Initial research was focused on the development of an artificial feeding system for the Asian citrus psyllid and testing this feeding system utility for screening peptides and dsRNAs as oral toxicants. The initial results led to the observation of significant toxicity of orally fed specific dsRNAs targeting and blocking the function of a specific psyllid gene (a total of eight psyllid genes were tested as dsRNA targets). This finding was the basis for initiation of a CRDF crowd-sourcing competition through innocentive’ to identify the best psyllid gene target to advance in a commercializable psyllid control strategy. Further analysis of this target was conducted in collaboration with Dr. Bill Dawson’s laboratory where the most active dsRNA was expressed in citrus phloem using a Citrus tristeza virus (CTV) expression vector. Plants expressing this dsRNA were used in various feeding trials and shown to induce up to 80% mortality in the adult psyllids feeding on individual leaves, but the level of toxicity was highly and positively correlated with the level of psyllid gene dsRNA in the leaf on which the Psyllid fed. In whole plant studies, where a population of ‘Candidatus ‘ Liberibacter asiaticus (CLas) positive psyllids was placed on the dsRNA producing citrus for 14 days and then removed and tested for the presence of CLas, the number of CLas positive psyllids was greatly reduced as compared to controls. Furthermore, eggs laid by these psyllids were allowed to hatch and the nymphs were allowed to feed, develop and emerge as adults. When these adults were tested for CLas a significant reduction in the number of emerged adults carried detectable amounts of CLas as compared to controls. Continued analysis of the effect of this dsRNA on the psyllid supports the interpretation that the dsRNA is adversely affecting the biochemical pathway that in which the targeted gene functions and the research is now being advanced to test the feasibility of using this RNAi approach in a commercially deployable psyllid control strategy. Two manuscripts are prepared for this research; however, there is also a patent application for this work and once complete, the manuscripts will be submitted.
December 2013 The objectives of this proposal are 1) to determine if a) leaf litter biodegradation treatments reduce Guignardia spp. pseudothecia and improve control afforded by routine fungicide applications; b) if biodegradation is affected by the current fungicide application practices; and c) whether the biodegradation treatments will affect current citrus best management practices (BMP); 2) to determine the seasonal dynamics of leaf litter inoculum load in varying management regime intensities and how environment affects pseudothecia production in the leaf litter; 3.) to test if the resistance to black spot in the leaves and fruit in sour orange is correlated and under simple genetic control through laboratory and field testing of progeny of sour orange crosses in both Florida and Australia. Small plots were set up an Immokalee area grove with 5% urea, CaCO3, Soil set, Compost Aid, Soil Set and Compost aid, and an untreated control. Leaves were collected and treated. The samples were collected and are still being processed. To look at the effect of bagasse, the Senior Biological Scientist and the post doctoral associate undertook the controlled experiment portion of this project and then repeated it. This involves an in vitro study of the decomposition of citrus leaves and inactivation of G. citricarpa by various amendments including bagasse including the microbial consortium of fungal and bacterial strains to aid in the decomposition of bagasse and of citrus leaves. Leaves and other components were collected at time zero and at 10 day intervals. At each collection time, leaves and other components were oven dried and weighed. Soil weight, leaf weight, and then the total weight of each box were recorded. Leaf infection by G. citricarpa was verified by plating. Data collection and analysis is ongoing. To begin tests for understanding the genetic and environmental conditions influencing pseudothecia (ascospores) production in P. citricarpa, a culture-based screening method was developed. This technique utilizes double agar culture plates to detect the presence of forcibly discharged ascospores in culture. This high throughput screening technique eliminates the need for individual microscopic examination of culture plates. It will be utilized as a first stage screen in isolate pairings under controlled environmental conditions to determine specific conditions required for pseudothecia production. There are reports of significant resistance to black spot in sour orange types, most notably ‘Chinotto’. The USHRL scion breeding program has a large population of seedlings from crosses between ‘Chinotto’ and the USDA mandarin hybrid selection 1-37-12, which are now fruiting, and have been evaluated for a number of fruit characteristics. Some of these approach commercial fruit quality and it is reasonable to expect that this population may be segregating for resistance to black spot. Ten seedlings of each of 20 selected Chinotto x 1-37-12 hybrids, the two parent genotypes, and the susceptible standard ‘Valencia’ have been developed at the USHRL and are ready for trial exposed to black spot inoculum. We are currently confirming earlier preliminary results in vitro and data collection is on-going. The subcontracting process with the University of Queensland is still on-going. In the mean time, methods have been developed or optimized to collect different spore stages from leaf litter. There have been two rounds of inoculations of citrus germplasm in Bundaberg. Results are expected as the fruit ripen.
Reduction/suppression of fruit infection in citrus trees using foliar applications of soluble copper novel bactericidal formulations in comparison with film-forming copper formulations 1) Standard copper formulations, Kocide 2000, Kocide 3000, Nordox and Champ were less effective than in previous seasons for canker control on Ruby red grapefruit in a season with above average spring and summer rainfall. For example, 2013 fruit canker incidence was ‘ 35% in the copper treatments and ~80% incidence in the UTC compared with 2012 at ‘ 20% incidence in the copper treatments and ~40% in the UTC. 2) Nordox for 5 sprays followed by Magna-Bon at the 100 ppm rate was not significantly less effective than the 1.33 lb of Nordox alone at 50% of the metallic copper per season. 3) Treatments of Nordox 30/30 or Nordox at 0.44 lb combined with Magna-Bon at the 100 ppm rate both at ~50% of the metallic copper per season were significantly less effective than the 1.33 lb rate alone. 3) Magna-Bon at 100 ppm and formulations of Copper Silica Nanoparticle (CuSiNG pH 4, MV1-CuSiNG pH 4, MV2-CuSiNG pH 4) were less effective treatments except than the CuSiNG pH 4 formulated with fixed quaternary ammonium (fixed Quat). 4) The lowest canker fruit incidence achieved with MV1-CuSiNG pH 7 plus Quat was similar to that of the best fixed copper treatments at ~20% of the metallic copper of the fixed copper formulations. 4) For ‘Hamlin’ orange fruit, despite average rainfall, canker incidence on fruit was 44% lower than in the 2012 season due to the overriding effect of HLB on tree vigor and susceptibility of foliage and fruit to infection. As a result, no significant differences among copper formulations were detected in incidence of fruit canker or percentage fruit drop due to canker. Reduction/suppression of inoculum and fruit infection in grapefruit using soil and trunk applications of insecticidal and non-insecticidal SAR inducers 1) Soil drenches of Actigard and season long rotations with Admire and Platinum were effective for suppressing canker and protecting fruit on young Ruby Red grapefruit trees. The control responses were comparable to those obtained with 21-day copper sprays. Best treatment was a rotation of Platinum 75G, Admire Pro and Actigard combined with season long sprays. These findings are congruent with the recommendation to use neonicotinoid insecticides in rotation for psyllid control (Rogers, 2012) and to integrate their use with protectant sprays of copper (Graham and Myers, 2013). 2) Alternatively, SAR inducers may be applied to the trunk as sprays if there is concern with repeated soil applications leading to risk of leaching or biodegradation in soil. Use of non-insecticidal Actigard also addresses potential concerns for neonicotinoids of non-target effects on beneficial insects. 3)Although not as effective as copper, SAR inducers have substantial disease control activity against scab but little or no activity against melanose. They may be useful to maintain control of fruit fungal diseases should copper applications lapse before the end of period of fruit growth as fruit are still susceptible to late season infection. 4) For ‘Hamlin’ orange fruit, despite average rainfall, canker incidence on fruit was 44% lower than in the 2012 season due to the overriding effect of HLB on tree vigor and susceptibility of foliage and fruit to infection. As a result, no significant differences among copper formulations were detected in incidence of fruit canker or percentage fruit drop due to canker. Residual copper activity on fruit as affected by different formulations of copper products, over time after application for young fruiting trees of Ruby red grapefruit. Results have been compiled and the conclusions will be included in the next progress report
Jan 29, 2014 The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. Guignardia mangiferae sequencing is complete and the assembly is continuing. The full mating type gene has been located and the information was used to better characterize the mating gene from G. citricarpa. A manuscript is in preparation with all of the data for figures collected. Materials and methods are being written. We are confirming some of the results in one section. Protocols for paraffin section of leaf tissue to look at the early infection process has been developed. We have found penetration-peg like structures at 36 hours. A preliminary inoculation experiment was undertaken and results are in progress. Inoculated twigs are undergoing the wetting and drying cycles after refining techniques with preliminary experiments. The ergosterol HPLC analysis protocols with the the standard ergosterol are being refined for in preparation for the first experiment.
A field trial was completed that examined the spatial distribution and movement of individual males and females in a plantation of a eukcalyptus relative, Corymbia torelliana. The trial included mark and re-capture studies. Results suggest that within-field distribution of males and females is not highly aggregated and this suggests that aggregation pheromones may not play a significant role in mate location and reproductive behavior of Diaprepes. Field pulation density suggests that Corymbia is an excellent host for Diaprepes. The relative susceptibility of Corymbia and Pongamia pinnata will be examined in greenhouse trials to determine their relative susceptibility to larval and adult Diaprepes. We have continued a series of olfactometer studies to determine the response of male and female Diaprepes adults to odor sources that include synthetic pheromone [methyl (E)-3-(2-hyroxyethyl)-4-methyl-2-pentenoate], carvacrol, linalool and other compounds previously shown to be active by gas chromatograph-coupled electroantennography. We have also collected volatile compounds in the head space over female Diaprepes onto molecular sieves and using SPME fibers for analysis by gas chromatograph-coupled mass spectrometry and by GC-coupled electroantennography in an effort to detect a female-produced pheromone in addition to the male-produced pheromone noted above. We have been unable to produce data in support of the presence of a female-produced pheromone to date. Two-way olfactometer assays were conducted to determine male and female (separately) preference for odors. Statistically significant (P<0.05, G-test) were observed for female response to male frass when presented with a choice between male frass and clean air with 85% of females choosing the olfactometer arm containing male frass. This response was eliminated when male frass was combined with female frass. Male response to male frass was not significant. However, both males and females preferred either clean air of male frass when presented with a choice of those options or male frass combined with carvacrol and linalool. Further tests will examine carvacrol and linalool as potential repellents.
Late summer/fall 2013: We completed field trials addressing pheromone carrier, timing, deployment pattern, and efficacy of mating disruption and leaf damage by CLM. We continue to see exceptional trap catch disruption and longevity of disruption using solid rubber pheromone dispensers (DCEPT CLM’) supplied by ISCA Technologies Inc. Studies to determine optimal dispenser placement showed that low placement (2 ft above ground) resulted in decreased disruption in the canopy top; disruption low in the canopy was unaffected by dispenser placement. These results suggest that overall movement of the pheromone molecule in the grove is downward. Therefore, we recommend placement of dispensers in the upper third of the canopy. Results from transect surveys suggest that female citrus leafminer moths are capable of moving >1 km from citrus and that such movement is related to wind direction. The practical implications of movement of gravid females is that the benefit of mating disruption and actual reduction of leafminer population, damage and canker incidence and severity will increase with the size of the area treated. Therefore, we have proposed large scale trials to begin in 2014 at three locations in southeastern and southwestern Florida in cooperation with ISCA Technologies and CRDF that would treat a total of approximately 3,000 acres of citrus. Experimental design would allow for assessment of the effect of neighboring untreated groves as a source of gravid females on CLM infestation and canker and should provide a definitive estimate of cost/benefit ratio for this new method of control of citrus pests and provide guidance regarding the feasibility and expected benefit of an area-wide management approach for this pest complex.
We have continued a series of olfactometer studies to determine the response of male and female Diaprepes adults to odor sources that include synthetic pheromone [methyl (E)-3-(2-hyroxyethyl)-4-methyl-2-pentenoate], carvacrol, linalool and other compounds previously shown to be active by gas chromatograph-coupled electroantennography. We have also collected volatile compounds in the head space over female Diaprepes onto molecular sieves and using SPME fibers for analysis by gas chromatograph-coupled mass spectrometry and by GC-coupled electroantennography in an effort to detect a female-produced pheromone in addition to the male-produced pheromone noted above. We have been unable to produce data in support of the presence of a female-produced pheromone to date. During this period, effort has been focussed on completion of olfactometer trials; results are not complete as of this writing. A field trial was completed that examined the spatial distribution and movement of individual males and females in a plantation of a eukcalyptus relative, Corymbia torelliana. The trial included mark and re-capture studies. Results are being analyzed and will be provided in our next progress report.
We continue to document the antennal responses of male and female Asian citrus psyllid to host and nonhost plant volatiles using gas chromatograph-coupled electroantennographic detection (GC-EAD). Dose-response curves for active compounds are being constructed and will be published. Active compounds will be used to study behavioral response using several bioassays under development at this location and with collaborators. A new bioassay suggests that the reason certain accessions of Poncirus trifoliata are resistant to the Asian citrus psyllid may be the result of the absence of host recognition factors or phagostimulants in the resistant plants compared with susceptible accessions of the same species. Studies are now underway to confirm or refute this hypothesis using specially constructed cages that provide psyllids access to foliage of P. trifoliata accessions without contact with the leaves of another accession but in the presence of volatile compounds from both. Feeding bioassays have also been devised that allow psyllids to feed on young leaves (flush) without exposure to plant volatiles or cuticular compounds that may influence behavior. In this way, we hope to elucidate the chemical signals that control or influence feeding and oviposition behavior.
We are evaluating and attempting to develop optimal RNA interference constructs to target Diaphorina citri, the psyllid vector of Ca. Liberibacter asiaticus, the causal agent of citrus greening. We are using two psyllid species for our work, D. citri and Bactericera cockerelli, the latter being the potato psyllid. Using B. cockerelli offers the opportunity to use herbaceous plants and make more rapid progress that can then be applied to citrus and D. citri. So far, 7 aMIRNAs targeting the BC-ATPase gene (pAMIRA1, pAMIRA1c, pAMIRA2, pAMIRA2c, pAMIRA3, pAMIRA3c, and pAMIRA2PE) and 4 pAMIRNAs targeting the GFP gene (pAMIRG1, pAMIRG1c, pAMIRG2, and pAMIRG2c, as controls) were constructed into the geminivirus viral vector, and cloned into the binary vector pCB301 so they can be used for transient agroinfiltration assays or even plant transformation. Our amiRNA Northern blot analysis showed that in Nicotiana benthamiana plants high amounts of artificial miRNAs (amiRNA) accumulated, while in Nicotiana tabacum plants we achieved only low amiRNA accumulation, and not all clones have given expected miRNA production. The same 11 aMIRNAs were also cloned into the agroinfiltration-compatible Tobacco mosaic virus expression vector. To test if the amiRNAs can be generated by the TMV viral vector, we performed miRNA Northern blot analysis to confirm the amiRNA accumulation in plants, and we are in the process of sequencing the small RNA populations from the different plants to assess the specificity of the inducing small RNAs produced by these two different systems. We are now using agroinfiltrated N. tabacum plants for psyllid feeding experiments to assess psyllid target mRNA knockdown by RT-real time PCR. We are also now optimizing the conditions and settings for RT-real time PCR by using different numbers of psyllids for RNA extraction and different amount of primers and probes in real time PCR. We will collect more quantitative real time PCR data and analyze if the strategy works for Bactericerca cockerelli before applying to Diaphorina citri. Using different agroinfiltration and plant viral systems to deliver amiRNAs will help us to identify the optimal versions to apply to citrus in order to target Diaphorina citri, and to obtain specific RNAi inducers and minimize potential off-target effects. We also believe that plant viruses will give higher levels of RNAi inducers than may be possible by transgenic approaches, we are testing this now. We are also evaluating direct agroinfiltration for transient expression of interfering RNAs in citrus plants. In initial experiments, the amiR-TAV-pCB301 constructs (expression driven by the geminivirus) were agroinfiltrated into different citrus plants, and then leaves tested by Northern blot analysis to assess for amiRNA accumulation. The first experiments showed no accumulation of the desired amiRNA products in citrus plants, we are now assessing more A. tumefaciens strains and citrus species in attempts to optimize this strategy.
Dormant sprays are currently an effective tactic for managing the Asian citrus psyllid (ACP), Diaphorina citri. Although effective for reducing ACP populations, dormant sprays for managing ACP may not always prevent resurgence of their populations in the spring. Furthermore, inadequate knowledge of ACP overwintering habits limits our ability to improve upon existing winter management techniques. The purpose of this study aims to identify overwintering preferences of ACP populations in commercial groves and surrounding areas. We have previously identified potential overwintering sites based on management practices and surrounding host vegetation. Since the inception of this project, we have found significantly more ACP in poorly managed groves compared with groves under any other management regime. The objective of the current study is to track psyllid movement between these sites (classified as well-managed, poorly managed, and abandoned) and surrounding alternate hosts throughout the winter to test the hypothesis that ACP move to and ‘shelter’ in poorly managed groves during winter months before dispersing to more actively managed groves in the spring. We are preparing to initiate mark recapture studies in several replicate locations each containing citrus groves representing the three management regimes identified above. We have completed site selection for the study and have begun purchasing required materials, including traps, flagging tape, immunomarking reagents, and reagents for ELISA analysis of psyllid samples.
Recently we have found that pre-exposure temperature alters the vector capacity of ACP. Thus, prevaling climatic conditions appear to influence the likliehood of Las transmission. Moreover, in cool temperatures (e.g. 15’C) ACPs exhibit altered coloration indicating up-regulation of melanization genes. This response is a typical insect response to fungal pathogens. Determining the underlying mechanisms of temperature-related changes in Las transmission efficiency by ACP should allow development of methods to artificially interfere with the Las transmission process. i.The objective of the current study is to determine the impact of cool temperatures, and expression of melanization genes, on ACP vector competence. In addition, we will investigate melanization gene expression in response to entomopathogenic fungal infection at different temperatures. The expected output of these additional experiments will be 1) identification of a specific target for disrupting ACP vector competence, and 2) a better understanding of the potential for use of entomopathogenic fungi as an overwinter biocontrol agent of ACPs. Experiments to evaluate ACP gene expression in response to simultaneous challenges of cool temperature and entomopathogenic fungi have been initiated. Cold and heat-acclimated insects obtained from assays conducted in climate controlled chambers with entomopathogenic fungi treatments will be used to evaluate changes in target gene expression. We have begun purchasing the materials for these srudies, including plants (Valencia), extraction kits, and RT-PCR reagents.
Five years harvest data (2008-2013) from our replicated trial in a 32 acre commercial grove of ‘Valencia’ in southwest Florida evaluating foliar nutrition has shown yield in HLB infected trees have increased each year and juice quality has been maintained. Preliminary economic analysis is showing the treatments with the highest fruit yield is not the most productive in dollar returns. This economic analysis takes into account the cost of the nutritional products to give the net return to the grower. The common ingredients in the most cost productive treatments during the past 5 years has been micro- and macro-nutrients, some with phosphite and salicylate. Juice maturity and quality factors don’t appear to be affected by HLB in trees receiving foliar nutritional therapy sprays. We have initiated contact with two growers in the Indian River with whom we are establishing foliar nutritional trials with grapefruit. We are close to completing the sixth and last year of a replicated field experiment evaluating individual and combined effects of insecticide sprays and foliar nutrients on ACP populations, HLB incidence, fruit yield and quality. Reports from the first 4 years were published in Pest Management Sci. (wileyonlinelibrary.com) DOI 10.1002/ps.3577 and The European Journal of Plant Pathology 137(4):727-742. The third annual nutrient + SAR (systemic acquired resistance) spray went out on fall flush October 31. ACP populations of 0.16 ‘ 0.11 and 0.13 ‘ 0.07 adults per tap triggered an application that same day of CloserTM SC at 5 fl oz acres in plots designated for vector control after which numbers declined to close to zero. The first dormant spray (Imidan 70@ @ a lb/ac) whet out 19 December in ‘insecticide’ and ‘insecticide + nutritional’ plots with the second dormant spray planned for January 22 with Danitol. The Threshold study in an ‘Earlygold’ block in Hendry County was harvested in the latter half of Nov. Trees in plots receiving monthly sprays (TRT 1) showed highest yields (96.24 ‘ 2.47 kg of fruit per tree) followed by TRT 2 (0.2 ACP/tap threshold), TRT 4 (untreated) and TRT 3(0.7/tap) (85.01 ‘ 5.74, 81.68 ‘ 1.61 and 77.27 ‘ 3.41 kg/tree respectively). No treatment effects on brix, acid, ratio or lbs solids per box were observed. Yields correlated to accumulated biweekly tap results (F = 13.14; df = 2, 14; P = 0.0009; r2 = 0.69). These results stress the importance of vector control even when almost 100% of trees are HLB infected. However, 12 sprays a year has not yet proven to be cost effective. The correlation between yield losses and ACP densities found in this study is being used in the elaboration of an economic injury level model to help optimize the number of insecticide sprays during the growing season based on ACP monitoring. A paper describing treatment effects on key beneficial arthropods was recently accepted by the Bulletin of Entomological Research. We continued sampling of two HLB infected groves in southwest Florida that have been treated with extra nutrient applications, foliar and under tree. One of these groves is the experimental nutrient plots at SWREC and the other is the Orange Hammock commercial grove in Felda. Using a fluorescent dye application we further studied phloem translocation photographically and spectrophotometrically, and determined that phloem translocation was enhanced in HLB affected trees that had the additional nutrient applications. Comparisons have been made with untreated HLB affected trees and with healthy untreated trees. Trees at the Orange Hammock location translocated dye at a rate comparable to healthy untreated trees. At SWREC the trees treated with two treatments (6 and 7) showed the most consistent phloem translocation.
The goal of this project is to determine overwintering habits of Asian citrus psyllid (ACP), including determining alternative hosts, to improve dormant season control strategies for ACP. In order to examine ACP population density over winter months during 2012/13 (objective 1), we sampled 40 citrus groves under different management regimes (conventional management, intermittent management, organic and abandoned). In the previous quarter, we reported the increase of ACP in groves under intermittent management (groves defined as grower use of insecticide treatment between 1 and 5 times per year). Analysis of additional abiotic and biotic data collected from these 40 groves over the same winter months showed minimal impact on ACP populations during winter. The abundance of ACP was affected by their proximity to urban areas, but was unaffected by natural and artificial edges including roads, forests and lakes. Interestingly, analysis of the nutrient content of leaf sampled from each grove and indicated that psyllid were positively associated with trees with higher concentrations of phosphate (P) and potassium (K). We are currently repeating this study for a second year to confirm the observation that ACP abundance increases in intermittently managed groves during the winter season. In conjunction with this study, we are also assessing the movement of ACP between groves using protein marking and recapture experiments to further investigate ACP host and grove selection during the winter months. This January, we will begin the second season of sampling to determine the vertical distribution of ACP within trees during winter (objective 3). Four conventional managed fields will be surveyed. Additionally, we will also begin year two of sampling to assess the seasonality of alternative host plant use by ACP (objective 2).
Understanding the interaction between the Asian citrus psyllid (ACP) Diaphorina citri and the greening pathogen, Candidatus Liberibacter asiaticus (Las), is critical to managing the spread of this disease. Recently, we found that pre-exposure temperature alters the vector capacity of ACP. Thus, prevailing climatic conditions appear to influence the likelihood of Las transmission. Moreover, at some temperatures ACPs exhibit altered coloration indicating up-regulation of melanization genes. In order to investigate these findings further, we have completed experiments examining how temperature may effect ACP gene expression and melanization. Psyllids exposed to a range of temperature and RNA was extracted from all individuals in these experiments. We are currently conducting qRT-PCR assays to quantify regulation of a number of heat shock proteins and melanization genes expressed within insects at these temperatures. Another objective of this project is to introduce Wolbachia (Wol) successfully as vector control agent into natural ACP populations; therefore, fundamental knowledge about the biology of the ACP native Wol strain wDi is extremely important. We continue to investigate whether differences in natural dispersion, infection rates and diversity of Wol exist in Florida ACP populations. After developing a highly sensitive molecular assay (qPCR), we found significant differences in Wol infection rates between south and central Florida. To assess the reasons for those differences, we are investigating the diversity of natural Wol strains by using 6 Wol gene loci and two ACP gene loci. Our initial results suggest two different variations of wDi present in Florida populations. In the next quarter, data collection and analysis to confirm identification of psyllid wDi will be completed and will continue the ongoing investigation of interactions between Wol and CLas in ACP tissues.
Citrus leaf miner (CLM) Phyllocnistis citrella (Stainton) was established in Florida in 1993. CLM is found throughout the year but increases during the flushes period of citrus. The larvae of this pest produce mines on the young leaves between the upper and lower epidermal layers eating the parenchymatic tissue which served as foci of infection for citrus canker. The objectives of this project are: 1) Improve effectiveness of CLM management strategies with the assessment of the use of pheromone traps for monitoring CLM populations. 2. Monitor susceptibility of CLM field populations exposed to intensive versus modest insecticide use; 3) Relate CLM and canker incidence and severity and 4) Provide recommendations for management of CLM and citrus canker disease. A CLM colony never exposed to insecticides (susceptible colony) is being kept at SWFREC facilities. Specimens from this colony (larvae and adults) are used to determine the LD50 and LD80 that will be further utilized to evaluate larvae and adult tolerance to some commonly used insecticides in CLM field populations. To this end, CLM larvae and adults from this colony are exposed during 48 h to different doses (0; 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 50; 100, 300, 600 and 1000 ppm) of the following insecticides Actara 25WG, Agri-Mek 0.15EC, Danitol, Delegate WG, Dimethoate, Intrepid 2F, Micromite 80WGS and Cyazypyr. So far, a LD50 of 2.81 (CL90: 1.27-5.79) ppm and a LD80 of 619.18 (CL90: 234.12-2278.01) of Actara active ingredient (a.i.) has been obtained for larvae (n = 681; .2 = 3.814; d.f = 5; Heterogeneity = 0.76). The estimated LD50 and LD80 for Agri-Mek also on larvae were 0.097 (CL90: 0.0058-0.152) and 2.62 ppm (CL90: 1.54-5.16) respectively (n = 743; .2 = 2.03; d.f = 5; Heterogeneity = 0.41); the estimated LD50 and LD80 for Delegate on larvae were 2.59 (CL90: 0.79-6.35) and 95.61 (CL90: 33.68-553.88) ppm respectively (n = 859; .2 = 10.501; d.f = 5; Heterogeneity = 2.10); the estimated LD50 and LD80 for Dimethoate on larvae were 1.56 (CL90: 0.27-16.34) and 497.45 ppm (CL90: 35.15-1.13E+06) respectively (n = 546; .2 = 13.07; d.f = 6; Heterogeneity = 2.18); and the estimated LD50 and LD80 for Micromite on larvae were 88.32 (CL90: 32.66-191.81) and 1800.5 ppm (CL90: 643.15-18419.00) respectively (n = 714; .2 = 10.31; d.f = 5; Heterogeneity = 2.06). After testing 309 larvae with Danitol, a dose of 600 ppm only yielded a larvae mortality of 52%. Due to the high a.i. rates used and low mortality values, no higher doses will be tested with this product. LD50 and LD80 for Cyazypyr on larvae, and for Delegate, Intrepid and Micromite on adults are being currently estimated (n = 186, n = 487, n = 703, n = 381 individuals already tested respectively). The number of individuals tested for each a.i. is planned to be increased to obtain more accurate results. During the last major flushing period of the year (second half of October), CLM larvae were collected from four commercial citrus groves where different ACP and CLM insecticide management strategies are followed: Bob Paul, Tanner Road, B9, Duda and PTG. Potential resistance development to Agri-Mek in CLM field populations was tested by obtaining mortality rates using the previously estimated LD80 for this product. In Bob Paul block CLM larvae mortality rates with Agri-Mek LD80 were of 0.76 (n = 33), in Tanner Road mortality rates with Agri-Mek LD80 were of 0.76 (n = 44), in B9 mortality rates were of 0.70 (n = 80), in Duda of 0.82 (n = 44), and in PTG of 0.80 (n = 50). In all cases mortality was similar to the that observed in the laboratory susceptible colony (0.80) indicating that no resistance to this product has been developed yet in the field populations tested. For the spring major flushing periods the number of individuals tested for this insecticide will be tested be increased. In addition, other a.i. will be similarly tested.
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