1. Please state project objectives and what work was done this quarter to address them: Year-1 Generate CTV infectious clones that express different FT3s or downregulate negative regulators of flowering to inoculate into Citrus macrophylla. Prepare different citrus genotypes for inoculation with the generated CTV vectors. Our focus in the 3rd and 4th quarter of this project was to monitor flowering in the Citrus macrophylla genotypes infected with the different CTV-FT3 (Arabidopsis and Hamlin FT3 (Hamlin FT3 had a consensus sequence and two variants as revealed by sequencing) constructs and CTV-RNAi (supposedly suppress negative regulators of flowering). Hamlin CTV-FT3 consensus sequence constructs did not induce flowering in C. macrophylla. On the contrary, the two Hamlin FT3 variants induced early flowering in Citrus macrophylla. All CTV-RNAi constructs targeting downregulating the expression of negative regulators of flowering failed to induce early flowering. CTV constructs expressing Arabidopsis FT3 failed to induce flowering in infected Citrus macrophylla. This prompted us to test the stability of Arabidopsis FT3 in the CTV vector via Revere transcription polymerase chain reaction with primers upstream and downstream of the the insertion site. The size of the RT-PCR product was slightly smaller than the plasmid PCR product. This suggested minor recombination that was confirmed by sequencing that revealed a 76 nts deletion in the sequence. The transgenic FT3 Carrizo lines rooted have not yet flowered under our growth chamber conditions and are not yet big enough to top them with other citrus genotype. 2. Please state what work is anticipated for next quarter: In the 5th quarter, we will reinfect citrus with the CTV-FT3 vectors that failed to flower to make sure of the result. 3. Please state budget status (underspend or overspend, and why): On budget
Research progress Sept 2022-December 20221. Near term field management(a) Develop methods to time management actionsIn the previous report, I discussed our efforts to understand the odors that appear to be attracting lebbeck mealybug to help develop better scouting and potentially an odorant lure in the future to make sampling to determine management easier. To date, the primary components of the tree parts as well as damage have been identified and pure isolates of these odors have been obtained. We are currently testing attraction to combinations of these odors and to individual odors to move closer to understanding what the mealybugs are attracted to. Because there is a strong recruitment of lebbeck mealybug to our traps around small wounds versus traps without wounds (Fig. 1), we anticipate that the odors associated with this damage will be the most attractive.(c) Evaluate promising materials in open grove settingIn the fall of 2022, we completed 2 field trials to look at longevity of several promising foliar applied insecticides. The first trial compared those known to have contact activity. We tested Agri-Flex (8.5 oz/a), Transform (2.75 oz/a), Voliam-Flexi (7 oz/a), Besiege (12.5 fl oz/a), AgriMek (4.25 fl oz/a), Actara (5.5 oz/a), Minecto Pro (12.5 fl oz/a), and Esteem (5 oz/a) (all with 0.25% NIS). Materials were applied to trees with flush in the grove and leaves that were soft but fully expanded brought back to the lab where they were challenged with mealybugs. In week 1, Agri-Flex had the greatest mortality with over 80% of juvenile mealybugs dying, closely followed by Transform (68%), and Voliam-Flexi (31.5%). All other chemistries were no different than the untreated control for mortality in the first week of application. Unfortunately there was no residual efficacy for any materials in weeks 1, 2, and 3 after application. In the second trial, we focused on foliar-applied insecticides with systemic activity and included some variation in rate as well as adjuvant used. Treatments consisted of: Movento 16 oz/a + NIS, Senstar + NIS, Sivanto + NIS, Movento 10oz/a + 435 oil, Senstar + 435 oil, and control. In the initial week of application, Movento + NIS had the highest mortality at 38%, Senstar +NIS, Sivanto + NIS both had 28% mortality, and the rest fell below this. This isn’t surprising as systemic materials need time to build up in leaves and most mortality was likely due to the adjuvant sticking to the insects and smothering them. One week after application, the Movento 10 oz + oil had increased mortality relative to the other treatments (37%) but no material showed the ability to kill 50% or greater of the nymphs at any point in the trial. This is not consistent with what we see in CUPS houses when these systemic materials have been applied. I believe the reason for this is that the trees we were testing have had HLB for several years and materials are not likely moving through the vascular system as they would in a health tree. In healthy trees, systemic materials are able to translocate through the canopy, and it is likely that the vascular plugging exhibited by HLB affected trees reduces this.II. Long term managementc. Determine what insecticide chemistries inhibit feedingWhile there has been some progress on this subobjective, it has been far slower than anticipated. Weve sent equipment for repair/ tune- up and are reworking the initial feeding interaction data.Working with the repaired equipment, we have been able to document the feeding waveforms, though with some noise, that we will need to move forward and complete documenting the feeding interactions. We can now see the E1/E2 ingestion pathway (Fig. 3). This must be fully documented and reviewed to move into the next step, which will be determining if we can interrupt the feeding via insecticides and at what rate we need to interrupt feeding, halt offspring production, and lead to adult mortality.d. Develop tools to minimize spreadWhile we were unable to complete the solarization and freezing studies we had planned this past term due to colony infestation by a predator impacting available insects to work with. However, we did make progress in understanding lebbeck mealybug dispersal, which is important for determining broader methods for reducing spread. In the late summer of 2022, we built a wind tunnel in which we can control the velocity of wind (Fig 3). In this wind tunnel, we can introduce plants from below with varying states of infestation and at varying distances. While still preliminary, we are finding lebbeck mealybug fairly easy to dislodge at low to medium wind velocities (Fig. 4). 10 minutes at a high velocity of wind (exact speed will be reported later, the anemometer is broken) is sufficient to dislodge 50% of crawlers, 30% of immatures, and 15% of adult females. The ability to dislodge easily in wind events helps to understand the rapid spread of this pest and underscores the important of management actions shortly after major weather events.II. Next quarter:We are continuing to test promising materials in open groves and are in the process of planning a test of a subset of materials in a CUPS house as we have learned that chemistries dont all behave similarly in CUPS to the open field.We will continue to document feeding interactions and plan to start insecticide trials in late spring to document influence of systemic chemistries on feeding, offspring production, and mortality.We will continue working towards identifying appropriate odors to use towards future scouting tools.Solarization and freezing tests for sanitation will be completed in spring 2023.III. Budget statusWe are behind on salary spending after having a vacant postdoc position for several months, however we are on track in other categories.
January 2023 1. Please state project objectives and what work was done this quarter to address them:The objectives are to 2) determine if the flush cycle and infection period for Z. citri-griseum have changed due to the influence of HLB on citrus physiology or changing environmental factors; 4) evaluate the potential promotion of greasy-green symptoms related to nutrition programs or to peel reactions like a chemical burn from different pesticide and combinations of pesticide tank mixes; and 5) evaluate if postharvest degreening treatments might be modified to adequately remove the green coloration while mitigating poor shelf life from anticipated longer degreening times. Most of the progress to date have been on objective 2. Two sites were established in grapefruit groves in the Indian River region. These sites were identified to have significant problems with the greasy green disorder affecting the grapefruit industry. In each block, twenty trees were selected for uniform canopy without excessive thinning or obvious sectoring from HLB and mapped, and ten flushes per tree were tagged to be observed for greasy spot symptoms later in the season. Every two weeks from the beginning of May, ten leaves and one fruit per tree are collected. The fruit diameter has also been collected since June. The leaves are cleared and 5mm disk samples are examined under the microscope to observe whether there is epiphytic growth. Our previous method of sampling fruit was not giving satisfactory results so we have moved to applying clear nail polish to the fruit surface to remove the fungal growth and observing it under the microscope instead. We had much better success with the nail polish technique. We are also molecularly confirming that the structures we were observing under the microscope were Zasmidium citri-griseum. We have done a conventional PCR, observed bands at the correct size and preparing the samples for sequencing. The microscopy samples are preserved, and we are using the winter season to collect the observations for analysis. We presented some preliminary phenology data at the Southeastern Professional Fruit Workers Conference held in Lake Alfred in November. From our first observations in May to October2022, the majority of flush was fully mature with significant peaks of younger flush in June and September. The red and white grapefruit had similar patterns of flush but the red grapefruit had a longer flush period in the fall than the white. Fruit growth increased in a typical exponential pattern until September when the growth rate was substantially reduced as expected. The average diameter in October was 8 cm. The fruit are currently asymptomatic and spores and mycelial growth were observed from August. We are not sure if we did not detect them earlier in the season was because they were not there or an artifact of the way we were attempting to visualize them. We will hopefully determine the extent of epiphytic growth and when the epiphytic growth is the most prevalent to compare with what is known from previous work. Ideally, this will allow us to adjust when applications occur to better control greasy spot as part of the greasy green disorder. 2. Please state what work is anticipated for next quarter: Samples will continue to be evaluated microscopically. We plan to start evaulating flush in the early spring, probably mid-February or early March. Samples will continue to be collected. More fruit samples have been promised for the greasy-green physiology experiments and it is expected that several experiments following up on the ones already undertaken will be conducted. 3. Please state budget status (underspend or overspend, and why): No over or underspend on budget currently
In Central Florida (Lake Placid grove), all data regarding tree height and canopy as well as canker and HLB incidence were collected. Also samples for time 0 nutrient analysis and auxin quantification were collected. Unfortunately, hurricane Ian destroyed many trees and produced a great amount of fruit drop, so Hamlin plots were harvested before we could finish our work for this year. In Southwest Florida (Duda farms) we have characterized all trees used (tree height and canopy volumes) and we started assessing HLB and canker incidence. For HLB, most of the trees we are using category 3 = 26-50% of the canopy with foliar disease symptoms, and category 4 = 51-75% of the canopy with foliar disease symptoms. Canker incidence is so far negligible. We have collected samples for leaf nutrient analysis and auxin quantification. We started treatments with the three advanced liquid Zn products (Zinkicide TMN111, FertiZink, and NuZinc Trees under treatments continue are showing greener canopies than controls. Control trees are presented some off-blooms still in September and October. These off-blooms have not been observed in the treated trees. Fruit drop is being recorded in Hamlin weekly. Unfortunately, hurricane Ian has increased fruit drop but since we are collecting data weekly, we are going to be able to reduce the fruit drop background noise and in any case, see if our treatments had a beneficial impact even under hurricane conditions in alleviating fruit drop. We have lost also some trees due to the high wind. Interestingly, all of the trees were lost in a control plot. Other treatments in the same grove with commercial Zn sulfate are showing the same effects. We do not have a clear explanation for this, but fortunately, treatments were not compromised and other trees in untreated plots are being used as new control replacements.To evaluate the oxidative properties of the FertiZink and NuZinc product formulations, Amplex Red based reactive oxygen species (ROS) assay was performed to measure the production of ROS. Neither of the formulations significantly produced ROS even at 28,000 ppm Zn. This results suggest that the formulations would not cause oxidative stress to plants upon foliar application. Dynamic Light Scattering (DLS) studies were conducted on two batches of FertiZink and NuZinc that were fresh and 1 year old at the time of evaluation. Measurements were conducted at 400ppm and 800pm Zn to replicate field spray application rate. The DLS studies of the products showed the presence of micron and sub-micron size particulates, confirming that the products are not in the nano-regime. We also noticed sedimentation of products during the DLS measurements, which resulted inconsistence readings. This inconsistency was apparent in both the 1 year old batch and the fresh batch. Assessment of the particle size will be performed through scanning electron microscopy. Work anticipated for next quarter: We anticipate that Hamlin fruit will be harvested. This has not done yet as Brix in general are around 8.2 (December 13, 2022), but slowly increasing. Treatments will continue in Valencia trees.Outreach: Alferez, F. Understanding and Managing Fruit Drop in HLB-Affected Citrus. Invited Seminar at American Society of Horticultural Science. January 12, 2023.
1. Please state project objectives and what work was done this quarter to address them:The objectives are to 2) determine if the flush cycle and infection period for Z. citri-griseum have changed due to the influence of HLB on citrus physiology or changing environmental factors; 4) evaluate the potential promotion of greasy-green symptoms related to nutrition programs or to peel reactions like a chemical burn from different pesticide and combinations of pesticide tank mixes; and 5) evaluate if postharvest degreening treatments might be modified to adequately remove the green coloration while mitigating poor shelf life from anticipated longer degreening times. Most of the progress to date have been on objective 2. Two sites were established in grapefruit groves in the Indian River region. These sites were identified to have significant problems with the greasy green disorder afftecting the grapefruit industry. In each block, twenty trees were selected for uniform canopy without excessive thinning or obvious sectoring from HLB and mapped, and ten flushes per tree were tagged to be observed for greasy spot symptoms later in the season. Every two weeks from the beginning of May, ten leaves and one fruit per tree are collected. The fruit diameter has also been collected since June. The leaves are cleared and 5mm disk samples are examined under the microscope to observe whether there is epiphytic growth. Our previous method of sampling fruit was not giving satisfactory results so we have moved to applying clear nail polish to the fruit surface to remove the fungal growth and observing it under the microscope instead. The fruit are currently asymptomatic and spores and mycelial growth were observed from August. We are not sure if we did not detect them earlier in the season was because they were not there or an artifact of the way we were attempting to visualize them. We will hopefully determine the extent of epiphytic growth and when the epiphytic growth is the most prevalent to compare with what is known from previous work. Ideally, this will allow us to adjust when applications occur to better control greasy spot as part of the greasy green disorder. Initial survey attempts of fresh fruit growers have not generated many responses about the greasy green disorder. Plans are underway to conduct in person interviews as the greasy green symptoms develop in November and December to get the information needed to inform experiments in the next season. 2. Please state what work is anticipated for next quarter: Field sampling will continue and samples will be evaulated microscopically. We will stop sampling at harvest and start again in the new year. Post-harvest work will be planned and started with the upcoming harvest season. In particular, we will be working to see what effects conventional degreening techniques have and how they can be adjusted. We will also be taking fruit peels on a subsample of the fruit to see how much mycelial growth is associated with the greasy green symptoms in the packinghouse. Interviews will be conducted with growers on the greasy green disorder to give us more information to inform experiments in the next season. 3. Please state budget status (underspend or overspend, and why): No over or underspend on budget currently
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 year 1 of the project, Vismax treated orange trees were significantly more resistant to citrus canker, caustive agent Xanthomonas citri, when the formulated peptide was applied as a dilute foliar spray or soil drench, 7 days prior to leaf inoculation. In year 2 of the project, inoculated greenhouse assays are being established to further probe Vismax-activated defenses, with greenhouse testing conducted by Dr. Megan Dewdney (U. Florida IFAS CREC). In Year 2 Q2 (June 16 2022 – Sept 15 2022), Canker trial #5 was completed on susceptible sweet orange saplings. The saplings were sprayed with Vismax foliar applications with four different surfactants with varied properties (pH, spread index), in an effort to identify compatible adjuvants that enhance Vismax plant protection activity. The results from this greenhouse trial indicated that Vismax foliar applications reduced canker severity both in the presence and absence of surfactant, and that commercially available surfactants were compatible with Vismax. Statistically significant reduction in canker disease severity were observed for Vismax applied with non-ionic blend surfactants within a pH range of 6 – 8.5. Currently, protocols are being prepared for phytophthora root rot trials. Canker trial results were communicated to the agronomic research and grower communities through several forums, including 3 annual conferences: Florida Citrus Mutual (Bonita Springs, FL; June 15-17, 2022), American Society of Plant Biologists (Portland, WA; July 9-13, 2022), and American Phytopathological Society (Pittsburgh, PA; Aug 6-10, 2022).
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 year 1 of the project, Vismax treated orange trees were significantly more resistant to citrus canker, causative agent Xanthomonas citri, when the formulated peptide was applied as a dilute foliar spray or soil drench, 7 days prior to leaf inoculation. In year 2 of the project, inoculated greenhouse assays further probed Vismax-activated defenses, with greenhouse testing conducted by Dr. Megan Dewdney (U. Florida IFAS CREC). The results of canker trial #5 indicated that Vismax foliar applications gave statistically significant reductions in canker severity both in the presence and absence of surfactant, and that commercially available surfactants were compatible with Vismax. A repeat inoculation of the trial on a subset of the same plants showed that the canker protection does not persist, and the treatment must be reapplied to effectively provide canker protection. Currently, a phytophthora root rot trial is underway, evaluating rates of drench-applied Vismax in combination with and comparison to Foliar-applied Vismax for their ability to promote resistance to phytophthora root rot in susceptible orange seedlings by comparing dry root mass and scoring roots and leaves for symptoms of phytophthora rot.
Note: postdoctoral researcher previously working on this project for a faculty position left in May and the new hire started in mid August, so some objectives are behind where we would like to be. I. Research progress June 2022-August 20221. Near term field management(a) Develop methods to time management actionsIn the previous report, I discussed developing a relative scouting method for lebbeck mealybug detection. To determine if we could use an element of host attraction to monitor for lebbeck mealybugs, we induced damage to infested trees on peripheral limbs and wrapped these locations in corrugated cardboard. All damage traps were paired with undamaged limbs in similar locations on infested trees. After 2 weeks, we counted more juvenile lebbeck mealybugs in the damaged locations than in other traps. We now suspect that we may be able to use the odors produced by trees in response to damage to attract mealybugs. To move this forward, we are identifying odors associated with various tree parts (mature leaves, flush, flower buds, and various stages of fruit, damage) to determine what odors are common to the two things we suspect that the mealybugs are attracted to from our work so far: immature/setting fruit and damage. Once we complete the analysis of the odors, we will work on determining what odor blends may be used as an attractant using lab studies. (b) Expand laboratory insecticide and adjuvant screening.No new data to report (c) Evaluate promising materials in open grove settingSpray trials are currently underway to determine optimal insecticides for managing lebbeck mealybug. While we are confident that early season control is key to protecting young trees, populations can and do establish throughout the year which may require management, in particular in CUPS, IPCs, and fruit intended for fresh market. We are currently testing a range of contact and systemic insecticides in the CREC research groves. These tests include efficacy testing at 0, 7, 14, 21, and 28 days after treatment to better understand the potential of residual to management population build up. The recent rains have delayed our second round of trials, which we anticipate starting again the week of September 19. These are tests that are not part of routine insecticide evaluations but are important to help growers understand what materials may be better options to incorporate into their programs. (d) Fire ant management as part of lebbeck mealybug managementNo new data this quarter- still working on publishing (e) Evaluate management options for IPCsNo work done this quarter. II. Long term managementa. 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 mealybugsData presented in previous quarter has encouraged a classical biological control researcher within UF to start working on the basic research needed to determine if any of the predators identified by our work could be targets for mass rearing and release for management. Their work is currently funded using their UF faculty start up package while they seek funding opportunities. b. Determine how to implement mealybug management concurrent with other pest management programsNo new data to report this quarterc. Determine what insecticide chemistries inhibit feedingFeeding interactions between lebbeck mealybug and citrus hosts is currently being documented, with the goal of starting insecticide assays in mid fall 2022. The new postdoc has taken up the data amassed in the past year to decipher the various interactions in the overall feeding process and compare those with other hemipteran pests. Once these behaviors are fully documented, we can determine optimal insecticide rates for killing feeding lebbeck mealybugs, which will reduce population growth and can be used to help develop optimal rates for use in IPCs. d. Develop tools to minimize spreadNo new data to report this quarter II. Next quarter:1b,c. and 2b We are continuing to test promising insecticides using field aging to determine duration of efficacy post-application. Adjuvant + Delegate trial was no completed as planned in the previous quarter in the absence of the postdoc, so that will be completed in the upcoming quarter. 2c. We will complete the feeding interaction documentation and move into insecticidal drench assays to look at impacts of insecticide on the feeding interaction. 2d. Develop protocol for sanitation using solarization and freezing (not completed last quarter as planned) III. Budget statusWe have had to request to move funds into our materials budget to cover increased gas/travel costs to the Lake Placid research site and for additional trees to maintain lebbeck mealybug colonies on. The mealybugs destroy plants at a faster rate than anticipated.
1. Please state project objectives and what work was done this quarter to address them:
1. Please state project objectives and what work was done this quarter to address them:
Year-1 Generate CTV infectious clones that express different FT3s or downregulate negative regulators of flowering to inoculate into Citrus macrophylla. Prepare different citrus genotypes for inoculation with the generated CTV vectors.
Our focus in the 2nd quarter of this project was to infect citrus with the CTV vectors generated in the first quarter and grow transgenic rootstocks to enable experiment set ups.
We were able to infect citrus with CTV vectors expressing FT3 of Hamlin sweet orange and Citrus clementina. In addition, the CTV RNAi vectors targeting different silencing suppressors also successfully infected citrus. We are monitoring the different vectors for their ability to induce flowering in citrus.
In addition, we were able to root different transgenic lines expressing Citrus clemetina FT3. These lines are growing in our growth chambers. Upon reaching the proper size, the transgenic plants will be topped with different citrus genotypes and infected with CTV RNAi vectors targeting the different flowering suppressors independently. The transgenic FT3 carrizo lines rooted did not yet flower under the growth chamber conditions.
2. Please state what work is anticipated for next quarter:
In the 3rd quarter, we will monitor the different FT3 vectors for there ability to induce flowering in Citrus macrophylla and the stability of the different inserts within the CTV vector. We will monitor the rooted transgenic plants for flowering.
3. Please state budget status (underspend or overspend, and why):
On budget
1. Please state project objectives and what work was done this quarter to address them:
1. Please state project objectives and what work was done this quarter to address them:
Year-1 Generate CTV infectious clones that express different FT3s or downregulate negative regulators of flowering to inoculate into Citrus macrophylla. Prepare different citrus genotypes for inoculation with the generated CTV vectors.
Our focus in the 2nd quarter of this project was to infect citrus with the CTV vectors generated in the first quarter and grow transgenic rootstocks to enable experiment set ups.
We were able to infect citrus with CTV vectors expressing FT3 of Hamlin sweet orange and Citrus clementina. In addition, the CTV RNAi vectors targeting different silencing suppressors also successfully infected citrus. We are monitoring the different vectors for their ability to induce flowering in citrus.
In addition, we were able to root different transgenic lines expressing Citrus clemetina FT3. These lines are growing in our growth chambers. Upon reaching the proper size, the transgenic plants will be topped with different citrus genotypes and infected with CTV RNAi vectors targeting the different flowering suppressors independently. The transgenic FT3 carrizo lines rooted did not yet flower under the growth chamber conditions.
2. Please state what work is anticipated for next quarter:
In the 3rd quarter, we will monitor the different FT3 vectors for there ability to induce flowering in Citrus macrophylla and the stability of the different inserts within the CTV vector. We will monitor the rooted transgenic plants for flowering.
3. Please state budget status (underspend or overspend, and why):
On budget
1. Please state project objectives and what work was done this quarter to address them:The objectives are to 2) determine if the flush cycle and infection period for Z. citri-griseum have changed due to the influence of HLB on citrus physiology or changing environmental factors; 4) evaluate the potential promotion of greasy-green symptoms related to nutrition programs or to peel reactions like a chemical burn from different pesticide and combinations of pesticide tank mixes; and 5) evaluate if postharvest degreening treatments might be modified to adequately remove the green coloration while mitigating poor shelf life from anticipated longer degreening times. Most of the progress to date have been on objective 2. Two sites were established in grapefruit groves n the Indian River region. These sites were identified to have significant problems with the greasy green disorder afftecting the grapefruit industry. In each block, twenty trees were selected for uniform canopy without excessive thinning or obvious sectoring from HLB and mapped, and ten flushes per tree were tagged to be observed for greasy spot symptoms later in the season. Every two weeks from the beginning of May, ten leaves and one fruit per tree are collected. The leaves and fruit are cleared and 5mm disk samples are examined under the microscope to observe whether there is epiphytic growth. We will hopefully determine the extent of epiphytic growth and when the epiphytic growth is the most prevalent to compare with what is known from previous work. Ideally, this will allow us to adjust when applications occur to better control greasy spot as part of the greasy green disorder. Initial survey attempts of fresh fruit growers have not generated many responses about the greasy green disorder. Plans are underway to potentially conduct in person interviews to get the information needed to inform experiments in the next season. 2. Please state what work is anticipated for next quarter: Field sampling will continue. Post-harvest work will be planned and started with the upcoming harvest season. Interviews will be conducted with growers on the greasy green disorder to give us more information to inform experiments in the next season. 3. Please state budget status (underspend or overspend, and why): No over or underspend on budget currently
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 year 1 of the project, Vismax treated orange trees were significantly more resistant to citrus canker, caustive agent Xanthomonas citri, when the formulated peptide was applied as a dilute foliar spray or soil drench, 7 days prior to leaf inoculation. In year 2 of the project, inoculated greenhouse assays are being established to further probe Vismax-activated defenses, with greenhouse testing conducted by Dr. Megan Dewdney (U. Florida IFAS CREC). In Year 2 Q1 (March 16 2022 – June 15 2022), Canker trial #4 was intiatiated with susceptible sweet orange saplings. The saplings were sprayed with Vismax foliar applications with four different surfactants with varied properties (pH, spread index), in an effort to identify compatible adjuvants that enhance Vismax plant protection activity. The saplings were inoculated and scored, and now this greenhouse trial has been completed. Data is under analysis for future reporting. Protocols are being prepared for subsequent citrus canker and phytophthora root rot trials in Year 2.
1. Please state project objectives and what work was done this quarter to address them: 1. Near term field management(a) Develop methods to time management actionsWe have completed one year of lifecycle documentation, finding as previously noted, that populations begin to grow concurrent with fruit production until mid-summer when we see some fluctuation and reduction in mobile life stages (crawlers, 2nd-3rd instar nymphs) concurrent with the heavy rains of June and July. We again see a rise in the juvenile life stages in the fall after rains and intense heat of late summer cease. While there is a fluctuation in the juveniles in these periods, the number of females with ovisacs remains constant suggesting that there is higher natural mortality during this period. Natural mortality could be due to consumption, physical removal from hosts by rain, and death due to regional temperatures (this last factor should be further evaluated). Concurrent with population development, we have been evaluating a method for population estimates that would be user-friendly if accurate. This method entails minor damage to a branch (e.g. pocket knife scraping) and covering the damage with cardboard to create a tight place for mealybugs to settle as they like these tight points and appear to have an attraction to damage. We are nearing completion of this test and will relate the data to field populations at those same locations. (b) Expand laboratory insecticide and adjuvant screening.Adjuvant screening: We evaluated 6 adjuvants relative to water and an insecticide control (spinetoram). The insecticide control was chosen based on efficacy for mealybugs in previous trials and being less impactful on beneficial organisms that are important for control of lebbeck mealybug. This design allows us to see the impact of adjuvant alone on mortality with promising materials moving forward to field trials in combination with insecticides. The goal of this work is to enhance efficacy of insecticides while maintaining populations of key predators. Several materials are promising based on this trial for use as adjuvants to enhance mortality of most mealybug life stages with minimal impacts on the primary predator of lebbeck mealybug. Incorporating adjuvants such as Clearsurf 90 (NIS), Trio, 800 Plus, and Suffoil and similar products into tank mixes should enhance efficacy of insecticides. 435 oil and Wake Up had 70% and 80% mortality respectively of mealybug destroyer juveniles and should not be used in the latter portion of the season when predators are more active and less direct damage should be occurring to fruit. Based on preliminary lab studies (data not shown), similar testing on agricultural detergents is worthwhile, however these will require detailed phytotoxicity studies prior to field use. (c) Evaluate promising materials in open grove settingA drench trial began on March 8, 2022 comparing Admire Pro, Platinum 75 SG, Belay, Sivanto Prime, Verimark, Aldicarb, and an untreated control for management of lebbeck mealybug. In the absence of a consistent field population, we are brought back field-aged treated expanded soft leaves to challenge with mealybugs in the laboratory every 2 weeks until no available soft leaves were available for a total of 6 weeks after application. At two weeks after application, Aldicarb yielded the greatest mortality with 67% of 2nd-3rd instar juveniles dead compared to 36% from Admire, 21% from Belay, and all other comparable to the untreated control. In week 4 mortality was similar across treatments, and in week 6, Aldicarb continued to have the highest mortality, with Admire, Belay, and Platinum performing better than the control. (d) Fire ant management as part of lebbeck mealybug managementNo new data this quarter- data analysis and manuscript preparation is underway. It is clear that fire ant management is important to lebbeck mealybug management and the most effective tools to keep fire ant populations down is through (e) Evaluate management options for IPCs No work done this quarter. II. Long term managementa. 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 mealybugsIn total, six different species of predators have been identified actively preying on lebbeck mealybug, including both generalists and mealybug specialists. Four other generalist predators found in Florida citrus have been confirmed to consume lebbeck mealybug in lab trials, and likely prey on mealybugs in groves as well. Results on these predators have been written up and are currently being submitted for publication. In addition to predators previously found in fields, we have now found at least two species of parasitoid that are promising for management. A taxonomic specialist identified them as Anagyrus dactylopii and Aprostocetus sp. Future work will focus on establishing a laboratory colony to better understand the potential of this predator for control in citrus groves. b. Determine how to implement mealybug management concurrent with other pest management programs No new data to report this quarter c. Determine what insecticide chemistries inhibit feedingContinuing baseline feeding interaction work. d. Develop tools to minimize spreadNo new data to report this quarter 2. Please state what work is anticipated for next quarter: Note: several of these goals were intended to be completed in the March-May quarter, however the postdoc working on this left for a faculty position and recruitment of a new postdoc was delayed until funding could be ensured. I am attempting to fill the position by the start of August 20221b c. Field testing of insecticides and promising adjuvants (for ovisac penetration). This will include combining adjuvants with Delegate to determine if the combination of adjuvant and insecticide results in increased mealybug instar and ovisac mortality. 2a. Further testing of predators for management in CUPS, field mesocosm studies of predators (bagged trials on infested trees) to determine efficacy in groves compared to controlled lab study. We have a planned open field release of mealybug destroyers with a grower in late June/early July. 2b. Field evaluations of management incorporating data from 1b, c, and d 2c. Continued documentation to develop robust feeding interaction understanding. Based on this, we can evaluate impacts of specific insecticides on this interaction (Can we block it? Can we kill the feeding adult? Can we kill her offspring?) 2d. Develop test to evaluate sanitation procedures for larger equipment (trucks, tractors). Develop protocol for sanitation using solarization and freezing. 3. Please state budget status (underspend or overspend, and why): With additional funding now secured, we are on track to complete this work.
The effect of six nematicide treatments on tree size and fruit yield was described in the previous report (4/02/2022). Here we describe the effects of 3 annual applications of aldicarb on trees at a second grove (Avon Park site) that was havested on April 4, 2022. After 3 years of treatment, we found no significant effects of the aldicarb on trunk girth (2943 vs 2971 cm2, untreated vs treated), tree height (4.97 vs 4.96 ft.), or cumulative fruit drop measured on 4 occassions (48.7 vs 45.2 fruit). Aldicarb-treated trees had 16% greater weight of harvested fruit per tree (3.8 vs. 4.4 lbs, untreated vs treated), but the results were not significant (P=0.63; r2=0.0%). The most notable aspect of this 3rd trial year was the virtual absense of fruit production – fewer than 9 boxes per acre. Both groves, this and that with the multiple nematicide trial, are noteworthy for their low amount of fruit production. The manager of this grove has agreed to pull some of the trees to determine if nematode damage is evident on roots deep in the soil, which might account for the poor tree performance despite the absence of nematodes in soil samples taken to a depth of 12 inches. This project is operating on a no-cost-extension to allow harvest data to be reported. An overview, final report of the project will be submitted in June. A manuscript summarizing the results of the trial is also being prepared.
May 2022Objective 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. In this project, we conducted a two year skirting and fungicide timing trial in a grove with a moderate severity of citrus black spot. We found that skirting had a non-significant effect on CBS incidence and severity. The fungicide timing programs did improve the CBS management but only in the second year when the disease intensity was higher. We can conclude that skirting does not provide additional benefit to a fungicide program and that the fungicide program as currently recommended is sufficient for CBS management in Florida. The opitmal timing of our fungicide programs had not been confirmed in Florida previously and were based on our best estimates from the literature from other countries, particularly Brazil. We were able to confirm that azoxystrobin mixed with difenoconazole is a good product for CBS management in Florida and South Africa. We had similar results with febuconazole as well where the activity was inconsistent between seasons. The reason is not easily determined. The mixture of fluopyram and tebuconazole did not perform well in either country. We had promising results with zinc polyoxin-D rotated with pyraclostrobin in Florida but was unable to try it in South Africa. We also had good results with the adjuvant Goodspray One with pyraclostrobin rotated with copper hydroxide and thyme oil rotated with copper hydroxide in the 2nd year. The product with only one year of data will need confirmation in another season. The results with two years of data will be used to make chemical recommendations in the Florida Citrus Production Guide. We screened our collection Phyllosticta citricarpa isolates and did not find any MAT1-1 isolates, meaning that there appears to be only one mating type here in Florida at this time. We also did not find the new pathogen P. paracitricarpa in our collection. In screening the collection, we did find a species previously not associated with citrus, P. hymenocallidicola, which was not pathogenic on citrus. It appears to be an endophyte on citrus but this was not confirmed. The species was poorly described in the literature and it was unclear as to whether it was a pathogen on the host it was originally described from Hymenocallis littoralis. We did find it was a pathogen on H. littoralis, causing leaf spots. We worked with colleagues from Cuba and were able assist the characterization their isolates. It was found that their isolates were very similar to those in Florida and also only had the MAT1-2 mating type idiomorph. It appears that these populations may be linked, but how is uncertain. This work was published in 2022. Further work from the South African group showed that whole genome analysis of isolates from Florida could detect isolate differences even among this clonal population. This allows for better differentiation among populations in larger population studies. This work was published in 2021. Further work on the global populations of CBS confirmed that Florida is a clonal population based on the number of genetic differences and that the Cuban population was indeed very close to the Floridian one. The next closest population is from eSwatini, but it is quite distant. Again China and Australia were identified to have the most population variation. The remaining populations were regions within South Africa, Argentina, Brazil. These populations had less diversity than China and Australia but had more diveristy than the North American population. The populations outside of North America and China were interconnected and relatively close to each other. This work shows that more collections of isolates needs to be done, especially in Southeast Asia to better understand how P. citricarpa has been moved globally, likely in plant materials.
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