup on the North-East canopy of trees (total = 40 clip cages per tree). For eight consecutive days, we recorded the status of the psyllid within each clip cage. By the eighth day, all inoculated and incubated psyllids were dead and infected with Ifr. In comparison, 50 % of psyllids inoculated but not incubated were dead and only 20 % of these psyllids were infected with Ifr. None of the control psyllids were killed or infected by Ifr. These results demonstrated that incubation of Ifr-inoculated adults for at least 24 h will ensure 100 % mortality and infection within 8 days of release in residential citrus trees under summer weather conditions in Texas. We are currently repeating these studies under cooler and wetter fall conditions that should enhance infection of ACP by Ifr. “Effects of Ifr spores on lacewing larvae”. During June of 2013, we compared predation by lacewing larvae on healthy or Ifr-inoculated adults of ACP. We collected lacewing larvae from residential orange trees infested with ACP and held them for 12 h on orange leaf disks in plastic vials. After 12 h, we offered four healthy ACP adults and then four Ifr-inoculated ACP adults (inoculated by dusting with Ifr formulation 24 h earlier) to lacewing larvae that were similar in size and development. We observed each lacewing larva and found they accepted and fed on all the healthy ACP adults and also all the Ifr-inoculated adults. None of the lacewing larvae showed visible signs of infection by Ifr and appeared healthy after being held for five days in leaf-disk vials and fed additional healthy ACP adults. This study demonstrated that lacewing larvae do not appear to be adversely affected by feeding on ACP adults inoculated with Ifr formulation.
Our long term goal is to develop a system for inoculating Asian citrus psyllid (ACP) with a native pathogenic fungus, Isaria fumosorosea (Ifr), and use these infected psyllids to rapidly ‘autodisseminate’ the pathogen to ACP populations in residential citrus. Specific research objectives for 2012-2013 were as follows: (1) Initiate Ifr-epizootics among ACP populations on dooryard citrus trees, (2) Evaluate seasonal and environmental effects on infection levels, (3) Determine the impact of pathogen on arthropod biological control agents. For objectives 1 and 2, we secured the permission of trailer park residents in the Rio Grande Valley to use their citrus trees for our field studies. To minimize the risk of spreading HLB, we agreed to: (1) inoculate and release only ACP adults collected from the study sites, and (2) use only trees infested by wild ACP. Since our last report submitted in April of 2013, the following trials were completed: “Quantify infection of Ifr-inoculated adults under field conditions and the effects of incubation treatments prior to field release”. To better assess what release rates of inoculated psyllids would be required to initiate epizootics within target trees, we conducted trials to quantify infection of Ifr-inoculated adults under field conditions and the effects of incubation treatments prior to field release. The rationale behind the incubation treatments were to develop a means of maximizing infection of inoculated psyllids after release on target trees. During June to September of 2013, we conducted field studies using orange trees at the ‘Victoria Palms’ trailer park in Donna, TX. Adult psyllids collected from orange trees at ‘Victoria Palms’ were inoculated with Ifr formulation and held (incubated) in humid vials with orange leaf disks for: 0 h, 24 h, and 48 h . Another set of adults were not inoculated and set aside as controls. Each psyllid was released into a ‘clip cage’ attached to a leaf on an orange tree at the trailer park. Groups of four clip cages (one cage for a control psyllid and one cage for each of the three types of inoculated psyllids) were attached to four apical leaves on the same stem. Ten groups of clip cages were setup on the North-East canopy of trees (total = 40 clip cages per tree). For eight consecutive days, we recorded the status of the psyllid within each clip cage. By the eighth day, all inoculated and incubated psyllids were dead and infected with Ifr. In comparison, 50 % of psyllids inoculated but not incubated were dead and only 20 % of these psyllids were infected with Ifr. None of the control psyllids were killed or infected by Ifr. These results demonstrated that incubation of Ifr-inoculated adults for at least 24 h will ensure 100 % mortality and infection within 8 days of release in residential citrus trees under summer weather conditions in Texas. We are currently repeating these studies under cooler and wetter fall conditions that should enhance infection of ACP by Ifr. “Effects of Ifr spores on lacewing larvae”. During June of 2013, we compared predation by lacewing larvae on healthy or Ifr-inoculated adults of ACP. We collected lacewing larvae from residential orange trees infested with ACP and held them for 12 h on orange leaf disks in plastic vials. After 12 h, we offered four healthy ACP adults and then four Ifr-inoculated ACP adults (inoculated by dusting with Ifr formulation 24 h earlier) to lacewing larvae that were similar in size and development. We observed each lacewing larva and found they accepted and fed on all the healthy ACP adults and also all the Ifr-inoculated adults. None of the lacewing larvae showed visible signs of infection by Ifr and appeared healthy after being held for five days in leaf-disk vials and fed additional healthy ACP adults. This study demonstrated that lacewing larvae do not appear to be adversely affected by feeding on ACP adults inoculated with Ifr formulation.
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