Other


Evaluating the role of greasy spot and peel disorders in the greasy green defect on citrus fruit

Report Date: 10/17/2022   Project: 21-012   Year: 2022

Evaluating the role of greasy spot and peel disorders in the greasy green defect on citrus fruit

Report Date: 10/17/2022
Project: 21-012   Year: 2022
Percentage Completion: 0.2
Category: Other
Author: Megan Dewdney
Sponsor: Citrus Research and Development Foundation

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    



VismaxTM: A novel peptide-based therapeutic for mitigation of citrus diseases, including HLB

Report Date: 09/15/2022   Project: 20-015   Year: 2022

VismaxTM: A novel peptide-based therapeutic for mitigation of citrus diseases, including HLB

Report Date: 09/15/2022
Project: 20-015   Year: 2022
Percentage Completion: 0.4
Category: Other
Author: Michelle Leslie
Sponsor: Citrus Research and Development Foundation

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). 



Developing near and long-term management strategies for Lebbeck mealybug (Nipaecoccus viridis) in Florida citrus

Report Date: 09/12/2022   Project: 20-002C   Year: 2022

Developing near and long-term management strategies for Lebbeck mealybug (Nipaecoccus viridis) in Florida citrus

Report Date: 09/12/2022
Project: 20-002C   Year: 2022
Percentage Completion: 0.82
Category: Other
Author: Lauren Diepenbrock
Sponsor: Citrus Research and Development Foundation

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.       



CTV-T36 vector as a tool to induce efficient flowering in citrus seedlings

Report Date: 08/26/2022   Project: 21-014   Year: 2022

CTV-T36 vector as a tool to induce efficient flowering in citrus seedlings

Report Date: 08/26/2022
Project: 21-014   Year: 2022
Percentage Completion: 0.2
Category: Other
Author: Choaa El Mohtar
Sponsor: Citrus Research and Development Foundation

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



CTV-T36 vector as a tool to induce efficient flowering in citrus seedlings

Report Date: 08/26/2022   Project: 21-014   Year: 2022

CTV-T36 vector as a tool to induce efficient flowering in citrus seedlings

Report Date: 08/26/2022
Project: 21-014   Year: 2022
Percentage Completion: 0.2
Category: Other
Author: Choaa El Mohtar
Sponsor: Citrus Research and Development Foundation

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



Evaluating the role of greasy spot and peel disorders in the greasy green defect on citrus fruit

Report Date: 07/12/2022   Project: 21-012   Year: 2022

Evaluating the role of greasy spot and peel disorders in the greasy green defect on citrus fruit

Report Date: 07/12/2022
Project: 21-012   Year: 2022
Percentage Completion: 0.1
Category: Other
Author: Megan Dewdney
Sponsor: Citrus Research and Development Foundation

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    



VismaxTM: A novel peptide-based therapeutic for mitigation of citrus diseases, including HLB

Report Date: 06/15/2022   Project: 20-015C   Year: 2022

VismaxTM: A novel peptide-based therapeutic for mitigation of citrus diseases, including HLB

Report Date: 06/15/2022
Project: 20-015C   Year: 2022
Percentage Completion: 0.33
Category: Other
Author: Michelle Leslie
Sponsor: Citrus Research and Development Foundation

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.   



Developing near and long-term management strategies for Lebbeck mealybug (Nipaecoccus viridis) in Florida citrus

Report Date: 06/11/2022   Project: 20-002C   Year: 2022

Developing near and long-term management strategies for Lebbeck mealybug (Nipaecoccus viridis) in Florida citrus

Report Date: 06/11/2022
Project: 20-002C   Year: 2022
Percentage Completion: 0.8
Category: Other
Author: Lauren Diepenbrock
Sponsor: Citrus Research and Development Foundation

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.     



Cover crops and nematicides: comprehensive nematode IPM across the grove landscape

Report Date: 05/26/2022   Project: 18-036C   Year: 2022

Cover crops and nematicides: comprehensive nematode IPM across the grove landscape

Report Date: 05/26/2022
Project: 18-036C   Year: 2022
Percentage Completion: 1
Category: Other
Author: Larry Duncan
Sponsor: Citrus Research and Development Foundation

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.  



Understanding the underlying biology of citrus black spot for improved disease management

Report Date: 05/25/2022   Project: 18-006   Year: 2022

Understanding the underlying biology of citrus black spot for improved disease management

Report Date: 05/25/2022
Project: 18-006   Year: 2022
Percentage Completion: 1
Category: Other
Author: Megan Dewdney
Sponsor: Citrus Research and Development Foundation

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.



Developing near and long-term management strategies for Lebbeck mealybug (Nipaecoccus viridis) in Florida citrus

Report Date: 04/13/2022   Project: 20-002C   Year: 2022

Developing near and long-term management strategies for Lebbeck mealybug (Nipaecoccus viridis) in Florida citrus

Report Date: 04/13/2022
Project: 20-002C   Year: 2022
Percentage Completion: 0.75
Category: Other
Author: Lauren Diepenbrock
Sponsor: Citrus Research and Development Foundation

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 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.

Findings through March 2022:

Similar to data from 2021, we have seen lebbeck mealybug populations grow alongside with fruit development. During bloom, we have also documented mealybugs arriving to flowers during bud swell, suggesting that there may be an odor-based attractant. We have seen similar recruitment of mealybugs to tree damage. Based on our observations, we have collected volatile odors from various stages of flower and fruit development as well as from mechanically induced damage. These will be used to determine if specific odors are attractive to lebbeck mealybug in ongoing research. This will help us to understand additional drivers of attraction of lebbeck mealybug to better target management.

The ongoing lure trial has proved unfruitful. Pheromone lures from our colleagues in Isreal were compared to traps baited with virgin females and blank traps. Only traps baited with virgin females attracted male mealybugs. This needs to be refined before it could be useful for local mealybug detection.

(b) Expand laboratory insecticide and adjuvant screening.

Adjuvant screening: 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 leaves with adult female mealybugs and ovisacs. Three adjuvants resulted in high mortality of mealybug adults (comparable to mortality from applications of Delegate (Spinetoram)), and 1 resulted in higher mortality of eggs within ovisacs.

(c) Evaluate promising materials in open grove setting

A 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 field population, we are bringing back field-aged treated expanded soft leaves to challenge with mealybugs. This test will continue for 90 days after application.

(d) Fire ant management as part of lebbeck mealybug management

Data collection has concluded for management of fire ants, sampling of ants associated with mealybug clusters, and cluster collection to determine impact of management on establishment of predators. While we are still exploring the data, we do see a clear impact of fire ant removal whereby a higher abundance of predators are found in ovisacs and mealybug clusters without fire ants present than clusters where fire ants are actively tending clusters. We do not see a the same association with any other ant species present in our research site, which suggest the importance of fire ant management for control of this pest. Additionally, treatments to remove fire ants result in lower numbers of mealybug clusters and ovisacs found during visual surveys.

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

In 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 feeding

Continuing baseline feeding interaction work. Documentation will continue throughout spring and summer, with continued funding insecticide assays should begin in summer/fall 2022.

d. Develop tools to minimize spread

Data have been submitted for publication and the manuscript is currently under review. Recommendations using isopropanol and steam to treat infested materials will be ready to share in spring 2022.

2. Please state what work is anticipated for next quarter:

Most of these are in the beginning stages and will be continued through the coming year

1b 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.

We plan to treat and field-age insecticides similar to the drench study throughout the spring, summer, and fall to determine efficacy on mealybugs in lab assays.

1d. Develop fire ant management recommendations based on 2021-22 data

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.

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):

On track, however some objectives are taking longer than anticipated and we have requested a continuation of current funding and additional funding through a 3rd year of work to complete our objectives.



CTV-T36 vector as a tool to induce efficient flowering in citrus seedlings

Report Date: 04/04/2022   Project: 21-014   Year: 2022

CTV-T36 vector as a tool to induce efficient flowering in citrus seedlings

Report Date: 04/04/2022
Project: 21-014   Year: 2022
Percentage Completion: 0.1
Category: Other
Author: Choaa El Mohtar
Sponsor: Citrus Research and Development Foundation

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 first quarter of this project was to generate the necessary CTV vectors to induce efficient early flowering in citrus1- Generate CTV expression vectors needed to induce efficient flowering in citrusFT3 cloning and sequence analysis:In previous work, we selected Citrus clementia FT3 gene to express from the CTV vector. In our current work, we selected to amplify the FT3 gene from Hamlin, an early flowering sweet orange variety.  We worked with flower and mature leaf tissue and found that the RT-PCR amplification from mature leaves revealed a very weak band whereas amplification from flower sepals revealed a strong band. Thus, we used the RT-PCR product from flower sepals to clone FT3 into 3 different positions within the CTV vector. The different positions of insertion within the CTV vector will enable differential expression of the FT3 protein. We successfully generated vectors that express Hamlin FT3 gene as an insertion between CPm and CP, replacement of p13, and insertion between p23-3’NTR. Sequence analysis revealed a consensus sequence among the different plasmids with minor variations in two plasmids. When compared with the Citrus sinensis FT3 genes theory sequence reported in the National Center for Biotechnology Information (NCBI), the cloned product was a hybrid between the two theory sequences. The variation in the FT3 gene cloned into the different CTV vector plasmids could produce variable efficiency in flower induction in citrus. We selected two divergent sequence as well as 6 consensus sequence vectors for amplification of virions in Nicotiana benthamiana before infecting into citrus.FT3 gene from Arabidopsis thaliana is robust in its ability to induce early flowering in different crop species including perennial hosts. Thus, A. thaliana FT3 gene was amplified by RT-PCR from the mature leaves of A. thaliana and cloned into the CTV vector in the position between CPm and CP. We have sequenced 6 CTV vector plasmids carrying A. thaliana FT3 ORF. Sequence analysis of the plasmids revealed 100% homology to A. thaliana FT3 ORF sequence (NCBI reference # AB027504.1).2- Generate CTV RNA interference (RNAi) vectors to depress targeted genesThere is a complex process to control flowering in plants, especially perennials. Environmental cues are perceived through receptors that instigate a complex cascade of reactions to prevent flowering and, at other times, promote flowering. They involve transcription factors, timing of gene expression level through microRNA, post translational processing, movement through the plant vascular system, and protein competition for the same ligand. As the silencing signal is mobile, to make citrus seedlings more conducive to flowering, we are directing citrus plant RNAi machinery to downregulate citrus genes that repress flowering.  We generated CTV RNAi vectors to target:A. The Terminal Flower Locus (TFL) from C. sinensis. TFL is a suppressor of flowering and competes with FT3 for the FD transcription factor. TFLs from different citrus species and relatives have high homology. Thus, the same CTV RNAi vector could prime the plant RNAi machinery to downregulate TFLs of different citrus species and wild relatives.B. The APETALA 2 (AP-2) transcription factor is an activator of flower repressing genes and a repressor of flower activating genes. Thus, downregulation of AP-2 should make citrus seedlings more conducive to flowering.C. A temperature dependent interaction between FT3 and a membrane bound phosphatidylglycerol (PG) occurs that prevents its uploading into the phloem sieve tube, limiting its movement into the apical meristem where it induces flowering.  We targeted the phosphatidylglycerol phosphate synthase 1 for downregulation by CTV RNAi vector to limit the effect of temperature on flower induction.D. Plasmodesmata are channels of communications between adjacent cells; however, they limit protein movement based on size. We are targeting two genes to increase size exclusion limit-1 and size exclusion limit-2 for downregulation by CTV vector either independently or together (3 vectors in total). ISEs from different citrus species and relatives have high homology. The aim is to improve the amount of FT3 loaded into the phloem which should help induce flowering more efficiently.3- Sequestration of microRNAMicroRNAs are noncoding RNAs transcribed by plants to control level and timing of mRNA of a set of genes. Plants also produce microRNA analogues which sequester microRNAs, preventing the mRNA of the targeted gene from slicing by the RNA-induced silencing complex (RISC). We are mimicking plant interventions against microRNA 156 and 157, which target inducers of flowering, by sequestering them through expression of analogues from the CTV vector. We have produced two CTV expression vectors which target sequestration of microRNA 156 alone or microRNA 156 and microRNA157 together.4- Propagation of transgenic rootstocksCTV vectors can accommodate foreign inserts for many years. However, in a few cases the foreign inserted sequences affect the replication or movement of CTV vector virions within the plant. If the Citrus or Arabidopsis FT3 open reading frame or protein affect CTV replication or movement, we are working on alternative approach to produce an efficient system that induces flowering in citrus by combining transgenic Carrizo rootstocks expressing FT3 (Soares et al., 2020) with CTV RNAi vectors to induce efficient flowering in citrus scions. For this purpose, we are rooting transgenic Carrizo rootstocks, which will be subsequently topped with different citrus scions, especially sweet orange scions.5- Propagation of different citrus genotypesWe planted seeds from different citrus genotypes, including Duncan grapefruit, Madam vinous sweet orange and Pineapple Sweet orange to be available for future bioassays.2. Please state what work is anticipated for next quarter:During the next quarter we aim to infect the different CTV vectors generated into C. macrophylla and test for stability of the vectors in citrus3. Please state budget status (underspend or overspend, and why):We are underspending because one person took a new job during the 1st quarter and we are planning to hire a replacement.



Cover crops and nematicides: comprehensive nematode IPM across the grove landscape

Report Date: 04/02/2022   Project: 18-036C   Year: 2022

Cover crops and nematicides: comprehensive nematode IPM across the grove landscape

Report Date: 04/02/2022
Project: 18-036C   Year: 2022
Percentage Completion: .99
Category: Other
Author: Larry Duncan
Sponsor: Citrus Research and Development Foundation

The trunk girth (cross-sectional area measured one inch above the rootstock union) before treatments began in 2019 was highly correlated with every subsequent trunk measurement, as well as subsequent root mass and fruit yield.  Consequently, it was used as a covariate when analyzing treatment effects on those variables.  The average untreated (control) trunk girth in December 2021 was numerically less than all other treatments, but there were no significant differences between the treatments at end of trial.  However, the growth of the trees in terms of area added and the rate of change was numerically greatest for trees treated with oxamyl. The proportional change in trunk girth during the trial for oxamyl-treated trees was 36% greater (P=0.02) than that of controls. The trees treated alternately with the Syngenta compound-Salibro, Nimitz-Velum, and Temik increased 18%, 9% and 7% more than controls, respectively, but the differences were not significant.  The fibrous root mass density of oxamyl treated trees was 2.27-fold that of untreated trees (P=0.001). Trees treated with combinations of Nimitz-Velum, Syngenta compound-Salibro or Temik had 50%, 36% and 15% more fibrous roots than untreated trees, respectively, but the differences were not significant.  The average tree heights of all treatments differed from the untreated trees (4.95 ft.) by less than 5%.  The harvested fruit from trees in the trial averaged just 35 boxes per acre in 2022.  There were no significant treatment effects on fruit yield.  The highest average yield (aldicarb) was 22% greater than that of the untreated trees and the lowest (oxamyl) was 30% below that of the untreated controls. Although nematicide treatments per se did not explain the variation in yield, the effect they had on the 3-year, cumulative numbers of sting nematodes were highly predictive.  While unrelated to trunk girth prior to nematicide treatments, the relationship between girth and cumulative nematode densities increased in significance each year, and was inverse and highly significant (r = -0.32, P=0.01) at the final measurement.  Similarly, there were inverse relationships between the cumulative sting nematode populations and fibrous roots (r= -0.39, P=0.003) and fruit yield (r= -0.29, P=0.03) measured during this reporting period.  The only variable more predictive of yield was the trunk girth. Stepwise multiple regression of yield against the trunk girth in 2019 (P=0.001), prior to treatments, combined with the cumulative sting nematode populations during the trial (P=0.01) explained 31% of the fruit yield in 2022.  The data suggest that trees responded positively to nematode management.  A second experiment to assess the effect of aldicarb on yield will be reported in the next (final) report when harvest data are available.  A manuscript is being prepared and those analyses and conclusions will be given in that report.     



Novel multi-metal systemic bactericide for HLB control

Report Date: 03/28/2022   Project: 18-020   Year: 2022

Novel multi-metal systemic bactericide for HLB control

Report Date: 03/28/2022
Project: 18-020   Year: 2022
Percentage Completion: 1
Category: Other
Author: Swadeshmukul Santra
Sponsor: Citrus Research and Development Foundation

Various multi-metal formulations of Mg-hydroxide and/or Zn-hydroxide were successfully loaded with Cu-chelates using a sol-gel strategy. The average hydrodynamic radius of the MM50C50M(Mg-Cu), MM25C75Z(Zn-Cu), and MM25C75M(Mg-Cu) formulations determined by DLS were 880, 580, and 450 nm, respectively. XRD results suggested the sucessful substitution of Cu in the MM25C75M Mg-OH brucite lattice structure. All multi-metal formulations demonstrated a superior or equivalent performance against model pathogens such as Xanthomonas alfafae relative to copper standards in vitro. Systemic movement results suggested signifcant uptake of Mg for all multi-metal formulations. XRF and AAS results suggest the systemic movement of Cu was also detected in the leaves, roots, and stems at significant levels that exceed the MIC of Cu. Approximately 24-hours post treatment, the Cu-Zn treatment was detected primarily in the stems and leaves. Within 72-hours after application signifcant increases of Zn and Cu was detected in the roots. All Zn-Cu and Zn control formulations signifcantly reduced Canker lesion sums as compared to the untreated controls in a series of greenhouse trials.  MM25C75M(Mg-Cu), MM25C75Z(Cu-Zn), and MM10C45M45Z(Mg-Cu-Zn) treated grapefruit trees in the first field trial were observed to have 0% of fruit with canker lesions present throughout this trial, which was significantly better than the untreated control (0.8%, 1.2%, 4.6%, and 8.2% at successive disease ratings). The yield of MM25C75M(Mg-Cu), MM25C75Z(Cu-Zn), and MM10C45M45Z(Mg-Cu-Zn) treated grapefruits was 50.6, 60.5, and 49 lb/tree respectively. The yield of MM25C75Z(Mg-Zn) treated grapefruits showed a statistic difference compare with untreated grapefruits (43.8 lb/tree). The mean fruit diameter for MM25C75M(Mg-Cu), MM25C75Z(Mg-Zn), and MM10C45M45Z(Mg-Cu-Zn) treated grapefruits of 86.9, 83.6, and 85 mm, respectively, was not significantly different from the untreated controls (85.8 mm). No significant differences were found in internal fruit quality based on brix acid ratio or total soluble solids.All experimental treatments significantly improved yield with respect to the untreated control. The untreated control group was observed to yield an average of 157 lbs/tree. The Kocide copper standard control groups reported an average of 171 lbs/tree. Although not statsicallystatistically different from the Kocide control, laboratory grade Cu-ZnO, laboratory grade TMN 113-MgSOL-Cu, and Bz1.1-Cu, and Bz1.1 reported average yields of 202, 203, 189, and 207 lbs/tree, respectively. Laboratory grade ZnS-Cu did outperform all materials significantly with an observed yield of 229 lbs/tree in this years trial. All Mg:Cu ratios evaluated in the MM25C75M(Mg-Cu) subtreatment group promoted yields statistically similar to each other and the Kocide control. 



VismaxTM: A novel peptide-based therapeutic for mitigation of citrus diseases, including HLB

Report Date: 03/15/2022   Project: 20-015   Year: 2022

VismaxTM: A novel peptide-based therapeutic for mitigation of citrus diseases, including HLB

Report Date: 03/15/2022
Project: 20-015   Year: 2022
Percentage Completion: 0.33
Category: Other
Author: Michelle Leslie
Sponsor: Citrus Research and Development Foundation

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 2 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 will further probe Vismax-activated defenses, with greenhouse testing conducted by Dr. Megan Dewdney (U. Florida IFAS CREC).  In Year 1 Q4 (Dec 16  2021 – March 15 2022), susceptible orange saplings were obtained and propagated for Canker trial #4. This trial will commence with treatments in Year 2 (Q1), and will include Vismax foliar rate testing with four different surfactants with varied properties (pH, spread index). Protocols are being prepared for subsequent citrus canker and phytophthora root rot trials.