1. Please state project objectives and what work was done this quarter to address them:
I. Near term field management
(a) Develop methods to time management actions
Working with 6 commercial citrus growers throughout central Florida, we have been developing a robust dataset to describe the seasonal phenology (life cycle) of lebbeck mealybug populations. Describing the seasonal life cycle of this pest helps determine key timepoints for management of its population in relation to tree development and/or time of year. In addition to intense sampling to describe the annual seasonal biology of lebbeck mealybug, we are also testing rudimentary pheromone traps to see if we can attract male mealybugs into traps. Reducing the number of males in a mating population can reduce offspring production by females (data shown in a previous report). We are hoping to determine if traps based on pheromones can help detect populations and their relative phenology in the future, which could enable growers to fine tune management actions. Starting in January 2021, we observed zero to very low mealybug population until late March 2021 from all citrus groves sampled in Florida. However, since onset of spring season (April 2021), we have been recording greater mealybug population densities in the citrus groves with mealybug populations peaking in late spring 2021 (June 2021), before a steady decline in population up till July 2021 (Figure 1). Immatures appear to reach a population peak in early Spring (May 2021), while crawler populations appears to reach a peak in late Spring 2021 (June 2021) (Figure 1). We also observed that mealybugs were more likely to be located around the calyx and peduncle of fruit set and developing fruits. They feed on the developing citrus fruit causing premature fruit abortion and/or fruit necrosis in 2 weeks which will result in yield loss (Figure 2).
Figure 2. Abundance of different life stages of lebbeck mealybugs inn central Florida
Figure 2: infested developing fruit over 2 week period
(b) Expand laboratory insecticide and adjuvant screening.
EPF data presented in previous report.
Adjuvant screening: Preliminary trials have been conducted with adjuvants alone to determine their lethality to lebbeck mealybug. A total of 10 adjuvants, including and 4 from Helena Agri Enterprises were mixed with DI water at label rates and sprayed until dripping on Volk lemon leaves with 2nd and 3rd instar mealybugs attached. 8 of the adjuvants resulted in significantly greater mealybug mortality over a 7-day period compared to a DI water control, and were comparable to the morality achieved by the insecticide Delegate (Spinetoram) applied at maximum label rate.
All adjuvants and Delegate were also tested against adults and larvae of Cryptolaemus montrouzieri to determine their potential impact on known predators of lebbeck mealybug. 3 of the adjuvants resulted in significantly higher C. montrouzieri larvae mortality compared to a DI water control, while none of the adjuvants resulted in higher C. montrouzieri adult mortality. The insecticide Delegate resulted in 35% mortality of C. montrouzieri larvae, and 100% mortality of C. montrouzieri adults, both significantly greater than a DI water control.
Next steps involve testing adjuvants against mealybug ovisacs and combining adjuvants with Delegate to determine if the combination of adjuvant and insecticide results in increased mealybug instar and ovisac mortality.
c. Evaluate promising materials in open grove setting
The insecticides Malathion and Assail (Acetamiprid) were tested on an ad hoc basis on heavily infested young citrus trees. Trees were part of a separate experiment and became naturally infested with lebbeck mealybug. Both Malathion and Assail were applied at maximum label rate, and the number of living mealybugs of all life stages at 3, 6, and 15 days post application were compared to the number of living mealybugs pre-application.
Both Malathion and Assail resulted in close to 100% mortality of adults and instars. However, in 35% of ovisacs, at least some eggs survived treatment and crawlers emerged in the following days. These results suggest that Malathion and Assail can be useful to knock down infestations of lebbeck mealybug, but that populations may rebound without additional treatments.
We have additional trials planned pending appropriate infestation levels at 3 locations.
(1d) Ant Management
We are testing a variety of methods to remove red imported fire ants (Solenopsis invicta) from citrus groves. Four experimental treatments were tested and compared to an untreated control: Chlorpyrifos drench followed by a Bifenthrin drench after 3 months, Clinch ant bait (Abamectin) applied every 3 months, Extinguish ant bait (S-methoprene) applied every 3 months, and spot treating ant colonies with hot water.
Fire ant abundance was assessed by counting the number of surviving colonies, and by determining presence/absence of foragers with cookie baits. Natural enemy abundance was assessed by dissecting mealybug clusters for predator larvae. Sampling for each of these methods took place every 2 weeks.
Preliminary data show that both insecticidal baits and hot water treatments significantly reduced fire ant colony abundance initially, while the chlorpyrifos drench had no effect on colony abundance. However, fire ant colony abundance rebounded in the hot water treatments after 3 months and has returned to close to the same levels as control plots. Colony abundance in both bait treatments has remained consistently low. Finally, although Chlorpyrifos had no effect on colony abundance, once bifenthrin was applied, colony abundance dropped and has continued to trend downwards, although this treatment still remains statistically similar to control plots.
Predator populations within mealybug clusters were unaffected by treatment, and numerous predators have been found in mealybug clusters across all treatment types.
Future work includes retreating hot water plots and continuing to monitor colony abundance for a total of 1 year. Ant forager identification and quantification is currently underway.
II. Long term management
a. Assessment of predator- what is currently in the system, can they be enhanced, how to implement use of predators alongside insecticide use for ACP and mealybugs
Predators currently present in the system:
Using molecular gut content analysis, we have identified a broader suite of predatory arthropods, including several species of spider, that consume lebbeck mealybug than anticipated. We are still extracting DNA from the remaining field-collected predators and anticipate having more to share with the regards to this sub area in our next report.
Predators available for purchase or present locally for potential augmentative control:
Predators who directly consume prey:
The commercially available predators Cryptolaemus montrouzieri, Orius insidiosus, Adalia bipuctata, Hippodamia convergens, and Chrysoperla carnea have been screened using no-choice assays to determine if they will feed on 2nd and 3rd instar lebbeck mealybugs, and on mealybug ovisacs. Additionally, several wild-caught and lab reared predators have also been screened, including the coccinellids Diomus austrinus, Coccinella septempunctata, and Olla v-nigrum; larva of the genus Ceraeochrysa (colloquially called trash bugs); and the earwig Euborellia annulipes.
For commercially available predators, only Cryptolaemus montrouzieri consistenty consumed both lebbeck mealybug instars and ovisacs. Chrysoperla carnea consumed larvae at comparable rates to C. montrouzieri, but only infrequently consumed ovisacs. Adalia bipuctata and Hippodamia convergens ate only a few instars, and no ovisacs. Finally, Orius insidiosus consumed virtually no instars, and no ovisacs.
For wild-caught and lab-reared predators, both Ceraeochrysa larvae and Euborellia annulipes consumed instars and ovisacs at high rates, and although not quite as consistently as C. montrouzieri. Diomus austrinus and Coccinella septempunctata consumed a few larvae, and only infrequently consumed ovisacs. Olla v-nigrum ate very few instars and no ovisacs.
From our results, only Cryptolaemus montrouzieri shows promise as a commercially available biological control agent for lebbeck mealybug, although Ceraeochrysa larvae and Euborellia annulipes may act as natural biological control in the grove. Chrysoperla carnea may be useful for controlling mealybug larvae, but not reproductive adult females.
Future work will consist of conducting choice assays with predators that did feed on lebbeck mealybug, as well as testing greater numbers of all predators to increase sample size.
Field trials:
Cryptolaemus montrouzieri were tested in the field against a lebbeck mealybug infestation on an ad hoc basis. A grower experienced a lebbeck mealybug infestation inside a Citrus Under Protective Screening (CUPS) grove and ordered Cryptolaemus montrouzieri to release to control the outbreak. Trees infested with mealybugs were marked and the infestation recorded as low or high. 10,000 C. mountrouzieri were released to cover an area of ~3,150 mature trees, ~100 of which were infested. Marked trees were reassessed 2 weeks after release.
After 2 weeks, most of the infested trees had visibly reduced mealybug abundance, and in many cases, all mealybugs had been consumed. In approximately half of infested trees, Cryptolaemus montrouzieri larvae could be seen feeding on mealybug clusters. The majority of mealybug clusters that remained undamaged either appeared to have died on their own accord or were composed of very few individuals. While preliminary, these results indicate that Cryptolaemus montrouzieri can effectively be used control infestations of lebbeck mealybug in enclosed settings.
Parasitoids:
Limited numbers of Anagyrus pseudococci, a commonly used parasitoid for commonly occurring mealybugs in greenhouses, were tested to determine if they would parasitize lebbeck mealybug adults and ovisacs. A. pseudococci adults were placed in arenas with adult lebbeck mealybugs, or ovisacs and left for 40-48 hrs. Surviving parasitoids were then transferred to adult Citrus mealybugs and given an opportunity to parasitize them, to act as a positive control. Mealybugs were checked for parasitism after 16 days. Most parasitoids died in arenas with lebbeck mealybugs, and none parasitized adults or ovisacs. However, half of those that survived went on to parasitize Citrus mealybug adults. These preliminary results suggest that A. pseudococci will not readily parasitize lebbeck mealybug, however further trials with more individuals and life stages of mealybugs are needed.
b. Determine how to implement mealybug management concurrent with other pest management programs
Ongoing population monitoring shows that, to date, there is no correlation between flush development and population development of lebbeck mealybug. We had anticipated that this pest, because it feeds in a similar manner to ACP, may have populations that were in sync with flush development, however flush phenology does not appear to be directly tied to mealybug population growth. What we do see, however, is a large increase in population concurrent with fruit set and development. Large populations have been documented developing at the calyx junction, often leading to fruit death and abortion of fruit well before physiological fruit drop. This is still an ongoing area of work; however, we have been able to confirm that fruit development is a key period to target management actions, and several growers have been successful in keeping populations of mealybugs down while protecting their fruit from heavy impacts of this pest by targeting appropriate, systemic materials during this time point. We still have several months remaining in this study, but the data generated so far have been crucial to understanding when to time management actions.
c. Determine what insecticide chemistries inhibit feeding
We are continuing to tease apart the feeding biology of lebbeck mealybug, which is the integral step to enable this sub area to be completed.
d. Develop tools to minimize spread
Killing crawlers with isopropyl alcohol
Different concentrations of isopropyl alcohol were tested to determine how lethal they are to 1st instar lebbeck mealybugs. 50%, 70%, and 90% solutions of isopropanol were sprayed onto mealybug crawlers placed on cloth swatches and compared to a DI water control. Both 1 spray at each concentration, and 2 sprays at each concentration were tested. Mortality was assessed after 5, 10, and 15 minutes. The test was repeated, this time assessing mortality at 30 min, 1hr, and 2hrs. For all concentrations of isopropanol, 1 spray resulted in significantly greater mealybug mortality compared to the control. However, several mealybug crawlers remained alive and active after a single spray for all concentrations. 2 sprays of each concentration resulted in almost 100% mortality or incapacitation at all time points.
Killing ovisacs with isopropyl alcohol
50%, and 70% solutions of isopropanol were also tested against lebbeck mealybug ovisacs to assess lethality. Single ovisacs were sprayed both 1 and 2 times with each concentration and compared to a DI water control. Mortality of the adult females was assessed at 0, 3, and 5 days, and ovisacs were held for up to 3 weeks to see if crawlers emerged.
Adult mortality was high by the end of 5 days for both concentrations and spray regimes and was significantly greater than the DI water control. However, most ovisacs remained viable, and crawlers emerged within several days for all treatments. Our results suggest that isopropanol is effective against adults but does not reliably penetrate the ovisac to kill eggs.
Next steps involve testing 90% isopropanol against ovisacs, and more thoroughly soaking ovisacs with each different concentration to determine if any amount of isopropanol will result in reliable egg mortality.
Using steam to kill adult mealybugs and ovisacs
Adult mealybugs and ovisacs may be accidentally transferred from infested groves on tools and equipment. This is a particular concern for Individual Protective Covers (IPCs), where ovisacs can easily be transferred to new young trees if IPCs are reused. Steam treatments may be an effective method of sanitizing equipment and killing both adults and ovisacs.
Using a steam cabinet on-station, we steam treated adults and ovisacs at 100, 120, and 130 degrees Fahrenheit for 5, 10, and 15 minutes to determine mortality. Previous preliminary experiments showed steam treatments at 130 degrees Fahrenheit for 15 and 30 minutes resulted in 100% mortality. We also tested ovisacs inside of a folded IPC to determine if ovisacs within bags would be more insulated from heat and moisture during steam treatments. For ovisacs in IPCs, 120 degrees for 10 and 15 minutes, 130 degrees for 5 and 10 minutes, 135 degrees for 5 minutes, and 140 degrees for 5 minutes were all tested.
Mortality was assessed at 0, 3, and 5 days after treatment, and ovisacs were held for an additional 2 weeks to see if crawlers emerged. For ovisacs and adults without an IPC, adult mealybug and ovisac mortality was not significantly different than the control at 100 degrees F for all time points. Crawlers emerged from all ovisacs at this temperature. At 120 degrees F for 5 minutes, mortality was also functionally 0%, and crawlers emerged. However, mortality rose to 100% at 120 degrees F for 10 and 15 minutes, and no crawlers survived. At 130 degrees F, mortality was 100% for all time points with no crawlers. Inside of an IPC, there were no crawlers and mortality was 100% at 130 degrees for 10 minutes, and at 140 degrees for 5 minutes. All other time points had functionally 0% mortality, and crawlers emerged.
It appears that IPCs insulate ovisacs and adults from steam treatments and require additional time or higher temperatures to sterilize adults and eggs. Currently, exposed ovisacs will be sterilized at 120 degrees for 10 minutes, and at 130 degrees for 5 minutes. Inside of an IPC, ovisacs will be sterilized at 130 degrees for 10 minutes, and 140 degrees for 5 minutes.
A final test will be conducted with ovisacs inside IPCs at 120 degrees to determine how much time is required to achieve 100% mortality at this temperature.
Future work will include sanitation procedures for large equipment and vehicles to develop recommendations for growers and scouts.
2. Please state what work is anticipated for next quarter:
Many of the updates provided in the previous sections are updates to ongoing research. These projects will continue to make progress over the next quarter.
3. Please state budget status (underspend or overspend, and why):
We are currently on track with our spending for this project.
Industry-grade multi-metal formulations containing 2 wt% metallic Cu were manufactured and supplied by our industry collaborator for further evaluation. The industry-grade Cu-Mg was compared with two different versions of lab-grade Cu-Mg, Cu-hydroxide industry standard and Cu sulfate pentahydrate at the same metallic Cu content for differences in particle size, surface charge, pH, and antimicrobial efficacy. The industry-grade formulation presented an average hydrodynamic radius of 207 nm with a broader size distribution (PDI 0.482) as compared to the lab-grade formulation which had a similar radius of 293 nm but smaller size distribution (0.174 PDI). In contrast, the surface charge of both formulations was found to be comparable, indicating that the primary particle for both formulations is stable in suspension after dilution. FTIR analysis was also conducted on the formulations to compare the interactions between particle and adjuvants. Both lab-grade and industry-grade presented almost identical spectra, which indicates that the industry-grade formulation’s components behave similar to that of the lab-grade formulation. The antimicrobial properties of both formulations were compared. The minimal inhibitory concentrations (of Cu) for both materials were the same when assessed on Xanthomonas alfalfae and Pseudomonas syringae at 32 ppm Cu and 64 ppm Cu, respectively. While Clavibacter michinganesis’ minimum inhibitory concentration for the industrial-grade formulation was one stage higher than that of the laboratory synthesized formulation. Both formulations outperformed copper sulfate and the Cu-hydroxide industry standard for all pathogens tested. A more quantitative assessment of the bactericidal potency of these materials will be performed and reported. Additionally, leaf residue of the formulations was imaged through scanning electron microscopy. From our overall assessments, it was observed that both the industry and laboratory synthesized multi-metal formulations were of similar particle size, chemical characteristic, and antimicrobial potency. Preliminary field trial of the industry-grade multi-metal formulation is underway from which efficacy and metal residue data will be reported in the near future. Additionally, we will be reporting on tissue sample characterization including sap to understand the systemic movement of the A.I. 2021 harvest data is tentatively scheduled to be collected for the week of Nov 15th.
Spring nematicide treatment effects on sting nematode and plant root mass in the nematicide comparison plots were evaluated from the 5th set of soil samples (June 2021) taken during the course of this trial. Compared to control plots, the oxamyl treatment reduced sting nematode populations by 73% (P=0.004). The Salibro, aldicarb, Velum Prime, Nimitz and Syngenta products had 61, 51, 44, 22, and 7 percent fewer nematodes, respectively, than in control plots. To date the overall percentage reduction in sting nematode population density by the products compared to untreated plots has been oxamyl (55%), Syngenta product (49%), Nimitz (42%), Salibro (41%), Velum (20%), and aldicarb (1%). The root mass density from treated plots did not differ significantly from untreated plots. Oxamyl had just 12% more roots than controls, which was the highest level for nematicide treatments, but well below its effect on roots the previous June and December when oxamyl increased root density (P=0.01) by as much as 47%. Tree heights in these plots were unaffected by any treatment, nor did they differ in a second trial measuring the effect of aldicarb on tree size.The perenial peanut plots performed during the monthly spring assessments as previously reported. The reduction of sting nematodes in peanut compared to native vegetation continued to be highly significant. Seasonality of sting nematode numbers was evident in these middles as well as in the tree rows of this trial and that of the nematicide comparisons – much higher population density in the winter samples both years compared to those taken in summer. Neither the peanut treatment or oxmyl treatment had any measurable effect on the low numbers of sting nematodes or the roots mass density in the tree rows.Chemical treatments will be applied in late September or early October for the final time. The trial will be completed when a third year of fruit on these young trees are harvested in Spring 2022 and an economic analysis of the profitability to date of the treatments is performed.
September 2021Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp. A skirting and fungicide timing trial was conducted during two different years (2019-2020 and 2020-2021) in a commercial citrus grove located in Southwest Florida. This study aimed to evaluate the optimal timing for fungicide application and investigate if skirting pruning helps reducing citrus black spot (CBS) incidence and severity under field conditions. It is a split-plot design with skirting as the main plot and fungicide treatments as the minor plot. Our preliminary results showed no statistical differences in disease incidence among the application timing treatments in both seasons. However, disease incidence by tree was much higher in 2020-2021 compared to the 2019-2020 seasons likely due to favorable environmental conditions. In the same study, evaluation of disease severity relied on the area fruit affected by CBS symptoms. There was no statistical difference between the fungicide treatments nested within skirting and non-skirting plots during the 2019-2020 season when the severity was evaluated by tree. However, in the 2020-2021 season, a statistical difference was found between fungicide treatments and untreated control when the disease pressure was substantially higher. In the non-skirted trees, only the Florida standard (FLS) treatment was statistically different from our untreated control. The Early and Late application treatments showed no statistical difference when compared to the untreated control or FLS. Within all skirted plots, fungicide timing treatments showed statistical differences compared to the untreated control, but not among them. Therefore, our results provide evidence for the best timing for the fungicide applications on the disease severity and confirms that tree skirting has a minor effect on disease control when we evaluate the data by tree. We are working on a new analysis to see if there is a significant difference among the treatments when we evaluate the incidence and disease data by fruit rather than tree. There may be a difference when we change the scale of how we analyze the data. We are also examining the weather data from both seasons to see what factors rainfall and temperature may have played. In April and May, we prepared the fungicide trial. Trees were selected for chemical application based on disease severity levels, confirming presence and quality of selected trees in the field, and labeling selected trees along with warning signs in the area. Fifteen treatments were selected. Each treatment has five replicates of three trees and were blocked according to disease severity according to randomized block design. Beginning from late May to currently, there has been a monthly (every four weeks) fungicide application the chemical treatments, and there will be a total of six applications of each treatment, from May to October. The September treatment will be applied next week. We will not be able to collect the data from this trial until the end of March, 2022 when the fruit symptoms will appear consistently. This trial was initially planned for 2020 but because of the timing of the shutdown of field activities by the University of Florida, we were unable to conduct the trial. Our goal is to be able recommend further fungicides or alternate products for improved CBS management. Evaluation of the second season of trials, in which different fungicide products were tested for their efficacy to protect citrus fruit from CBS infection, was recently concluded. Products tested include Enable (Indar) and Luna Experience sprayed on their own as well as Copper hydroxide sprayed in alternation with either Amistar Top or Headline (Cabrio). Fungicides were applied every 4 weeks from October 2020 until March 2021, in a Valencia orange orchard with a history of CBS. Data analysis is currently under way. An additional 8 South African and 8 global (from Argentina and Swaziland) isolates have been sequenced. As the next-generation sequencing data becomes available, the analysis and results are continuously updated to include the new data. DNA from 16 isolates (Argentina, Australia, Brazil and China) passed QC and is in the process of being sequenced. More isolates from Brazil and China are being cultured for DNA extraction and sequencing. Sixty-five P. citricarpa isolates (36 from South Africa, representing the five provinces where CBS is found, and 29 from other countries, Argentina, Australia, Brazil, China, Eswatini and USA) have been sequenced. Sequence data for 6 isolates, 1 from China, 1 from Brazil and 4 from USA are in the process of being generated. The sequencing data for the 65 isolates were analysed in the same manner as previously described, namely read mapping and variant calling, and in silico genotyping. Both analyses approaches showed the same patterns in terms of genetic distribution of isolates. Isolates from China are the most genetically distinct, while there are different degrees of genetic connectivity between isolates from Argentina, Australia, Brazil, Eswatini, USA and those form South Africa, and corresponds with previously published results (Carstens et al., 2017). Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.). Our studies on the diversity of Phyllosticta spp. associated with citrus in Florida have continued. Our collection of P. citricarpa isolates were obtained from citrus fruit in different areas under quarantine from 2010 to 2020. These isolates were previously screened by morphology to remove nonpathogenic P. capitalensis isolates. We have now screened the majority of the remaining 202 isolates using molecular techniques to determine if we have cryptic species that may have been misidentified as P. citricarpa. To date we have screened 168 isolates by amplifying and DNA sequencing the tef-1a (Translation elongation factor-alpha) locus. This screen has revealed that two isolates (Gc-6 and Gc-7) match to a Phyllosticta species not previously reported in association with citrus. The best sequence match based on ITS and tef-1a is to a species reported previously as a pathogen on a member of the Amaryllidaceae family (Hymenocallis littoralis) in Australia. Numerous inoculations of citrus have determined that these isolates do not cause citrus black spot. Multilocus analysis and leaf inoculations on Hymenocallis species have been conducted. These studies indicate that these isolates are P. hymenocallidicola and infection studies indicate that they are pathogenic on spider lilly (Hymenocallis sp.). Vegetative and sexual compatibility assays (sandwich mating) were repeated between the two isolates. The results confirm that Gc6 and Gc-7 isolates are heterothallic and belong to the same mating type, as neither isolate was a capable of producing pseudothecia in solo cultures or in paired cultures with one another. Characterization of the tef-1a sequence from the remaining isolates in our collection screening and further characterization of these new species is continuing to obtain robust information on the diversity of Phyllosticta species and determine the presence of cryptic species in Florida.We screened an additional 12 P. citricarpa isolates for mating-type (MAT1-1 and MAT1-2). Only the MAT1-2-1 mating type has been detected in new isolates collected in 2020. We conclude that the MAT1-1 mating type is still absent in the Floridian population. To determine the phenology of fruit susceptibility inoculation studies of citrus fruit (Meyer lemon) were performed in a quarantine greenhouse at the Florida Department of Agriculture and Consumer Services (DPI) in Gainesville. A total of 97 fruit were used in this experiment. Of these 97 fruits, 25 served as controls and 72 were inoculated with the Gc-12 isolate of P. citricarpa. Disease assessments were performed weekly for a full calendar year. A total of 50 fruit produced symptoms in this period. All 50 were from fruit inoculated with the Gc-12 isolate, and no symptoms were observed in the control treatment. Therefore, from all inoculated citrus fruit, 69.4% produced symptoms, and 30.5% remained asymptomatic. The indications from this one experiment suggest that citrus fruit are susceptible at all stages of their development regardless of their maturation time. A second trial was planned for 2021 to determine the period of fruit susceptibility to P. citricarpa but the trees only recently flowered and the fruitlets are not yet ready for inoculation.
Vismax technology contains a peptide that directly activates the plant’s immune system, providing broad-spectrum prevention and suppression of a wide range of fungal and bacterial diseases, including Citrus Greening/Huanglongbing (HLB). The objective of the 2-year project is to determine whether Vismax treatment promotes resistance to other major citrus diseases, specifically citrus canker and phytophthora root rot in greenhouse assays in collaboration with Dr. Evan Johnson (University of Florida, IFAS CREC). In Year 1(Q2), Canker trial #2 was completed. The trial included foliar and soil application methods for Vismax, 7 days prior to inoculation of sweet orange with X. citri. Rate testing of foliar applications (1X, 2X, and 4X concentration) was performed. Trial was successful with controls performing as expected, and results were consistent with Canker Trial #1. Vismax reduced canker symptoms at all rates tested, with statistical significance (p-value <0.05) for the 2X and 4X foliar applications, and soil drench application. Raw data and report were received, and results were shared with key stakeholders (CRDF Board of Directors in August 2021, Growers partnering in field trials). Within the next quarter, the initial Phytophthora trials will be completed, with generation of reports. The trials utilize both traditional and Rhizotron imaging, and include foliar and soil applications of Vismax, as well as a Ridomil Gold (mefenoxam) control. In addition, Canker Trial #3 will be established on sweet orange, with expanded soil drench applications to include rate testing. Future trials will test application timing/length of protective effect of Vismax applications against canker.
This project will end at the end of October, 2021,so this is the second to last report. There are two objectives in this
project, and the progress of each objective is listed here:Objective 1. Evaluation of blended juice using released
HLB-tolerant sweet orange/mandarin cultivars via analyses of sensory and consumer acceptance. In this quarter, we did
the sensory and consumer study for Valencia blended with Sugar Belle at the end of May. In this study, 100% Sugar
Belle® juice, 100% Valencia juice, 90% Valencia and 10% Sugar Belle® blended juice, 50% Valencia and 50% Sugar
Belle® blended juice, and the same 100% commercial NFC orange juice product were prepared and consumed by 61
sensory panelists. Panelists were pre-screened based on their orange juice consumption frequency to ensure they are
familiar with orange juice. Compared to 100% commercial NFC orange juice, 50% Valencia and 50% Sugar Belle®
blended juice was rated significantly higher in overall appearance, overall liking and flavor liking. In the subsequent
evaluation on sensory attributes (i.e. sweetness, bitterness and sourness etc.) that are closely related to fruit quality, 50/50
and 90/10 blended juices received higher ratings on sweetness, lower ratings on sourness and bitterness, which indicated
Valencia orange juice blended with Sugar Belle® increased the consumer preference. In addition, from the sensory
aspect, 90/10 Valencia/Sugar Belle® blended juice were preferred the most by consumers. Surprisingly, when panelists
were asked their general opinion on their willingness-to-pay of 100% orange juice and mandarin and orange blended juice
without tasting any specific samples, it was observed that they expected to pay higher price for juice containing higher
ratio of orange juice. For example, the 100% orange juice receiving the most votes on $3.49 per 52 FL oz. More
information will be collected in the next quarter to explain the mismatch between willingness-to-pay expectation for known
ratio of orange juice in blended juices and the results obtained from the real tasting environment. Even though the
acceptance of tangerine juice is currently limited, it was noteworthy that the Valencia orange juice and Sugar Belle® juice
blends had a better performance in sensory evaluation than that of pure orange juice. In addition, there were considerable
number of consumers holding neutral opinion on juice quality and willingness-to-pay of blended orange and mandarin juice
indicating the potential improvement on acceptance and consumption of mandarin juice in the future. We wrote an article
for the Citrus Industry and this article will be published in September based on our findings from this quarter and the
previous quarters. Objective 2. Identify more tolerant cultivars resembling the quality of Valencia for the juice market, and
identify a chemistry definition of consumer accepted orange flavor. All the analytical work has been completed in the last
quarter, so in this quarter we focused on data analysis and writing a manuscript. We investigated citrus fruit flavor and
identify key flavor compounds in a diverse group of citrus fruits through multivariate statistical analysis. Volatile and
non-volatile chemicals were determined by MS spectrometric techniques while the rating of sensory attributes was
collected from a trained sensory panel. The results of Pearson Correlation showed the extent of positive/negative
correlation between the sweet attribute and other sensory attributes in different groups of citrus fruits. Based on the
correlation between chemicals and sensory attributes, sugars were highly correlated with the sweet attribute while
flavonoids and limonoids were highly correlated with the bitter attribute. Organic acids showed a strong correlation with the
sour attribute and demonstrated contribution to the citrusy attribute. Terpenes were highly associated with the terpene-like
attribute while octanal, decanal, ethyl hexanoate and ethyl octanoate were demonstrated to be highly correlated with the
citrusy attribute. The correlations between linalool, citronellol, and 1-octanol and the fruity/floral attribute were also
observed. In addition, pathway enrichment analysis demonstrated a close correlation between the biosynthesis of
terpenoids and steroids pathway and the identified key flavor compounds in sweet orange-like mandarin hybrids.
The purpose if this project is to reveal the mechanisms of bactericide uptake and transport in citrus plant and establish a theoretical basis for developing technologies to improve the efficacy of bactericides, which is helpful to provide potential solution to the development of effective chemotherapeutic tools for HLB management. Three application methods, foliar spraying, tree trunk injection and root administration, for bactericides (oxytetracycline and streptomycin) currently suggested to be used in citrus HLB management have been employed in this study with greenhouse and field tests. The data obtained in the test will be used to: 1) compare the delivery efficacy of the bactericides applied by the three application methods into the citrus plants; 2) to compare the distributions and concentrations of the bactericides in different citrus tissues and; 3) to compare the effect of citrus variety and age on the delivery of bactericides applied by the three methods and up taken concentrations of bactericides by the plants. This project officially started on December 1 2018. This is the 9th quarterly progress report covering March 1,to May 31, 2021 During this period, we have conducted and/or completed the following work/research tasks: 1) Completing the analyses of the extracted samples 2) Processing of the data from the field trials.3) Write the final report for this project.4) Preparation of manuscripts from the data obtained from the greenhouse and field experiments..
June 2021Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp. We collected data from the large spray timing and skirting trial in March. We evaluated 50 fruit each for disease severity on approximately 125 trees in 32 rows. We made significant progress on the incidence analysis of the data and are close to finalizing the analysis. We found that fungicide program significantly reduced the black spot incidence compared to the control. The greatest reduction was from the Florida standard timing with applications from May to September. Skirting had no significant effect on the disease incidence but the interaction between fungicide timing and skirting did have a significant effect. The Florida standard timing with skirting was the best performing treatment. For the late fungicide timing (May to October), there was a slight decrease in incidence with the addition of skirting. It should be noted that the 2019-2020 season was light for black spot incidence and severity. The minor plots were re-randomized within the main plots and we were able to get the trial re-flagged just in time for the early spray (delayed by 2 weeks but no rain occurred from the first of April until after the early application). All applications were made on time in the spray trial. Data was collected from the trial and has been entered into the computer (~188,000 entries) and is being verified for accuracy. As noted previously, there was far more disease this year than 2020. We are examining the weather data from both seasons to see what factors rainfall and temperature may have played. Pre-treatment data were collected at the end of March for the CBS spray trial. We have 15 treatments included in the trial. One application has been completed of a planned 6 applications with the 2nd scheduled for this week. The second season of trials in which different fungicide products are tested for their efficacy to protect citrus fruit from CBS infection, is currently underway in South Africa. Products being evaluated include Enable (Indar) and Luna Experience sprayed on their own as well as Copper hydroxide sprayed in alternation with either Amistar Top or Headline (Cabrio). Fungicides are applied every 4 weeks from October 2020 until March 2021. The fungicides are being tested in a ‘Valencia’ orange orchard with a history of CBS. The trials will be evaluated at the end of August 2021. An additional 8 South African and 8 global (from Argentina and Swaziland) isolates have been sequenced. As the next-generation sequencing data becomes available, the analysis and results are continuously updated to include the new data. DNA from 16 isolates (Argentina, Australia, Brazil and China) passed QC and is in the process of being sequenced. More isolates from Brazil and China are being cultured for DNA extraction and sequencing. Sixty-five P. citricarpa isolates (36 from South Africa, representing the five provinces where CBS is found, and 29 from other countries, Argentina, Australia, Brazil, China, Eswatini and USA) have been sequenced. Sequence data for 6 isolates, 1 from China, 1 from Brazil and 4 from USA are in the process of being generated. The sequencing data for the 65 isolates were analysed in the same manner as previously described, namely read mapping and variant calling, and in silico genotyping. Both analyses approaches showed the same patterns in terms of genetic distribution of isolates. Isolates from China are the most genetically distinct, while there is different degrees of genetic connectivity between isolates from Argentina, Australia, Brazil, Eswatini, USA and those form South Africa, and corresponds with previously published results (Carstens et al., 2017). Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.). Studies to determine what Phyllosticta spp. associated with citrus are present in Florida are on going. From 125 screened isolates using DNA sequencing of the translation elongation factor-alpha (tef-1a) locus, three isolates grew distinctly from species previously reported in Florida. Within the three new isolates identified, only two were screened using the internal transcribed spacer (ITS) and the actin (ACT). The result demonstrated that both isolates (Gc-6 and Gc-7) are from distant lineage from. P. citricarpa. and. P. capitalensis.a nd grouped with. P. hymenocallidicola, a. Phyllosticta. species not previously associated with citrus. An assay was conducted on citrus fruit (Valencia and Meyer lemon) using isolates Gc-6, Gc-7, Gm33 (P. capitalensis), Gc-12 (P. citricarpa), and water (control) to determine if both isolates are pathogenic to citrus or not. The result showed that only isolate Gc-12 produces symptoms on citrus fruits, demonstrating that. P. citricarpa. appears to be the only plant pathogenic species associated with citrus in Florida and isolates Gc-6 and Gc-7 are non-pathogenic species in citrus. Moreover, a pathogenetic test was performed in. Hymenocallis littoralis. leaves.because .P. hymenocallidicola. was originally described from this host. Leaves were inoculated using two methods (fungal plug and spore suspension), and similar symptoms (brown to reddish spots) were observed as in the only documented report. Koch’s postulates for this disease had never been done. The screening and further characterization of these new species is continuing to obtain robust information on the diversity of Phyllosticta species and determine the presence of cryptic species in Florida.Samples (fruit, twigs, leaves) were collected in the spring of 2021 from groves for mating-type screening using the conventional PCR primers described in Wang et al. (2016) to determine if previously undetected MAT1-1 idiomorph is present or not. Isolates are being purified so the study is ongoing to determine if MAT1-1 is still absent in the Floridian population or if it has entered Florida and the pathogen is reproducing sexually. Thus far, no isolate has been identified with MAT1-1.To determine the phenology of fruit susceptibility inoculation studies of citrus fruit (Meyer lemon) were performed in a quarantine greenhouse at the Florida Department of Agriculture and Consumer Services (DPI) in Gainesville. A total of 97 fruit were used in this experiment. Of these 97 fruits, 25 served as controls and 72 were inoculated with the Gc-12 isolate of P. citricarpa. Disease assessments were performed weekly for a full calendar year. A total of 50 fruit produced symptoms in this period. All 50 were from fruit inoculated with the Gc-12 isolate, and no symptoms were observed in the control treatment. Therefore, from all inoculated citrus fruit, 69.4% produced symptoms, and 30.5% remained asymptomatic. The indications from this one experiment suggest that citrus fruit are susceptible at all stages of their development regardless of their maturation time. A second trial will be conducted in 2021 to determine the period of fruit susceptibility to P. citricarpa.
The effect of five multi-metal bactericides containing Cu as minor component, Cu-Mg (MM25C75M), Cu-Zn (MM25C75Z), Cu-ZnO (MM20C80Z), Cu-ZnS (MM17C83Z), Cu-Mg-Zn (MM17C17Mg66Zn) and four respective controls without Cu (i.e. coated Mg, Zn, ZnO, ZnS) was evaluated on seedlings to determine metallic uptake, movement and potential change in plant tissues. The rate of foliar application was 1600ppm for Zn and Mg and 400 ppm for Cu. Metal content in seedlings were determined 14hrs after treatment. Residues on the leaves were imaged using scanning electron microscopy. Seedlings were thoroughly washed following a UF-IFAS recommended published protocol and elemental analysis of tissue samples (leaf, root and stem) was performed. Preliminary results showed that Zn and Cu moved systemically to the root system 14 hrs post foliar application. It appears that the treatment moved through phloem vascular system. We have noticed that there is a difference in uptake and mobility of ZnO and ZnS particles within the plant vascular system. Further investigation is needed to understand the rate of uptake and metal movement. We could not reliably differentiate the movement of Mg due to the presence of large amount of Mg in untreated controls (background). Analysis of the infrared spectroscopic signature of the tissues suggested a slight change in composition for some of the formulations, but further investigation is needed to confirm. To determine the application frequency, we have initiated time-based studies. However, we experienced delay in data collection due to seedling unavailability from the CREC greenhouse facility. Additional characterization of all the above industry-grade formulations are being carried out including DLS, Zeta potential, infrared spectroscopy and nanoscale imaging. We have identified certain characterization limitations with the agri-grade material using these tools. We will report our finding in the next reporting period. Our industry collaborator on this project has provided a batch of agriculture-grade NEW Cu product containing 2% Cu for potential registration, which is being investigated within the scope of this project.
1. Please state project objectives and what work was done this quarter to address them: Objective: Determine whether Vismax treatment promotes resistance to other major citrus diseases, specifically citrus canker and phytophthora root rot in greenhouse assays with Dr. Evan Johnson Progress (Year1 Q1; 3/4 – 6/11/2021)a. Canker trial #1: Status = Complete. Trial included foliar and soil application methods for Vismax, 7 days prior to inoculation of sweet orange with X. citri. Rate testing of foliar applications (1X, 2X, and 4X concentration) was performed. Trial was successful with controls performing as expected. Raw data and preliminary report received.b. Phytophthora trial #1 (traditional method; potted trees): Status = in progress, propagule counts. Trial includes foliar and soil applications of Vismax, and Ridomil Gold control applications.c. Phytophthora trial #2 (traditional method; Rhizotron): Status = in progress, imaged weekly for root growth. Trial includes foliar and soil applications of Vismax, and Ridomil Gold control applications.d. Trials were visited by EE personnel in May 2021 (no cost to grant)2. Please state what work is anticipated for next quarter:a. Completion of Phytophthora trials #1 and #2, including data anlaysisb. Second canker trials to be repeated to confirm the results of Canker trial #1c. Disease-free seedlings on order for repeat of Phytophthora trials. 3. Please state budget status (underspend or overspend, and why):Status = on budget ($14,462 of $30,600 budgeted in Year 1 – March 1, 2021- Feb 28, 2022) $14,462 paid to University of Florida on 04/05/2021 per University of Florida – Elemental Enzymes Ag & Turf LLC agreement to initiate trials
April 2021The objectives for this proposal are 1) Conduct field trials of new products and fungicide programs for PFD management as well as validation trials for the Citrus Advisory System (CAS); 2) Investigate the reasons for the movement of Postbloom fruit drop (PFD) to new areas and recent major outbreaks; 3) Evaluate methods for initial inoculum reduction on leaves so that early fungicide applications could be more effective and identify the constituents of the flower extracts using omics techniques. The two validation trials for CAS have been laid out in the Fort Mead area and a small amount of disease has been observed on early bloom in at least one site. We hope for at least one positive site. There were 3 weekly applications made and 2 applications according to the PFD-FAD. The fungicide trial had one application made on May 8th after a rain period. There were no triggers for disease this year at the site and this was the most likely infection period. Fruit data should be recorded in June or early July. A manuscript is in preparation for all the years of CAS validation. The majority of analysis for the leaf wetness models is now completed and manuscript preparation is continuing. We compared the output of leaf wetness sensors to combinations of predictive models for accuracy and sensitivity. As for all models, reliability was heavily influenced by the quality of the weather station data. Reasonable accuracy was found for one, three, or four models in combination but with just two models, the predictive capacity was poor. We are in discussions for how this information could be used to improve CAS predictions. We are working on evaluating PFD risks via an analysis for prediction accuracy. So far, we have tested floral extracts of pinhead, popcorn, and open citrus flowers. All floral extracts stimulated Colletotrichum abscissum conidial germination, and stimuli were compared among these treatments. We analyzed the sugar composition of the floral extracts. Then, we prepared sugar solutions with the same sugar concentration and composition to that of the floral extracts. The germination stimulus of the sugar solutions was higher than the water control, but less than the floral extracts. We tested the stimulus posed by leaf extracts prepared with new and old leaves on the pathogen. Leaf extracts were as good as floral extracts in stimulating the pathogen. As leaves are easier to obtain and available year-round, we decided to investigate the leaf extracts further, to pinpoint what portion of it was responsible for the stimulus considering polarity. We found that the highly polar portion of the leaf extract was responsible for the stimulus. An untargeted metabolomic analysis revealed that there are thirteen main components of the high-polarity portion of the leaf extract. These components will be tested soon.We have been in conversation with the USDA and found out that they have only just repaired their wind tunnel and had slightly more access to the facilities. In the mean time, as we were unsure how soon we could use the equipment, we decided to build a similar tunnel at CREC. We have done a complete set of experiments with the new tunnel with inoculated leaves and flowers with and without rain. We are analyzing the data and adding it to what we observed with the USDA wind tunnel. The manuscript is in preparation and we may do a few follow up experiments as questions arise during the analysis
1. Please state project objectives and what work was done this quarter to address them: This report is for the continued support of the Southern Gardens Citrus Diagnostic Laboratory that provides testing for citrus greening for researchers, growers, and homeowners. The current report is for the 3rd quarter of year 2 of a 2-year project. For the 3rd quarter of year 2, a total of 10,230 samples were processed and assayed. This brings the total for the project-to-date to 48,550 samples assayed, or 3,550 over the budgeted amount for the whole project (8% over). The projection for the full 2-year period is for the lab to have processed ~23% more than the budgeted amount. There is a growing trend for sample submitters to request copy number determination instead of just a Ct value (52 % of the samples were provided with copy number for this quarter, 59% the previous quarter). This is likely due to the fact that the majority of the samples being submitted are coming from researcher trials where quantitative data are desired. All of the samples for the current period were plant samples. No ACP samples were submitted. 2. Please state what work is anticipated for next quarter: It is anticipated that a higher than budgeted amount of samples will continue to be submitted. This is based on an increase in the number of trials being established that require extensive sampling and to several researchers bringing in backlogged samples. 3. Please state budget status (underspend or overspend, and why): Given that we are running 8% above the budgeted amounts for the whole project (123% over if prorated for 7 quarters), it is likely that we will be over the budgeted number of samples for the project. As has been done in the past, we will wait until the end of year 2 of the project and adjust the final invoice either up or down depending on the total number of samples run during the project. If the number of samples exceeds the budgeted amount, the final invoice will be Increased upwards to cover the cost of the consumables. If the number of samples is below the budgeted amount, the final invoice will be reduced to reflect the reduced amount of consumables used.
Work done this quarter:(1b) Adjuvant screeningPreliminary trials have been conducted with adjuvants alone to determine their lethality to lebbeck mealybug. A total of 9 adjuvants, including and 4 from Helena Agri Enterprises were mixed with DI water at label rates and sprayed until dripping on Volk lemon leaves with mealybugs attached. 8 of the adjuvants resulted in significantly greater mealybug mortality over a 7-day period compared to a DI water control. (1c) Evaluate promising materials in open grove setting: We began a field trial at a 10-acre commercial citrus planting that was heavily infested in 2020. First insecticide applications occurred as a pre-bloom prophylactic spray of a systemic material, with a second spray planned in the end of April.(1d) Ant Management · We are testing a variety of methods to remove red imported fire ants (Solenopsis invicta) from citrus groves. Removing the ants can allow natural enemies to prey on and parasitize lebbeck mealybug without interference, and provide more effective biological control. Our research goals are:1. Reduce abundance of fire ants associated with lebbeck mealybug (Nipaecoccus viridis) in central FL citrus groves. · 2. Determine long-term efficacy of different treatment types at reducing fire ant abundance · 3. Assess time and cost of different treatment types · 4. Determine effects of ant treatment on predators abundance within trees and within N. viridis clusters. We are collaborating with the King Lab from the University of Central Florida, and are conducting the experiment in the grove of a local citrus grower. Four experimental treatments will be tested and compared to an untreated control: Chlorpyrifos drench, Clinch ant bait (Abamectin), Extinguish ant bait (S-methoprene), and spot treating ant colonies with hot water. Fire ant abundance will be assessed by counting the number of surviving colonies, and by determining presence/absence of foragers with pecan sandie baits. Natural enemy abundance will be assessed by dissecting mealybug clusters for predator larvae. Sampling for each of these methods will take place every 2 weeks for a total of 3 months post-treatments. Currently, all treatments have been applied and we are conducting follow-up surveys to determine their efficacy. (1e) Evaluate management options for IPCsWe recently completed the study to evaluate several commercially available entomopathogenic fungi (EPF) products as a potential tool to control lebbeck mealybug infestation on young citrus trees within individual protective covers (IPCs). Findings of the study indicate that EPF can cause death of mealybugs and EPF conidia are viable on citrus trees within IPCs up to 42 days after treatment. Thus, EPF can offer control of mealybug for up to 1.5 months after treatment. In February, we began infesting trees in our research planting to run a similar comparison of topical insecticides for clean-up, with pretreatment drenches planned prior to the next set of trees to be infested. Additionally, a controlled greenhouse comparison of drench materials including aldicarb is planned once trees develop sufficient leaf mass to enable testing of residual post application of all chemistries used in addition to impact on mealybugs. We ran a methods trial and found that malathion lasted 2-3 times longer under IPCs compared to open field settings, suggesting that materials sprayed in IPCs that are known to have rapid UV breakdown may persist much longer and remain effective longer under IPCs. (2a) Predator assessmentsPredatory insects have been reared from locally infested plant materials the past two years, with the majority of predators emerging being parasitic flies, which consume a variety of piercing-sucking pest insects, mealybug destroyers, and one species of lacewing. A primer has been developed and evaluated to enable the detection of mealybugs in the guts of predators. Post-feeding retention time in a known predator was assessed and we feel confident that this tool will now enable screening of field caught arthropods that may play a role in the suppression of lebbeck mealybug. The commercially available predators Cryptolaemus montrouzieri, Orius insidiosus, Adalia bipuctata, and Hippodamia convergens have been screened using no-choice assays to determine if they will feed on larval lebbeck mealybugs, and on mealybug ovisacs. Additionally, several wild-caught predators have also been screened, including Harmonia axyridis and larva of the genus Ceraeochrysa (colloquially called trash bugs). Both adult and larval C. montrouzieri readily feed on lebbeck mealybug larvae and ovisacs, as do larval Ceraeochrysa. Adult Orius insidiosus, early instar Adalia bipunctata, and adult and larval Harmonia axyridis do not feed on lebbeck mealybug larvae or ovisacs. Adult Hippodamia convergens and Adalia bipunctata do feed on some lebbeck mealybug larvae, but appear to attack and kill the larvae more than fully consuming them. Neither feed on ovisacs. From our results, only Cryptolaemus montrouzieri shows promise as a commercially available biological control agent for lebbeck mealybug, although Ceraeochrysa larvae may act as natural biological control in the grove. (2d) Develop tools to minimize spread· Killing lebbeck mealybug with isopropyl alcoholWe tested different concentrations of isopropyl alcohol to determine how lethal they are to 1st instar lebbeck mealybugs. 50%, 70%, and 90% solutions of isopropanol were sprayed onto mealybug crawlers placed on cloth swatches, and compared to a DI water control. Both 1 spray at each concentration, and 2 sprays at each concentration were tested. Mortality was assessed after 5, 10, and 15 minutes. The test was repeated, this time assessing mortality at 30 min, 1hr, and 2hrs. For all concentrations of isopropanol, 1 spray resulted in significantly greater mealybug mortality compared to the control. However, several mealybug crawlers remained alive and active after a single spray for all concentrations. 2 sprays of each concentration resulted in almost 100% mortality or incapacitation at all time points. · Using steam to kill adult mealybugs and ovisacsAdult mealybugs and ovisacs may be accidentally transferred from infested groves on tools and equipment. Steam treatments may be an effective method of sanitizing equipment and killing both adults and ovisacs. Using a steam cabinet on-station, we steam treated adults and ovisacs at 100 and 120 degrees Fahrenheit for 5, 10, and 15 minutes to determine mortality. Previous preliminary experiments showed steam treatments at 130 degrees Fahrenheit for 15 and 30 minutes resulted in 100% mortality. Mortality was assessed at 0, 3, and 5 days after treatment, and ovisacs were held for an additional 2 weeks to see if crawlers emerged. At 100 degrees F for all times points, adult mealybug and ovisac mortality was not significantly different than the control. At 120 degrees F for 5 minutes, mortality also functionally 0%. However, mortality rose to 100% at 120 degrees F for 10 and 15 minutes. Currently, we are running trials on adults and ovisacs treated at 130 degrees F for 5 and 10 minutes, and preliminary results show 100% mortality. Work planned for next quarter(1b) Adjuvant screening will continue to determine optimal adjuvants to work in synergism with insecticide sprays.(1c) We will continue treating and scouting the grove we have started a management comparison trial at.(1d) The ant management project will continue throughout the upcoming quarter with the addition of monitoring for predatory insect establishment.(1e) IPC management management trials will continue, looking towards conventional materials for management and spray penetration by tractor mounted sprayers.(2a) Working with FDACS, we have permission to deploy sentinel infested materials to screen more broadly for predators that may be present in the system which we missed with haphazard rearing from infested grove sites.(2c) Feeding mechanisms will continue to be worked out using EPG with the future plan to determine if we can interfere with their feeding via chemical intervention leading to reduced offspring production.(2d) We will continue evaluating sanitation options and are planning to look at vehicle sanitation in the next quarter.
Work done this quarter:(1b) Adjuvant screeningPreliminary trials have been conducted with adjuvants alone to determine their lethality to lebbeck mealybug. A total of 9 adjuvants, including and 4 from Helena Agri Enterprises were mixed with DI water at label rates and sprayed until dripping on Volk lemon leaves with mealybugs attached. 8 of the adjuvants resulted in significantly greater mealybug mortality over a 7-day period compared to a DI water control. (1c) Evaluate promising materials in open grove setting: We began a field trial at a 10-acre commercial citrus planting that was heavily infested in 2020. First insecticide applications occurred as a pre-bloom prophylactic spray of a systemic material, with a second spray planned in the end of April.(1d) Ant Management · We are testing a variety of methods to remove red imported fire ants (Solenopsis invicta) from citrus groves. Removing the ants can allow natural enemies to prey on and parasitize lebbeck mealybug without interference, and provide more effective biological control. Our research goals are:1. Reduce abundance of fire ants associated with lebbeck mealybug (Nipaecoccus viridis) in central FL citrus groves. · 2. Determine long-term efficacy of different treatment types at reducing fire ant abundance · 3. Assess time and cost of different treatment types · 4. Determine effects of ant treatment on predators abundance within trees and within N. viridis clusters. We are collaborating with the King Lab from the University of Central Florida, and are conducting the experiment in the grove of a local citrus grower. Four experimental treatments will be tested and compared to an untreated control: Chlorpyrifos drench, Clinch ant bait (Abamectin), Extinguish ant bait (S-methoprene), and spot treating ant colonies with hot water. Fire ant abundance will be assessed by counting the number of surviving colonies, and by determining presence/absence of foragers with pecan sandie baits. Natural enemy abundance will be assessed by dissecting mealybug clusters for predator larvae. Sampling for each of these methods will take place every 2 weeks for a total of 3 months post-treatments. Currently, all treatments have been applied and we are conducting follow-up surveys to determine their efficacy. (1e) Evaluate management options for IPCsWe recently completed the study to evaluate several commercially available entomopathogenic fungi (EPF) products as a potential tool to control lebbeck mealybug infestation on young citrus trees within individual protective covers (IPCs). Findings of the study indicate that EPF can cause death of mealybugs and EPF conidia are viable on citrus trees within IPCs up to 42 days after treatment. Thus, EPF can offer control of mealybug for up to 1.5 months after treatment. In February, we began infesting trees in our research planting to run a similar comparison of topical insecticides for clean-up, with pretreatment drenches planned prior to the next set of trees to be infested. Additionally, a controlled greenhouse comparison of drench materials including aldicarb is planned once trees develop sufficient leaf mass to enable testing of residual post application of all chemistries used in addition to impact on mealybugs. We ran a methods trial and found that malathion lasted 2-3 times longer under IPCs compared to open field settings, suggesting that materials sprayed in IPCs that are known to have rapid UV breakdown may persist much longer and remain effective longer under IPCs. (2a) Predator assessmentsPredatory insects have been reared from locally infested plant materials the past two years, with the majority of predators emerging being parasitic flies, which consume a variety of piercing-sucking pest insects, mealybug destroyers, and one species of lacewing. A primer has been developed and evaluated to enable the detection of mealybugs in the guts of predators. Post-feeding retention time in a known predator was assessed and we feel confident that this tool will now enable screening of field caught arthropods that may play a role in the suppression of lebbeck mealybug. The commercially available predators Cryptolaemus montrouzieri, Orius insidiosus, Adalia bipuctata, and Hippodamia convergens have been screened using no-choice assays to determine if they will feed on larval lebbeck mealybugs, and on mealybug ovisacs. Additionally, several wild-caught predators have also been screened, including Harmonia axyridis and larva of the genus Ceraeochrysa (colloquially called trash bugs). Both adult and larval C. montrouzieri readily feed on lebbeck mealybug larvae and ovisacs, as do larval Ceraeochrysa. Adult Orius insidiosus, early instar Adalia bipunctata, and adult and larval Harmonia axyridis do not feed on lebbeck mealybug larvae or ovisacs. Adult Hippodamia convergens and Adalia bipunctata do feed on some lebbeck mealybug larvae, but appear to attack and kill the larvae more than fully consuming them. Neither feed on ovisacs. From our results, only Cryptolaemus montrouzieri shows promise as a commercially available biological control agent for lebbeck mealybug, although Ceraeochrysa larvae may act as natural biological control in the grove. (2d) Develop tools to minimize spread· Killing lebbeck mealybug with isopropyl alcoholWe tested different concentrations of isopropyl alcohol to determine how lethal they are to 1st instar lebbeck mealybugs. 50%, 70%, and 90% solutions of isopropanol were sprayed onto mealybug crawlers placed on cloth swatches, and compared to a DI water control. Both 1 spray at each concentration, and 2 sprays at each concentration were tested. Mortality was assessed after 5, 10, and 15 minutes. The test was repeated, this time assessing mortality at 30 min, 1hr, and 2hrs. For all concentrations of isopropanol, 1 spray resulted in significantly greater mealybug mortality compared to the control. However, several mealybug crawlers remained alive and active after a single spray for all concentrations. 2 sprays of each concentration resulted in almost 100% mortality or incapacitation at all time points. · Using steam to kill adult mealybugs and ovisacsAdult mealybugs and ovisacs may be accidentally transferred from infested groves on tools and equipment. Steam treatments may be an effective method of sanitizing equipment and killing both adults and ovisacs. Using a steam cabinet on-station, we steam treated adults and ovisacs at 100 and 120 degrees Fahrenheit for 5, 10, and 15 minutes to determine mortality. Previous preliminary experiments showed steam treatments at 130 degrees Fahrenheit for 15 and 30 minutes resulted in 100% mortality. Mortality was assessed at 0, 3, and 5 days after treatment, and ovisacs were held for an additional 2 weeks to see if crawlers emerged. At 100 degrees F for all times points, adult mealybug and ovisac mortality was not significantly different than the control. At 120 degrees F for 5 minutes, mortality also functionally 0%. However, mortality rose to 100% at 120 degrees F for 10 and 15 minutes. Currently, we are running trials on adults and ovisacs treated at 130 degrees F for 5 and 10 minutes, and preliminary results show 100% mortality. Work planned for next quarter(1b) Adjuvant screening will continue to determine optimal adjuvants to work in synergism with insecticide sprays.(1c) We will continue treating and scouting the grove we have started a management comparison trial at.(1d) The ant management project will continue throughout the upcoming quarter with the addition of monitoring for predatory insect establishment.(1e) IPC management management trials will continue, looking towards conventional materials for management and spray penetration by tractor mounted sprayers.(2a) Working with FDACS, we have permission to deploy sentinel infested materials to screen more broadly for predators that may be present in the system which we missed with haphazard rearing from infested grove sites.(2c) Feeding mechanisms will continue to be worked out using EPG with the future plan to determine if we can interfere with their feeding via chemical intervention leading to reduced offspring production.(2d) We will continue evaluating sanitation options and are planning to look at vehicle sanitation in the next quarter.
March 2021Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp. We collected data from the large spray timing and skirting trial in March. We evaluated 50 fruit each for disease severity on approximately 125 trees in 32 rows. We made significant progress on the incidence analysis of the data and are close to finalizing the analysis. We found that fungicide program significantly reduced the black spot incidence compared to the control. The greatest reduction was from the Florida standard timing with applications from May to September. Skirting had no significant effect on the disease incidence but the interaction between fungicide timing and skirting did have a significant effect. The Florida standard timing with skirting was the best performing treatment. For the late fungicide timing (May to October), there was a slight decrease in incidence with the addition of skirting. It should be noted that the 2019-2020 season was light for black spot incidence and severity. From initial scouting, it appears that black spot severity is greater this year and there may be more differences among the treatments that will hopefully support the conclusions from the first year. The minor plots were re-randomized within the main plots and we were able to get the trial re-flagged just in time for the early spray (delayed by 2 weeks but no rain occurred from the first of April until after the early application). All applications were made on time in the spray trial. We will be collecting the data at the end of March, 2021. We were unable to set up the second planned fungicide trial this year because of the COVID-19 shut down. We will be collecting pre-treatment data at the end of March and plan to move forward with the trial. The second season of trials in which different fungicide products are tested for their efficacy to protect citrus fruit from CBS infection, is currently underway. Products being evaluated include Enable (Indar) and Luna experience sprayed on their own as well as Copper hydroxide sprayed in alternation with either Amistar Top or Headline (Cabrio). Fungicides are applied every 4 weeks from October 2020 until March 2021. The fungicides are being tested in a ‘Valencia’ orange orchard with a history of CBS. The trials will be evaluated at the end of August 2021. An additional 8 South African and 8 global (from Argentina and Swaziland) isolates have been sequenced. As the next-generation sequencing data becomes available, the analysis and results are continuously updated to include the new data. DNA from 16 isolates (Argentina, Australia, Brazil and China) passed QC and is in the process of being sequenced. More isolates from Brazil and China are being cultured for DNA extraction and sequencing. We aim to sequence 6 isolates from each of the 5 South African provinces where CBS is found (North West, Limpopo, Mpumalanga, Kwa-Zulu Natal and Eastern Cape) and 6 each from the other countries (Argentina, Australia, Brazil, China, Swaziland and USA) by end of March. Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.). AStudies on the diversity of Phyllosticta spp. associated with citrus in Florida has progressed. Our collection of P. citricarpa isolates were obtained from citrus fruit in different areas under quarantine from 2010 to 2020. These isolates were previously screened by morphology to remove nonpathogenic P. capitalensis isolates. We are now screening the remaining 202 isolates using molecular techniques to determine if we have cryptic species that may have been misidentified as P. citricarpa. To date we have screened 125 isolates by amplifying and DNA sequencing the tef-1a (Translation elongation factor-alpha) locus. This screen has revealed that two isolates (Gc-6 and Gc-7) match to a Phyllosticta species not previously reported in association with citrus. The best sequence match based on ITS and tef-1a is to a species reported previously as a pathogen on a member of the Amaryllidaceae family (Hymenocallis littoralis) in Australia. Numerous inoculations of citrus have determined that these isolates do not cause citrus black spot. Multilocus analysis and leaf inoculations on Hymenocallis species are ongoing. These studies will allow us to conclusively determine the identity of these two isolates. Vegetative and sexual compatibility assays (sandwich mating) were performed to determine the mating type of the two isolates. The results showed that Gc6 and Gc-7 isolates are heterothallic and belong to the same mating type, as neither isolate was a capable of producing pseudothecia in solo cultures or in paired cultures with one another. The screening and further characterization of these new species is continuing to obtain robust information on the diversity of Phyllosticta species and determine the presence of cryptic species in Florida.We screened an additional 26 isolates of our P. citricarpa isolates for mating-type (MAT1-1 and MAT1-2). During this period we have screened an additional 26 new isolates and, as shown in Figures 1, only the MAT1-2-1 mating type is detected in new isolates collected in 2020. We conclude that the MAT1-1 mating type is still absent in the Floridian population.To determine the phenology of fruit susceptibility inoculation studies of citrus fruit (Meyer lemon) were performed in a quarantine greenhouse at the Florida Department of Agriculture and Consumer Services (DPI) in Gainesville. A total of 97 fruit were used in this experiment. Of these 97 fruits, 25 served as controls and 72 were inoculated with the Gc-12 isolate of P. citricarpa. Disease assessments were performed weekly for a full calendar year. A total of 50 fruit produced symptoms in this period. All 50 were from fruit inoculated with the Gc-12 isolate, and no symptoms were observed in the control treatment. Therefore, from all inoculated citrus fruit, 69.4% produced symptoms, and 30.5% remained asymptomatic. The indications from this one experiment suggest that citrus fruit are susceptible at all stages of their development regardless of their maturation time. A second trial will be conducted in 2021 to determine the period of fruit susceptibility to P. citricarpa.
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