Objective 1: Assays of non-bearing trees soil drenched with the chelated copper formulations Magna-Bon (MB) and Copper Quik (CQ) confirm that copper concentration in leaves of non-sprayed field trees is well above nutritional concentrations. HLB preventative treatments of MB and CQ were applied at 3 month intervals during two growing seasons at 1,2,4,5 or 10 oz/tree in 8 oz of water as soil drenches to visually asymptomatic 1 and 2 year old Valencia trees on Volkamer lemon and Swingle citrumelo, respectively. The solid plantings, located in Sebring and Avon Park, were surrounded by adult trees with 100% incidence of HLB symptoms. After two seasons, leaf samples for determining PCR status of trees in each trial were collected in December 2013 and analysis completed in early 2014. In the 4 year old block of Valencia on Swingle, PCR positive trees across all treatments ranged from approximately 40 to 80% incidence and in the 3 year old block of Valencia on Volkamer lemon ranged from 8-15% incidence. Incidence of PCR positive trees drenched with MB or CQ drenches was not significantly different from untreated trees. Soil copper treatments will be continued in the 3 year old block of Valencia on Swingle for one more season. Second year PCR results are pending from a trial at USDA Picos farm in Ft. Pierce of MB, CQ and copper or zinc phosphite drench treatments of Hamlin on Swingle trees treated since planting.
The application of low concentrations (1/4 rate) of Citrus Fix (2, 4-D) and Max-Cel (Cytokinin) with or without ProGibb (GA) very 45 days to Hamlin and Valencia tree canopies at two locations in central Florida for the 2014-15 growing season was completed in October. Trees were again sampled in late fall for phloem development at four locations in the scaffold system (root flare, trunk, small scoffolds and leaf main veins). Fruit drop and fruit per tree data were collected for Hamlin plots and similar Valencia data is being collected now. In one Hamlin test 2, 4-D + Cytokinin with or with out GA had less fruit drop than the Control trees, 11.5 and 13.1, respectively, versus 17 % drop for the Control. Root densities and canopy condition were evaluated and will be reported when the Valencia harvest is finished.
Entomopathogenic nematodes: Amending soils to increase biological control of insect pests In soil columns with moisture gradients, Steinernema diaprepesi migrated toward drier soil and Steinernema sp. toward wetter soil. Both species survived longest at water potentials to which they were attracted (reported previously). Total proteins were extracted from each species and separated by 2D-PAGE Gel electrophoresis after 48 h in wet (18% moisture) or dry (6%) sand. Proteins that were expressed differentially were identified using LC-MS-MS. We hypothesize that a membrane protein(s) senses and initiates responses to moisture levels differentially in each species. In S. diaprepesi, the expression of muscle proteins including paramyosin, actin and LET-99 required for the proper orientation of spindles after the establishment of polarity varied between wet and dry soil. Some energy enzymes, possibly helping the nematode migrate to preferred soil moisture, also varied between the two conditions. Interestingly, we found that some of these proteins in Steinernema sp. varied oppositely in the two conditions, suggesting that it uses some of the same mechanisms to respond differently than S. diaprepesi to soil moisture. Ongoing research seeks to resolve the roles of these and other proteins in pathways by which these nematodes sense and respond to soil water potential. Corporate officers of e-Nema the world’s largest producer of entomopathogenic nematodes toured Florida’s citrus industry for three days. Growers and distributors from the east coast and the central and western flatwoods met with the German EPN producers to describe the market potential for products containing Steirnernema riobrave and S. scapterisci for weevil and mole cricket control, respectively. As a result of these interactions, e-Nema is producing prototype products and attempting to scale up the process to provide a product for managing root weevils in late 2015 or early 2016. While not an objective of the current project, a product with S. riobrave will provide an important supplement to any management options resulting from our research. Catenaria sp. was isolated from a Bartow orchard, cultured and added along with Heterorhabditis indica to the fourth ‘generation’ in the food web experiment as described in the previous report submitted three months ago. Our hypothesis is that the increased complexity will result in EPN communities more congruent with naturally occurring communities. This is an ongoing study to recapitulate naturally occurring EPN communities in response to defined soil physical properties. Plant parasitic Nematodes: Characterizing a new nematode pest and the prevalence of resistance breaking populations of the citrus nematode. Harvested plots in the Fort Pierce trial. There were highly significant (P=0.01) inverse relationships between the dagger nematode population density monitored during the previous two years and both the number of boxes of fruit per plot and the fibrous root mass density. Fruit yield at the highest nematode densities were half those at the least infested plots. Although yields were 25% higher in treated than in untreated plots, the differences were not significant (P=0.26).
We maintained the new colony of Diaprepes abbreviatus, and the nematodes Heterorhabditis bacteriophora, H. floridensis and S. riobrave in anticipation of field experiments during cold events. Selected a site for trials on the UF Gainesville campus to best achieve temperatures low enough to test the proof of concept in the field. No suitable conditions occurred by December 31, trials did occur in January and February 2015 to be included in next report.
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.
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