To examine the general effects of double strand RNA (dsRNA) uptake on D. citri fitness and biology, we have conducting dsRNA feeding experiments on D. citri adults of a CLas-free laboratory colony. dsRNA of genes that exist (e.g., V-type proton ATPase [v-ATPase]) or do not exist in D. citri (a bacterial GFP gene) were used for this study. Briefly, primers targeting each DNA fragment were designed and synthesized with T7 promoters tagged (5 ends). PCR were carried out and purified amplicons were used for dsRNA synthesis using the MEGAscript RNAi kit (Ambion, Austin, TX). dsRNA treatments were conducted by feeding D. citri with 20% sucrose solutions supplemented with 50 ng/ l of dsRNA and 0.5% of green food coloring dye, using previously described methods (Wuriyanghan, Rosa, and Falk 2011). The insects were separated by gender and 25 to 30 insects were used for each Gender x Treatment. For the negative control, blank elution buffers were added to the 20% sucrose solution. To determine whether the insects were feeding, we included a treatment group in which the insects did not have access to any diet. All insects were kept at a growth chamber and their mortality was recorded daily. Our preliminary data showed that insects that did not receive any diet had the lowest survivorship, indicating that D. citri in the other treatments were feeding on the sucrose diets. In females, the survivorship of both the GFP-dsRNA and v-ATPase-dsRNA treatment was lower than the buffer-only group. No significant difference was detected between the survivorship of the two dsRNA treatments. In males, all treatments other than the no-diet treatment had similar survivorship. Overall, these findings suggest that male and female D. citri may respond differently to the same dsRNA treatments; whether such difference is related with their physiology or behavior deserves further investigation. The reason why GFP-dsRNA and v-ATPase-dsRNA treatment resulted in similar effect on D. citri survivorship (particularly in females) will also be studied further. In the future, we will also be testing how similar RNAi treatments may influence D. citri s immune system and ability to acquire or transmit CLas.
July 2016 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. In the large field trial, there was a greater amount of G. citricarpa DNA found in 2015 leaf litter so that while there was more G. mangiferae than G. citricarpa, it was less than 10 times. In 2014, there was no pattern in the number of leaves with Guignardia structures over time in any treatment but in 2015, the % leaves with structures increased until the third collection date and the started to decline. There was greater G. citricarpa DNA in the control whereas for G. mangiferae there was more DNA in the soilset treatment. The soilset treatment had the lowest disease incidence in 2015 (1st year trt) and 2016 (2nd year). The third year treatment was applied and will be assessed next spring. We have been working on a complete analysis of the first two years of data but this is not yet completed. The bagasse field trials confirmed the laboratory experiments that bagasse increased the leaf decomposition rate compared to nothing or urea. Greater soil moisture also accelerate leaf decomposition. The manuscript preparation is still continuing but the Co-PI had no further updates. Collection of leaf samples from the grove in Immokalee has continued biweekly. Each batch of samples contained 40 samples of 25 leaves collected below 40 trees. Leaves were examined under microscope to check for fructification of Phyllosticta spp. Leaf portions without fructification were discarded and the remainder were immersed in 0.02% tween20 to collect conidia and ascospores. Conidia and ascospores produced in leaf litter were quantified, weather data were collected from FAWN. Data collection is continuing and some of the qPCR data is being processed. In 2014, very little G. citricarpa DNA was found overall while G. mangiferea was high but, substantially more G. citricarpa DNA was detected in the 2015 collections. In general, conidia are always present but ascospores are related to the level of leaf decay. Because there was an increase in pathogen presence in 2015, we have decided to continue sampling since levels were very low in 2014. There appears to be more asexual structure formation in the spring of 2016 than 2015 and greater conidia production. In 2016, fewer ascospores were observed in the spring than in 2015 but in general the overall trends were similar. In Australia, confirmation of the ascospore and conidia production results continues. Sampling of leaf litter in two groves in the Queensland mandarin growing region was completed in April and samples are being processed. Inoculations of fruit are complete and preliminary symptoms have been confirmed on susceptible fruit but the less susceptible fruit are still incubating, including the promising accections. They repeated the fungicide work to confirm previous results. In 2015, mulch was the best treatment to reduce the amount of leaf litter under trees. The high volume fungicide applications did slightly reduce decomposition of the leaf litter but may not be significant. These results were confirmed in the 2016 trials.
Major accomplishments 1. We identified an optimal 2-component blend (1.9:1 formic:acetic acid) and an optimal 3-component blend (3.5:1.6:1 blend of formic acid:acetic acid:p-cymene) for maximal probing by ACP of a wax substrate. This could be used as a phagostimulant for attract-and-kill strategies for trapping psyllids. 2. We found that CLas-infected ACP probed significantly more often compared with healthy ACP on our phagostimulant blend. 3. This is the first report of a behaviorally active semiochemical blend that could be used for psyllid trapping strategies. 4. We determined that the presence of a thick, well-developed fibrous ring around phloem tissues of mature leaves acts as a barrier to frequent or prolonged phloem ingestion by D. citri from citrus leaves. This may have an important role in limiting or preventing CLas acquisition and/or transmission by D. citri, and could be used for identification and development of resistant citrus cultivars. 5. Our findings were reported in four peer-reviewed articles in prestigious journals including Chemical Senses, the Journal of Chemical Ecology and PLOS Computational Biology. Publications related to this project: Hall, DG, George J, Lapointe SL 2015. Further investigations on colonization of Poncirus trifoliata by the Asian citrus psyllid. Crop Protection 72:112-118. George J, Robbins PS, Alessandro RT, Stelinski LL, Lapointe SL 2016. Formic and acetic acids in degradation products of plant volatiles elicit olfactory and behavioral responses from an insect vector. Chemical Senses 41(4). doi: 10.1093/chemse/bjw005 Lapointe, S.L., J. George, and D.G. Hall. 2016. A phagostimulant blend for the Asian citrus psyllid. J. Chem. Ecol. 42(9): 941-951. DOI 10.1007/s10886-016-0745-4. Willett DS, George J, Willett NS, Stelinski LL, Lapointe SL 2016. Machine learning for characterization of insect vector feeding. PLOS Comp Biology 12(11): e1005158. Doi:10.1371/journal.pcbi.1005158 Manuscript submitted: George, G., E. Ammar, D. G. Hall, S. L. Lapointe. 2017. Sclerenchymatous ring as barrier to phloem feeding by Asian citrus psyllid: evidence from electrical penetration graph and visualization of stylet pathways. PLOS ONE (submitted 10/26/2016)
CRDF quarterly report July 2016 Electrical Penetration Graph (EPG) is a powerful tool for monitoring the cryptic feeding behaviors of piercing-sucking insects that feed on plant xylem and phloem tissues. While the insect probes, ingests, and salivates within the plant, characteristic voltage waveforms are produced that, in conjunction with histological studies, allow researchers to determine feeding patterns associated with pathogen acquisition and inoculation. We have shown that EPG can be used for detecting resistance in trifoliate citrus genotypes. Waveform annotations of feeding behavior of Asian citrus psyllids on trifoliate and citrus cultivars showed reduced phloem feeding on trifoliate accessions compared to trifoliate/citrus hybrids and citrus cultivars. Phloem feeding is required for acquisition and transmission of CLas. The observed low incidence of phloem feeding on P. trifoliata and trifoliate hybrids suggests a mechanism to explain the observed tolerance of citrus genotypes in the field, despite demonstrated susceptibility to the pathogen by graft inoculation. Poncirus trifoliata may possess physical traits that confer resistance to transmission by interfering with the vector’s ability to attain the phloem. Psyllid feeding may be hindered by physical barriers to stylet passage conferred by fibrous rings of sclerenchyma cells associated with vascular tissue in P. trifoliata. The presence of sclerenchymatous cells associated with the phloem may be a valuable trait for citrus breeding to produce scions that reduce or prevent ACP feeding and reduce CLas transmission. To study the role of the fibrous rings on ACP feeding behavior on emerging and fully expanded leaves of susceptible citrus, we recorded xylem and phloem feeding by EPG on adaxial (upper) and abaxial (lower) surface of young and old leaves. ACPs were allowed to feed on upper or lower surfaces of young and old leaves of Valencia (Citrus sinensis) for 21 hours. We monitored ACP feeding behaviors (np-non probing, c-intercellular stylet penetration, d-phloem penetration, e1-phloem salivation, e2-phloem ingestion, g-xylem ingestion). The waveforms produced from leaf tissues were manually annotated based on prior studies. Leaves fed upon during the EPG runs were sectioned and examined by flourescence microscopy to visualize the stylet paths and to correlate stylet position with EPG wavefoms. EPG waveform annotations showed that ACP feeding behavior on younger and older leaves was modified by the presence of sclerenchymatous cells associated with the phloem. Significantly longer phloem feeding was observed on upper surface of young leaves compared with lower and upper surfaces of older leaves. Although fewer phloem penetrations (d-e1-e2) were observed on younger leaf surfaces, they resulted in longer, sustained bouts of phloem feeding. This could be attributed to the reduced presence of sclerenchymatous rings in younger leaves that facilitates access to phloem compared with older leaves. Histological correlations showed that the fibrous rings are better developed in lower surface of older leaves, which may have resulted in decreased phloem feeding by ACPs. These findings have a significant role in understanding the feeding behavior of ACPs on young and older leaves of susceptible cultivars, and how it affects the acquisitions and transmission of HLB pathogen. Publications related to this project: George, J, PS Robbins, RT Alessandro, LL Stelinski, SL Lapointe. 2016. Formic and acetic acids in degradation products of plant volatiles elicit olfactory and behavioral responses from an insect vector. Chem. Senses 41 (4): 325-338. Lapointe, SL, J George, DG Hall. 2016. Phagostimulants for the Asian citrus psyllid (Hemiptera: Liviidae). J. Chem. Ecol. (accepted 6/29/16).
The objective of this research project is to investigate and develop a potential non-phytotoxic, environmentally-friendly film-forming ACP repellent solution for preventing HLB infection. In the last reporting period,OS-SG 6, 10, 11, 12 and 13 were studied for plant safety and rainfastness and these formulations were delivered to our collaborators for ACP infection trials. Preliminary results obtained from ACP infection trials on 02/14/2016 and 05/14/2016 revealed all of the formulations demonstrated lower infection rates compare to the control treatment. Among the formulations, OS-SG 10, 12 and 13 revealed minimal infection rate, while OS-SG 6 and 11 formulations displayed a moderate infection rate. In the last report period, OS-SG 15 formulation was proposed as improved version of OS-SG 12 and 13 formulations. In this report period, the formulation was optimized further to have high colloidal stability in aqueous solution, high surface coverage and moderate rain-fastness properties. The colloidal stability of the formulation was checked via measuring %Transmittance (%T) of the supernatant collected from the solution left undisturbed. The formulation revealed less than %50 transmittance up to 4 hours which was found to be comparable or better compare to commercial control -Surround WP- which is currently commercially available for growers. Safety analysis and plant leaf surface coverage of OS-SG 15 formulations were conducted using Cleopatra orange sp (common citrus variety) as a model plant. The formulations were sprayed at the application rate of 0.5 lbs/gallon (recommended rate for the commercial control) based on active content. The formulations revealed high plant leaf surface coverage at the application rate which was comparable to the commercial control. Phytotoxicity studies were conducted using a Panasonic Environmental Test Chamber (Model MLR- 352H) to control light intensity, humidity and temperature cycling to simulate summer conditions (85% RH, 32 Celsius). OS-SG 15 formulation did not cause any plant tissue damage at the applied rates, matching the commercial control. For next reporting period, the film adherence of the formulation will be tested using a rain-fastness experiment comparing the results to the commercial control and optimized version will be selected for future ACP trials.
The objective of this project is first to identify a Bacillus thuringiensis (Bt) crystal toxin with basal toxicity against Asian citrus psyllid (ACP). The toxicity of the selected toxin will then be enhanced by addition of a peptide that binds to the gut of ACP. This peptide addition to the toxin is expected to enhance both binding and toxicity against ACP. The identification of Bacillus thuringiensis strains with basal toxicity against ACP was conducted by means of a series of bioassays using trypsin-activated toxin as described in previous reports. Seven isolates showed promise with ACP mortality at 500ug/ml relative to control treatments. A single strain was selected for further analysis and individual toxins expressed by this strain were identified by LC-MS/MS analysis. Two individual toxins were shown to have toxicity to ACP in bioassays. Electron micrographs of ACP fed on the wild type ACP-active toxins confirmed damage to the midgut epithelium associated with ACP mortality. Modification of one of the selected ACP-active toxins with gut binding peptide 18 has been completed. Work is now underway to identify the optimal expression strategy for the modified toxin constructs.
Background information The objective of this project is to quantify the relative effect of copper (Cu), windbreak (Wb) and leafminer control (Lc) on the spatial and temporal progress of Asiatic citrus canker (ACC) under conducive conditions for epidemics and disease loss. The experiment is set up in a 10 ha plot planted with Valencia sweet orange grafted on Rangpur lime located in the municipality of Xambre, Paran , Brazil. The different treatments are the combination of up to three control measures (Cu, Wb, Lc) or none. The presence or absence of windbreak represents a plot. The presence or absence of copper sprays and leafminer control represents a subplot. Each subplot is composed of 112 trees. Each of the eight treatments has three replicates. Cu treated plots are being sprayed with Kocide (35% metallic copper) at 1 kg metallic copper/ha every 21 days. Lc is being performed with application of abamectin at 150 ml/ha every 21 days. Casuarina is used as a natural Wb around the plots. Disease evaluations started in December 2013 and include percentage of trees, leaves and fruits with ACC symptoms, and fruit yield. In the second season, the assessment of fruit drop was included. The present CRDF funding will cover the period of November 2015 to October 2016. Previous results – Trees and leaves: Up to February 2016 (25 after ACC epidemics begun), incidence of trees with citrus canker reached near 100%, except for trees with complete management (85%) or with the combination of copper sprays and leaf miner control (76%). Incidence of leaves increased since November 2015 and reached 5.6% for unmanaged trees and 1.1% for trees under complete management. – Fruits and yield: In the first harvest (September 2015), the incidences of symptomatic fruits from trees treated with the tree measures and none were 3.8 and 58.5%, respectively. Production of trees revealed the same trend observed for other assessments. Fruit yield of trees under complete management (40 kg/tree) was 186% higher than control trees (14 kg/tree). Latest results (June 2016 28 months of epidemics) – Trees and leaves: incidence of trees with citrus canker have not changed significantly since last report. All treatments reached near 100% incidence, except the ones with Cu+Wb, which have currently 81 to 86% of the trees with symptoms of citrus canker. Incidence of leaves continued to increase and reached 41.1% for unmanaged trees and 2.0% for trees under complete management. – Fruits and yield: As of February 2016, the incidence of fruits with citrus canker on the trees was 50.1% for the unmanaged trees and 4.5% for trees under complete management. Fruit drop per tree due to canker has increased and reached 96.1 and 18.6 for unmanaged and managed trees, respectively. All other treatments are showing intermediate results. Overall, relative treatment performances in the second season is similar to the first season. The highest disease control and reduction of fruit loss is being achieved with Cu+Wb. The second harvest is planned for August 1st 2016.
We finished the experiment in the climatic chamber where we tested Asian citrus psyllid (ACP) flight capability depending of ambient temperature and relative humidity. We determined that temperature was the major driver of ACP dispersal, with a minimum temperature for flight comprised between 16 and 18 C, whereas relative humidity did not have a significant effect on ACP dispersion. We changed the fan of our new wind tunnel in order to increase the speed of the air flow. We are now studying potential thresholds for for flight initiation. Psyllids will be tested at increasing wind speed and we will determine the proportion of ACP attempting to fly. Our objectives are to determine the optimal wind speed at which ACP prefer flying and the maximum wind speed threshold beyond which psyllids avoid flying. We also developed a pressure chamber to measure psyllid dispersion depending of controlled pressure changes. We found that psyllids responded to barometric changes rather than to different stable pressures. Psyllids were not more active at 1009 mbar as compared with 1022 mbar. However, if the barometric pressure was decreased during the experiment, ACP did not attempt flying. We are currently conducting an experiment where we investigate ACP response to citrus odor in olfactometer depending on pressure changes. Due to the fact that ACP were less prone to fly when pressure was dropping, we hypothesized that psyllid may be less responsive to citrus odor during pressure changes. We found that similarly to flight behavior, psyllid response to citrus odor was similar when pressure was stable. However, when pressure changed (either increasing, or decreasing) psyllids were less responsive to citrus odor in the olfactometer. Finally, we are preparing a field experiment, where we will look at psyllid dispersion depending on abiotic factors under field conditions. We first checked in the lab that the immunomarking method with albumin was still working. We will soon spray a block of citrus trees with egg protein. Five concentric circles of yellow sticky trap will be deployed up to 250 m from the sprayed area. Psyllids will be collected on a daily basis and, at the end of the week an ELISA will be conducted to determine if captured ACP will be positive for albumin. If they are positive, it will indicate that ACP departed from the sprayed area and moved to the sticky trap. We will correlate number of psyllids captured on sticky traps with temperature, relative humidity, barometric pressure, wind speed and wind direction.
The first objective of this study was to develop a simple and fast tool to determine insecticide resistance in Asian citrus psyllid (ACP) and implement it for field monitoring of resistance in Florida citrus groves. For this experiment, we developed a timed bottle bioassay. LC50 and LC95 estimates were determined for eight commonly used insecticides against a laboratory susceptible ACP population 24 hours after treatment. Insecticide resistance diagnostic times were determined for dimethoate (45 minutes), fenpropathrin (45 minutes), imidacloprid (45 minutes), bifenthrin (45 minutes) and flupyradifurone (60 minutes). Also, using the diagnostic time, we surveyed two central Florida groves for five major insecticides. There was no resistance detected for any of the tested insecticides. The next objective of this study was to monitor ACP populations for insecticide susceptibility in Florida. We collected ACP adults from three field populations in Polk and Orange county. The susceptibility of ACP was evaluated using carbaryl, dimethoate, fenpropathrin, spinetoram, bifenthrin, imidacloprid, aldicarb and chlorpyriphos. These insecticides represent several modes of action and are among those that are currently used to manage ACP in commercial groves in Florida. The method used was a topical application technique previously developed in our lab. At least five adult insects were treated in five replicates at the LD50, LD75 and LD95 diagnostic doses. Mortality of LD50 ranged between 36% to 56%; mortality of LD75 ranged between the 40% to 83%; mortality of LD95 ranged between 80% to 100% for both field and laboratory populations. There was no significantly resistance levels in the three field populations tested. The final, objective of this study was to develop effective insecticide rotation schedules based on the understanding of fundamental resistance mechanisms in the field. We have investigated three different rotation modules using dimethoate, adamectin, fenpropathrin, diflubenzuron and imidacloprid which have five different modes of action. There were three rotation models, one positive control and one negative control. Each treatment had four replicates. Before application we used a leaf dip bioassay to determine susceptible levels of ACP populations. We monitored ACP adults, eggs and nymphs weekly and determined when insecticide applications should be made based on a threshold of adults = 2, eggs =5 and nymphs =5 per per average sampling per sample date. We have already sprayed two insecticide applications for this study. Results indicated susceptibility levels of the field population being tested were not significantly different from our laboratory suspectable population at the onset of this experiment. This experiment remains currently in progress.
Two Zinkicide formulations (film forming plate structure or nanoparticle structure) provided better citrus canker control in a grapefruit field trial compared to industry standard copper-based products when applied at the same pounds metallic rate. The project was extended to cover a second year of field trials because the timing of funding did not align with the canker management season. The Zinkicide formulations remained effective against Xanthomonas at much lower rates than copper in culture media, so half and quarter rates were tested in the second year of the field trial. All rates of the plate forming Zinkicide and the half rate of the nanoparticle Zinkicide continued to be more effective than copper. However, the quarter rate nanoparticle Zinkicide formulation lost some efficacy and was equally effective as copper. This was surprising since the nanoparticle Zinkicide formulation was about 4 times more effective than the plate structure Zinkicide. It is hypothesized that this results form systemic movement. The entire applied rate of the plate structure Zinkicide would remain on or in the leaf keeping an effective concentration. If the nanoparticle Zinkicide moves systemically as supported by preliminary greenhouse soil drench experiments, the concentration of Zinkicide would be diluted by systemic movement. The loss of effective dose at the leaf surface suggests that the dose used in HLB trials was underestimated. In continuing projects funded by the USDA, we have adjusted our Zinkicide application rates to adjust for dilution from systemic movement in an attempt to get effective concentrations distributed throughout the plant vascular system. HLB field trials were applied at the half rate described above, which likely suffered from the same dilution problems. No significant changes in bacterial titer was observed with this low rate of Zinkicide. However, significant increases in fruit size were observed in Zinkicide treated trees, suggesting that some beneficial effects of Zinkicide on HLB were observed at these low rates. Ongoing trials funded by the USDA are using increased rates to attempt to match greenhouse efficacy observed with soil drenches on citrus canker and HLB. Without the relatively rapid canker field data, the application rate problem that limited control, but is suggestive of systemic movement, would have been overlooked. Commercialization and registration of Zinkicide is actively being pursued by Dr. Santra’s lab and an industry licensee.
Valencia harvests were completed and fruit/tree, fruit drop and leaf drop data collected. Juice analyses data is being analyzed. Fruit drop data was 22 % and 19 %, respectively, for Hamlin and Valencia trials at Babson Park. Drop % were 22 % and 20 % at Sebring and 18 % and 15 % at Ft. Meade, respectively, for Hamlins and Valencias. No differences were found between the low concentration PGR treatments and Controls at any of the sites for either healthier or more declined trees. The reduced fruit drop in treated trees after the first year did not occur in the second year. Leaf drop was heavier in September than later in the fall or winter for Hamlin trees and leaf loss was heavier for Valencias in December than later in the winter or spring. No differences were found between PGR-treated and Control trees in the amopunt of leaf drop. At each site there was no difference in the fruit per tree between the Control and PGR treatments in the second year after one year of treatments.
The low concentrations (1/4 rate) of Citrus Fix (2, 4-D) and MaxCel (Cytokinin) with or without ProGibb (GA) every 45 days to Valencia orange trees in central Florida for the 2015-16 growing season did not reduce fall leaf drop nor preharvest fruit drop of HLB infected trees. Total leaf drop per tree averaged 400 leaves for the Control trees and the treated trees had 7 to 17 % more for the 3 treatents (MaxCel + 2, 4-D, GA treatments and combined). Most of the difference was in the accumulated summer leaf drop, August count. A similar result occured with the Hamlin trial. Percentage fruit drop was near 15 % for the Control and all the treatments.. The fruit per tree in the second year after one year of treatments was plus or minus 4 % of the Control for all the treatments and not significant. Fruit quality data was collected and will be presented in the next report..
The evaluation of the up-graded on-line ‘Citrus Flowering Monitor System’ continued with extensive grower and extension use and in making advisories to growers. No adverse comments were received and the system worked very well in collecting data for grower advisories. The totally abnormal weather pattern this past fall and early winter, essentially no cold induction before January 1st, has made advising and deciding on flower bud enhancement and spray scheduling a large challenge for the Florida citrus industry. Although we only have one year of data, it looks like 5 to 10 % open flowers is reached about 21 to 27 days before full bloom. Therefore, open flowers for PFD invasion was about March 3 to 9 for the first wave. This was the same date for stopping psyllid sprays that were not bee friendly. From May 3 to 9 flowers with petals were available to sustain PFD innoculum. The first estimates of vegetative flush were 7 to 10 days earlier than the 5-10 % open flower date suggesting a one week window for flush spray coverage with more effective chemicals for psyllid control.
April 2016 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. In the large field trial, there was a greater amount of G. citricarpa DNA found in 2015 leaf litter so that while there was more G. mangiferae than G. citricarpa, it was less than 10 times. In 2014, there was no pattern in the number of leaves with Guignardia structures over time in any treatment but in 2015, the % leaves with structures increased until the third collection date and the started to decline. There was greater G. citricarpa DNA in the control whereas for G. mangiferae there was more DNA in the soilset treatment. The soilset treatment had the lowest disease incidence in 2015 (1st year trt) and 2016 (2nd year). The third year treatment was applied and will be assessed next spring. The bagasse field trials confirmed the laboratory experiments that bagasse increased the leaf decomposition rate compared to nothing or urea. Greater soil moisture also accelerate leaf decomposition. The manuscript preparation is still continuing. Collection of leaf samples from the grove in Immokalee has continued biweekly. Each batch of samples contained 40 samples of 25 leaves collected below 40 trees. Leaves were examined under microscope to check for fructification of Phyllosticta spp. Leaf portions without fructification were discarded and the remainder were immersed in 0.02% tween20 to collect conidia and ascospores. Conidia and ascospores produced in leaf litter were quantified, weather data were collected from FAWN. Data collection is continuing and some of the qPCR data is being processed. In 2014, very little G. citricarpa DNA was found overall while G. mangiferea was high but, substantially more G. citricarpa DNA was detected in the 2015 collections. In general, conidia are always present but ascospores are related to the level of leaf decay. Because there is an increase in pathogen presence in 2015, we have decided to continue sampling since levels were very low in 2014 In Australia, confirmation of the ascospore and conidia production results continues. They continue to sample leaf litter in two groves in Queensland mandarin growing region. Inoculations of fruit are underway in the field but no symptoms from this year. Confirmatory inoculations of promising germplasm accessions are incubating and will be ready soon. They are repeating the fungicide work to confirm previous results. As in 2015, mulch was the best treatment to reduce the amount of leaf litter under trees. The high volume fungicide applications did slightly reduce decomposition of the leaf litter but may not be significant. Our manuscript for the mating type work has been fully accepted and is currently available as a first look at http://apsjournals.apsnet.org/doi/pdf/10.1094/PHYTO-12-15-0338-R
April 2105 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: Manuscript preparation continues for the DMI fungicides. The spore germination experiments for Cannonball and Vanguard are near completion and the data will be analyzed soon. The assays for the SDHI fungicides have been completed and the data are being analyzed and manuscript prepared. Molecular characterization of the SDHB, C, D is underway. Objective 2: CBS was assessed by two methods. On 21 Dec 2015, 21 Jan 2016, and 18 Feb 2016, fruit on trees were assessed by holding a m^2 frame to both sides of three center trees in plots and counting the total number of fruit within. The number of symptomatic fruit with CBS lesions was counted within the same the same square meter. Percentage of fruit with CBS was calculated from the number of symptomatic fruit divided by the total number of fruit. The number of fruit on ground was assessed on 29 Jan 2016. Dropped fruit was counted under the canopy on the ground of the middle three trees within plot and scored for presence or absence of CBS lesions. The number of fruit with CBS lesions were added to the number of fruit without CBS lesions to get a total fruit drop and then the percent fruit with CBS was calculated. Objective 3: No update for this objective was given by Co-PI in charge despite requests. Objective 4: The volatiles from ClO2 in liquid or crystal form were tested to see if they inhibited mycelial growth of G. citricarpa. Both liquid ClO2 and powder ClO2 volatiles at 100 L/L inhibited mycelial growth of G. citricarpa. Liquid ClO2 inhibited mycelial growth by 73%, and powder ClO2 inhibited mycelial growth by 79%. Liquid ClO2 or powder ClO2 volatiles at 50 L/L did not inhibit mycelial growth of G. citricarpa. Another essential oil containing product, Genysis (0.4%v/v) was added to media and the effect on mycelial growth was observed. The product Genysis inhibited mycelial growth of G. citricarpa by 81% after 14 d incubation. We are making preparations for the final report.
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