Research targeting the development of a new Asian citrus psyllid (ACP) control strategy based on blocking salivary sheath formation continues to advance. We now have identified two enzymatic pathways that degrade the the salivary sheaths based on compositional knowledge and are moving to test the use of these strategies in plants. We have also finished the initial screening of the peptide library provided by Torrey Pines Institute for Molecular Studies. These results have identified peptide mixtures with activity affecting psylllid sheath formation and psyllid survival. These are being advanced into the second phase where individual peptides are identified. We continue to move ahead to identify defined peptide(s) that have adverse affects on the ACP that will be used to construct a synthetic gene(s) encoding the specific peptide(s). These will be used to construct transgenic plant and for topical application strategies to block ACP feeding processes.
Research targeting the development of a new Asian citrus psyllid (ACP) control strategy based on blocking salivary sheath formation was advanced in two main areas. First, we have developed a novel technique to isolate (to purity), fully intact ACP sheaths having both flange and sheath body as a single complete unit (previously our techniques could only isolate the body portion, excluding the flange and we have shown that they are compositionally different). Also, we are now able to isolate at an estimated >90% efficiency, intact sheaths, whereas previously our sheath harvest yield we estimated at <10% efficiency for partial sheath harvests. By isolating full sheaths, at high yield, we are able to effectively determine the complete structural composition of the sheaths by molecular and chemistry approaches. We are presently substantiating our previous GC, IR, and protein analyses that suggested the sheath structure was primarily composed of carbohydrates with some protein components, using these same techniques with the full sheaths. Also, using a barrage of enzymatic analyses, we have identified single gene products that prevent sheath formation. At present we now can both chemically and biological inhibit the formation of, and degrade, ACP sheaths. Currently, we are moving forward with definitive sheath compositional analyses and to application of sheath inhibitory mechanisms in in vivo systems. Second, using our artificial diet bioassay, we are presently de-convoluting a decapeptide amino acid mixture library developed by Torrey Pines Institute for Molecular Studies. Our diet assay is being used as a vehicle to screen and identify specific peptides responsible for biological activity against the sheath and/or psyllid. At present we have been able to determine certain specific activities associated with specific amino acids at defined positions of these decapeptides. Interestingly, we have been able to show both positive and negative impacts on the psyllid with different position specific amino acids. During the next year, defined peptide(s) will be identified that have adverse affects on the ACP that will be used to construct a synthetic gene(s) encoding the specific peptide(s) for use in transgenic plant construction and for topical application strategies to block ACP feeding processes.
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 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. 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 (Coo2, GSTS1 and S4e) either constitutively (35S promoter) or in a phloem-specific manner. 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 lines with the D. citri-specific gateway vectors for evaluation and use by the Florida citrus industry. Currently we have several lines containing gateway vectors with the vacuolar ATP synthase subunit G or S4e transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter. We have regenerated at least one line for each of the constructs of interest, and we are in the process of in vitro micro propagation to produce additional copies of each line. We also regenerated additional transformed lines, and beginning to characterize the transgene expression to select lines for micro propagation. We are beginning transformation of additional lines with the other transcript/promoter combinations.
We collected dead, preserved D. citri psyllids, and psyllid RNA samples from Florida, China, Brazil, and Taiwan. We are still attempting to obtain D. citri RNA samples from Pakistan, India, Mexico and Central America. We extracted total RNAs and from these we have isolated the small RNAs which result from virus infections. These have been purified and we prepared small RNA libraries for Illumina next generation small RNA sequence analysis. Samples have been submitted for RNA sequencing and we are waiting for the sequence data. We also have obtained permits and now have started a colony of D. citri within the UC Davis Contained Research Facility. As viruses are discovered we will attempt to transmit them to our D. citri and ultimately engineer them for RNAi studies.
We surveyed the occurrence of volatile compounds in the headspace over six species in the family Rutaceae that differ in their susceptibility to the Asian citrus psyllid. Some compounds were unique and others were common to several species. A manuscript (Robbins et al., “Volatile profiles of young leaves of Rutaceae spp. varying in susceptibility to the Asian citrus psyllid (Hemiptera: Psyllidae)” will appear in the next number of the Florida Entomologist. The list of volatile host plant compounds is being systematically surveyed by electroantennogram (EAG) and coupled gas chromatograph-electroantennogram detection (GC-EAD) We tested volatiles by EAG and GC-EAD identified from the headspace of citrus flush as well as compounds identified from the cuticle of female psyllid. Cuticular compounds were tested against the antennae of both male and female psyllids. In these preliminary studies, none of the heavy weight cuticular compounds were found to stimulate the antennae of either sex. We plan to continue examining other cuticular compounds to test for evidence of activity. We discovered that breakdown products of two common volatiles isolated from the headspace over citrus flush were highly stimulatory to the antennae of both sexes of the Asian citrus psyllid. This is a novel discovery since no degradation products of citrus volatiles have ever been found to stimulate the antennae of the psyllid. We have identified these degradation products by GC-Mass Spectrometry and are now testing them for behavioral activity.
Xambr’ Brazil Plots: Designed to examine the effect of windbreaks, copper sprays to reduce infection, and leafminer treatments to determine their individual and combined effects on control of citrus canker. Windbreaks were completed and plants were be established in Mid April 2010, but severe winds damaged the windbreaks during two storm events. These windbreaks have been reinforced and rebuilt. From July through September 2012, we continued replanting citrus trees damaged by high winds and continued replacing windbreak damaged screens and poles. Windbreak and citrus plants will be of sufficient size to begin the experiment in March 2013. New windbreak trials established in Saint Lucie County. Preliminary disease data was obtained from sampling trees adjacent to the weather stations. Results indicated that disease incidence and severity across the field was related to proximity to the wind breaks, with least disease near the wind breaks. Wind speeds were least near wind breaks. Programmable leaf wetness controller (PLWC) to examine canker bacterial survival in the field. Software was written, debugged, is complete, and the control program is working well. Development of leaf wetness sensors that function across the full range of wetness has eluded scientists for many years. We have designed and constructed sensors based on completely new technology and continue to debug them prior to deployment. During this quarter we have explored the use of polymeric tissue and a conduit, that is water sensitive and change shape when it comes in contact with water or an electric charge. Unfortunately the distortion was quite large and not always in the same direction (dependent on where the water hit). We also tested the current change through the strip between wet and dry and we are currently analyzing the data. Project publications: Bock, C. H., Graham, J. H., Gottwald, T. R., Cook, A. Z., and Parker, P. E. 2010. Wind speed effects on the quantity of Xanthomonas citri subsp. citri dispersed downwind from canopies of grapefruit tree infected with citrus canker. Plant Di Bock C.H., Graham, J.H., Gottwald, T.R., Cook, A.Z., and Parker, P.E. 2010. Wind speed and wind-associated leaf injury affect severity of citrus canker on Swingle citrumelo. Eur J. Plant Path 128:21-38 Bock, CH, Parker, PE, Cook, AZ, Graham, JH and Gottwald, TR. 2001. Infection and decontamination of citrus canker and inoculated the surfaces. Crop Protection 30:259-264. Hall, D.G., Gottwald, T.R. and C.H. Bock. 2010. Exacerbation of citrus canker by citrus leafminer Phyllocnistis citrella in Florida. Florida Entomologist. Florida Entomologist 93:558-566. Bock, C.H., Gottwald, T.R. and Parker, P.E. 2011. Distribution of canker lesions on the surface of diseased grapefruit. Plant Pathology (Accepted). Bock, C.H., Cook, A.Z., Parker, P.E., Gottwald, T.R., and Graham, J.H. 2011. Some characteristics of the dispersal plume of bacteria of Xanthomonas citri subsp citri in wind-driven splash downwind of canker-infected grapefruit tree canopies. (Plant Disease). Bock C.H., Graham J.H., Cook A. Z., Parker P.E., and Gottwald T.R. 2012. Predisposition of citrus foliage to infection with Xanthomonas citri subsp. citri. (Submitted to Plant Disease).
In this final study, we tested if widely-used neonicotinoid insecticides can prevent Las acquisition and/or inoculation on citrus plants. In a first experiment, we evaluated the effects of insecticide mode of application and citrus branch age on pathogen acquisition by ACP nymphs and adults, and found that both foliar (contact effect) and drench (systemic effect) applications of neonicotinoids were effective in preventing acquisition. The results were described in detail in the previous report. Recently, a second experiment was carried out to test if the same insecticide treatments (foliar and drench applications of neonicotinoids) can prevent Las inoculation by ACP on citrus nursery trees. Healthy sweet orange nursery trees [Citrus sinensis (L.) Osbeck grafted on ‘Rangpur’ lime] with or without young shoots were submitted to the following treatments: a) foliar application of imidacloprid (200 SC ‘ 0.24 ml c.p/1 L water), for contact action; b) drench application of thiametoxam (250 WG ‘ 1 g c.p./plant, diluted in 50 ml water), for systemic action; and c) untreated plants (water only; positive control for estimating inoculation efficiency after exposure to infective psyllids). Each of these test plants were exposed to 10 psyllid adults that had been previously submitted as 4th instar nymphs to a 10-day AAP on Las-infected plants, followed by 10 days on intermediate citrus healthy seedlings to conclude the latency period. The groups of 10 psyllids were confined inside clear plastic cup cages on a single apical branch (young or mature) of the test plants for a 72-h IAP. The mortality rate on test plants of each insecticide treatment was recorded at 1, 3, 24, 48 and 72 h after the beginning of the IAP. The experiment was repeated three times, using different source plants and insects for Las acquisition. Each trial was considered as a block in a randomized block design, with 6 treatments and 5 replicates (test plants). The inoculated test plants were evaluated by qPCR up to 12 months after the IAP. Transmission to test plants was detected in all treatments. By pooling the data of the three transmission trials, significant proportions of infected test plants were observed in treatments with systemic (drench application) (13.3%) and contact insecticides (foliar application) (6.7%). The transmissions rate to untreated plants (positive controls) was 26.7 and 13.3% for inoculations in mature and young branches, respectively. Regarding insect mortality, there was no effect of branch age on the effect of insecticide treatments (F = 0.72; P > 0.79). However, there was significant variation in mortality depending on the application mode (F = 10.2; P > 0.0001). The foliar application (contact insecticide) resulted in higher psyllid mortality, with a light knockdown effect (6% mortality) in the first hour after application, increasing to 60, 76, 82 and 84% at 3, 24, 48 and 72 h, respectively. The drench application (systemic action) showed no knockdown effect (F = 10.2; P > 0.30), and caused mortality rates lower than those observed for the foliar application in all evaluations. Except for the first hour (no knockdown effect), the mortality caused by drench application differed from those on the untreated controls, but its control efficiency estimated based on Abbott’s formula was <45% after 72 h. Despite the significant effects on psyllid mortality, the results of these two experiments indicate that the insecticides and application modes tested here do not prevent Las inoculation by D. citri to healthy citrus trees, although they can drastically affect bacterial acquisition.
Ultra High Performance Liquid Chromatography – Pesticide Residue Analysis (Sept 2012 Update) Following scheduling delays by the company performing the installation, the new LC-MS-MS was installed in late August. Additional service calls have continued to be made through September to correct leaks in the lines so that the equipment functions properly. At the end of September, the installation technician returned and provided 2 days of instrumentation and software training and we have now begun running preliminary residue samples.
Research identifying enzymatic pathways that degrade the the salivary sheaths was advanced and multiple enzymes within the two classes known to degrade the salivary sheaths have been evaluated. Natural gene products already produced in plant systems have been shown to function in sheath degradation. This suggests it may be possible to engineer plants to block sheath formation, thus making them resistant to psyllid feeding. Novel method of pure sheath isolation has been perfected using a soluble membrane through which the psyllids feed. Subsequent purification of pure sheath material was shown to be possible through differential filtration. This method allows purification of sheath material that is not contaminated by any other chemicals and therefore provides more evidence showing the building blocks used by the psyllid to synthesize sheaths.
Two experiments were concluded by USDA-ARS on protecting young citrus from HLB using different ACP management programs. The results were reviewed in the July 2012 quarterly report. Briefly, an intensive insecticide program was evaluated in each experiment: eight annual calendar applications of traditional hard insecticides, hereafter referred to as the ‘complete program’. The results of the experiments indicated that up to eight monthly applications per year of hard pesticides applied on a calendar schedule were ineffective for getting young citrus into production without becoming diseased. Of probable importance is that the grove where the two experiments were conducted was subjected to a minimal psyllid management program and contained many trees infected by HLB, thus the ACP management programs we evaluated might have been more effective if psyllids in the surrounding areas had been more aggressively controlled and HLB-infected trees removed to reduce inoculum loads. It is possible that better ACP and resulting HLB control in the young trees could have been achieved under the complete program if it had included a scouting component – either as a substitute for timing sprays on calendar dates or for determining if additional insecticide sprays were needed. UF (Stansly) also concluded two experiments. In one test, the efficacy of four treatments for protecting young citrus resets from HLB was studied: (1) nutrition alone, (2) insecticides alone, (4) nutrition plus insecticides, and (4) untreated control. Resets (‘Valencia’) were planted in July 2010. At that time 92-95% of the surrounding trees were PCR positive for HLB. The nutritional regimen was adapted from that of production manager Maury Boyd. Insecticide treatments included dormant sprays during the winter, and additional sprays were made when numbers of ACP exceeded a threshold, initially 0.5 ACP but later 0.2 ACP per tap sample. Resets were subjected to regular drenches of the following insecticides: bifenthrin, thiamethoxam, and imidacloprid. The resets were drenched 3 times during 2010, 4 times during 2011, and 4 times during 2012. Foliar insecticides (spinetorum, chlorpyrifos, phosmet, fenpropathrin, dimethyl phosphate, spirotetramat, abamectin + thiamethoxam, or dimethoate) were applied to the resets 3 times during 2010, 7 times during 2011, and so far 2 times during 2012. The resets were PCR-assayed for Las (Li primers, Ct threshold of 32) 4 times in 2011 and most recently in Feb 2012. By 19 months after planting, averages of 16 to 45% of the resets tested positive for the pathogen (Ct-values of 27 to 33), and there were no significant difference among treatments with respect to percentages of trees infected and titers of the pathogen. In a second UF experiment, a new citrus planting (‘Hamlin’) was planted during May 2010 and subsequently drenched with insecticides (Verimark, Admire, Plantinum) at different rates and rotational schedules. The trees were treated 2 times in 2010, 4 times in 2011, and twice in 2012. All treatment regiments significantly reduced the number of adult and juvenile ACP on all sample dates with differences amongst the treatments only being observed with the nymph counts on 13 July 2011. HLB incidence has been lower in the treated plots with the lowest levels being seen when Verimark at the higher rate was used. At 24 months after planting, 63% trees tested positive for HLB in plots of trees not drenched with insecticides while fewer (from 11 to 26%) trees tested HLB-positive in plots of trees that were drenched.
Entomopathogenic nematodes: Amending soils to increase biological control of insect pests Developed molecular probe to quantify Phytophthora nicotiana. Measured 6-fold greater amounts of the pathogen (P=0.0001) in samples obtained from Advanced Citrus Production System (ACPS) plots compared to conventional plots. Samples were obtained quarterly between July 2011-May 2012. This result is consistent with 2-fold more D. abbreviatus trapped in ACPS plots (P=0.02) during the same period, because D. abbreviatus exacerbates root infection by Phytophthora spp. We confirmed in soil assays, results from aqueous tests showing that spores of Paenibacillus sp. detach readily from Steinernema diaprepesi at pH<6.0, but not at neutral pH. Initiated laboratory bioassays to investigate effects of soils from ACPS and conventional plots on nematophagy by fungi and subsequent EPN efficacy against D. abbreviatus larvae. Plant parasitic Nematodes: Characterizing a new nematode pest and the prevalence of resistance breaking populations of the citrus nematode. Developed molecular probe to distinguish Xiphinema citricolum vs X. laevistriatum, two morphologically indistinguishable dagger nematodes encountered in citrus orchards. Began producing plasmids that will serve as positive controls for those primers. Sampled 6 orchards near Ft. Pierce and detected trace amounts of X. vulgarum in just two samples, juvenile Xiphinema sp. which cannot be identified to species in 4 samples. All samples had Phytophthora nicotianae and P. palmivora propagules above treatment thresholds and all samples were positive for the EPN Heterorhabditis indica. Discussed results with Denise Dunn who processes large numbers of samples for DuPont and was informed that X. vulgarum had declined greatly in DuPont samples, during this period. Will resume sampling in mid-autumn when populations are likely to increase.
Citrus production is facing increasing acreage losses due to disease threats arising from pests like the Asian Citrus Psyllid and Citrus Greening (Huanglongbing, HLB) Disease. Some growers in Florida have experienced positive results at combating the physiological damage and yield loss commonly associated with HLB, even in fields with up to 95% of trees with confirmed HLB disease. A mixture of micro- and macro-nutrient, along with some agents to fight against pathological diseases, applied in a oil-based foliar spray solution may have promise at boosting citrus tree vigor to assist in the combating HLB disease. Preliminary field and laboratory studies performed at TAMU-Kingsville Citrus Center focused on the impacts of Ca applications at reducing Asian Citrus Psyllid (ACP) populations. In the field, composts higher in calcium (Ca) levels led to higher Ca content in citrus leaves, and subsequently reduced ACP populations feeding on these trees compared to other compost sources. In a controlled lab study, foliar sprays containing Ca led to fewer ACP eggs laid and decreased adult ACP survival on ‘Rio Red’ grapefruit leaves. From these initial investigations a large field study was initiated in May 2011 on 6-7 year old ‘Rio Red’ grapefruit trees located at the Citrus Center South Farm. Varying foliar micro- and macro-nutrients treatment mixtures were applied as a means to supplying increase tree vigor to fight against citrus pest disease symptoms. 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. Foliar treatments contained various chelated nutrient sources in solutions that were formulated by Metalosate’: Trt 1= Control (N and oil only); Trt 2 = K; Trt 3 = Ca; Trt 4 = Crop Up: (Mg, B, Cu, Fe, Mg, Zn); Trt 5 = Crop Up+ K; Trt 6 = Crop Up + Ca; Trt 7 = K + Ca; Trt 8 = Crop Up + K + Ca. All treatments received 2% 435 Oil; 50 lb/Acre N; RequieM 25 EC; and Serenade (strain of Bacillus). Results: We observed a positive correlation between lower ACP infestations in trees receiving additional calcium application, regardless of whether the macro-nutrient is applied to the soil or to foliage. However, all foliar treatments that included supplemental calcium additions significantly reduced the ACP population in field trials from 16 to 25% compared to equivalent treatment excluding Ca. The lowest ACP infestation levels on citrus leaves were observed in foliar spray treatments that included the Crop-Up plus Ca treatment, followed by the Ca only treatment. Benefits to Citrus Industry: With an increased threat of HLB disease and other insect related diseases facing Texas citrus growers, we anticipate that this data may allow us to find another aspect of decreasing this threat to the industry with minimal use of bio-pesticides.
The questions to be addressed by this research project are 1) how does Imidicloprid move in the sandy soils of south Florida, 2) how long does imidicloprid persist in sandy soils, and 3) how much Imidicloprid leaches below the root zone of citrus trees. Isotherms relating soil Imidicloprid concentrations in soil solutions with Imidicloprid concentrations in the solid phase of sandy soil were conducted using Immokalee fine sand. The concentrations added to the soil were 2, 4, 6 and 8 mg ml-1 and represent the range of concentrations in the soil estimated after soil drenching. Imidicloprid was found to penetrate to a depth of 45 cm and have greatest affinity in the 0-15 cm soil depth due to higher soil organic matter at that depth. These data were supported by soil partition coefficients (Kd) of 1.68 mg of Imidicloprid. in one liter of soil solution at the 0-15 cm depth and decreases to 0.33 and 0.25 mg l-1 at 15-30 and 30-45 cm, respectively. A laboratory study found that Imidicloprid decreased in soil concentrations due to biodegradation at a rate of 0.013 .g g-1 d-1, thus one milligram of Imidicloprid would persist in the soil for more than 27 years. Two field studies were conducted the spring and summer of 2012. Initial analysis of soil samples from the spring study indicate that Imidicloprid concentrations decreased by 90% in 10 days at the 0-15 cm depth by tree uptake and recommended microsprinkler irrigation. At the 0-15 cm depth. Imidicloprid concentrations reached the 15-30 cm depth in 5 days after application and persisted at that depth for approximately 15 days. During this initial application study, adult psyllid populations per tree decreased from approximately 1.7 for both treated and non-treated trees to 0.1 psyllids per tree for the treated trees at 22 days after application. Psyllid populations on treated trees were below 0.1 psyllids per tree from 22 to 50 days after application.
In this project we are studying the influence of various factors on transmission efficiency of Candidatus Liberibacter asiaticus (CLas) by the Asian citrus psyllid (ACP), Diaphorina citri. Here we report partial results of the last study, on the effects of systemic insecticides on pathogen acquisition and or inoculation. In this final study, we are testing if widely-used neonicotinoid insecticides can prevent CLas acquisition and/or inoculation on citrus plants. In a first experiment, we evaluated the effects of insecticide mode of application and citrus branch age on pathogen acquisition by ACP nymphs and adults. CLas-infected sweet orange nursery trees [Citrus sinensis (L.) Osbeck grafted on ‘Rangpur’ lime] with and without young shoots were submitted to the following treatments: a) foliar application of imidacloprid (200 SC ‘ 0,24 ml c.p/1 L water), for contact action; b) drench application of thiametoxam (250 WG ‘ 1 g c.p./plant, diluted in 50 ml water), for systemic action; and c) untreated plants (water only; positive control for estimating acquisition efficiency after exposure to source plants). Two hours after treatments a) and c), and 10 days after treatment b), about 50 3rd-instar ACP nymphs were confined on the youngest branch of each nursery tree using sleeve cages, during a 72-h acquisition access period (AAP). As a negative control for checking possible vector infectivity before exposure to the treated nursery trees, ACP nymphs and adults from the same rearing batch were submitted to a similar AAP on untreated healthy nursery trees. Insect mortality was assessed right after the AAP; the surviving insects were kept on healthy citrus seedlings for 10 days and then tested by real-time (RT) PCR for infectivity with CLas. The experiment had a completely-randomized block design, with 2 factors (‘application mode’ and ‘citrus branch age’) (4×2) and 3 replications per treatment. Each replication was represented by a group of 50 nymphs confined on the youngest branch of a nursery tree. A similar experiment was carried out with 1-week old adults. No infective psyllids were detected by RT-PCR after the AAP on infected nursery citrus trees that had been treated with either foliar or drench applications of the insecticides, or on the untreated healthy trees (negative control), in both experiments. In contrast, 47.2% of the nymphs and 32.5% of the ACP adults were able to acquire the pathogen during the same AAP on untreated infected citrus trees with young shoots. On untreated infected nursery trees without young shoots, lower but relatively high acquisition rates were detected for adults (23.7%), whereas no nymphs acquired the pathogen. The average mortality rates of psyllid adults (<30%) and nymphs ('50%) were surprisingly low after the 72-AAP on treated plants, although significantly higher than those observed on untreated plants ('8%) in the experiments with adults (F=10.9, P=0.002) and nymphs (F=13.6, P=0.001). Citrus branch age did not influence the mortality rates caused by the insecticides for adults (F=1.99, P=0.19) and nymphs (F=2.98, P=0.11). Despite the low mortality rates, these results show that both insecticide treatments (foliar and drench applications) prevent pathogen acquisition by the vector, possibly because of sub-lethal effects on feeding behavior. We will now check for the presence of CLAS (by RT-PCR) in the test seedlings exposed to the surviving psyllids after the AAP, to make sure that no transmission event occurred in these insecticide treatments. A second experiment is being carried out to test if the same insecticide treatments (foliar and drench applications of neonicotinoids) can prevent CLas inoculation by ACP on citrus nursery trees with or without young shoots. Results and conclusions of this second experiment will be presented in the final report.
We have previously conducted experiments to evaluate the effect of chemical plant growth regulators (PGRs) on acquisition of the citrus greening pathogen, Candidatus Liberibacter asiaticus (Las), by the Asian Citrus Psyllid (ACP). Previously, we reported the results of bioassays in which psyllids were allowed to feed on Las-infected or healthy citrus plants treated with the PGR, prohexadione calcium, for 28 or 14 d acquisition access periods (AAPs). Subsequently, we have conducted assays to evaluate the effect of prohexadione calcium acquisition during a 7 d AAP. Three independent experiments were conducted in temperature-controlled environmental chamber as described previously. ACP from each treatment were then collected and analyzed with qPCR. Results of these analyses indicated that ACP feeding on PGR-treated, infected plants acquired Las at a higher rate than did ACP held on untreated, Las-infected plants. Subsequent experiments will be conducted to determine the rate of acquisition following a 24 h AAP with adult ACP and during nymph development.
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