Other


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.   



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

Report Date: 12/21/2021   Project: 18-006   Year: 2021

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

Report Date: 12/21/2021
Project: 18-006   Year: 2021
Percentage Completion: 0.9
Category: Other
Author: Megan Dewdney
Sponsor: Citrus Research and Development Foundation

September 2021Objective 1:  Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.Objective 3:  A MAT-1-1 isolate may enter Florida and allow for the production of ascospores.  The industry needs to know if this happens, as it will affect management practices.  Additionally, the existing asexual population may be more diverse than currently measured.  If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits.  We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification.  We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp.  A skirting and fungicide timing trial was conducted during two different years (2019-2020 and 2020-2021) in a commercial citrus grove located in Southwest Florida. This study aimed to evaluate the optimal timing for fungicide application and investigate if skirting pruning helps reducing citrus black spot (CBS) incidence and severity under field conditions. It is a split-plot design with skirting as the main plot and fungicide treatments as the minor plot.  Our preliminary results showed no statistical differences in disease incidence among the application timing treatments in both seasons. However, disease incidence by tree was much higher in 2020-2021 compared to the 2019-2020 seasons likely due to favorable environmental conditions. In the same study, evaluation of disease severity relied on the area fruit affected by CBS symptoms. There was no statistical difference between the fungicide treatments nested within skirting and non-skirting plots during the 2019-2020 season when the severity was evaluated by tree. However, in the 2020-2021 season, a statistical difference was found between fungicide treatments and untreated control when the disease pressure was substantially higher. In the non-skirted trees, only the Florida standard (FLS) treatment was statistically different from our untreated control.  The Early and Late application treatments showed no statistical difference when compared to the untreated control or FLS. Within all skirted plots, fungicide timing treatments showed statistical differences compared to the untreated control, but not among them. Therefore, our results provide evidence for the best timing for the fungicide applications on the disease severity and confirms that tree skirting has a minor effect on disease control when we evaluate the data by tree.  We are still working on a new analysis to see if there is a significant difference among the treatments when we evaluate the incidence and disease data by fruit rather than tree.  We have been delayed because our statistical consultant has been difficult to contact but hope that it will be easier in the new year.  There may be a difference when we change the scale of how we analyze the data.  We are also examining the weather data from both seasons to see what factors rainfall and temperature may have played. In April and May, we prepared the fungicide trial. Trees were selected for chemical application based on disease severity levels, confirming presence and quality of selected trees in the field, and labeling selected trees along with warning signs in the area. Fifteen treatments were selected.  Each treatment has five replicates of three trees and were blocked according to disease severity according to randomized block design. Beginning from late May to currently, there has been a monthly (every four weeks) fungicide application the chemical treatments, and there will be a total of six applications of each treatment, from May to October. The final chemical application was conducted in October. The trees are periodically observed for symptoms and ripening but data collection is anticipated to occur in March.  Additionally, trees are being relabelled due to label weathering, to ensure organization and visibility in preparation for disease rating. Evaluation of the second season of trials, in which different fungicide products were tested for their efficacy to protect citrus fruit from CBS infection, was recently concluded. Products tested include Enable (Indar) and Luna Experience sprayed on their own as well as Copper hydroxide sprayed in alternation with either Amistar Top or Headline (Cabrio). Fungicides were applied every 4 weeks from October 2020 until March 2021, in a Valencia orange orchard with a history of CBS. Data analysis is currently under way. An additional 8 South African and 8 global (from Argentina and Swaziland) isolates have been sequenced. As the next-generation sequencing data becomes available, the analysis and results are continuously updated to include the new data. DNA from 16 isolates (Argentina, Australia, Brazil and China) passed QC and is in the process of being sequenced. More isolates from Brazil and China are being cultured for DNA extraction and sequencing. Sixty-five P. citricarpa isolates (36 from South Africa, representing the five provinces where CBS is found, and 29 from other countries, Argentina, Australia, Brazil, China, Eswatini and USA) have been sequenced. Sequence data for 6 isolates, 1 from China, 1 from Brazil and 4 from USA are in the process of being generated. The sequencing data for the 65 isolates were analysed in the same manner as previously described, namely read mapping and variant calling, and in silico genotyping. Both analyses approaches showed the same patterns in terms of genetic distribution of isolates. Isolates from China are the most genetically distinct, while there are different degrees of genetic connectivity between isolates from Argentina, Australia, Brazil, Eswatini, USA and those form South Africa, and corresponds with previously published results (Carstens et al., 2017). Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.). Our studies on the diversity of Phyllosticta spp. associated with citrus in Florida have continued. Our collection of P. citricarpa isolates were obtained from citrus fruit in different areas under quarantine from 2010 to 2020. These isolates were previously screened by morphology to remove nonpathogenic P. capitalensis isolates. We have now screened the majority of the remaining 202 isolates using molecular techniques to determine if we have cryptic species that may have been misidentified as P. citricarpa. To date we have screened 168 isolates by amplifying and DNA sequencing the tef-1a (Translation elongation factor-alpha) locus.  A few isolates were rescreened to confirm conclusions but the data is being organized and prepared for publication.  The screen revealed that two isolates (Gc-6 and Gc-7) match to a Phyllosticta species not previously reported in association with citrus. The best sequence match based on ITS and tef-1a is to a species reported previously as a pathogen on a member of the Amaryllidaceae family (Hymenocallis littoralis) in Australia. Numerous inoculations of citrus have determined that these isolates do not cause citrus black spot. A thrid leaf inoculation on Hymenocallis species are underway but preliminary evidence appears to show that the results will be the same. These studies indicate that these isolates are P. hymenocallidicola and infection studies indicate that they are pathogenic on spider lilly (Hymenocallis sp.). Vegetative and sexual compatibility assays (sandwich mating) confirm that Gc6 and Gc-7 isolates are heterothallic and belong to the same mating type, as neither isolate was a capable of producing pseudothecia in solo cultures or in paired cultures with one another. Characterization of the tef-1a sequence from the remaining isolates in our collection screening and further characterization of these new species is continuing to obtain robust information on the diversity of Phyllosticta species and determine the presence of cryptic species in Florida.We screened an additional 12 P. citricarpa isolates for mating-type (MAT1-1 and MAT1-2). Only the MAT1-2-1 mating type has been detected in new isolates collected in 2020. We conclude that the MAT1-1 mating type is still absent in the Floridian population. To determine the phenology of fruit susceptibility inoculation studies of citrus fruit (Meyer lemon) were performed in a quarantine greenhouse at the Florida Department of Agriculture and Consumer Services (DPI) in Gainesville. A total of 97 fruit were used in this experiment. Of these 97 fruits, 25 served as controls and 72 were inoculated with the Gc-12 isolate of P. citricarpa. Disease assessments were performed weekly for a full calendar year. A total of 50 fruit produced symptoms in this period. All 50 were from fruit inoculated with the Gc-12 isolate, and no symptoms were observed in the control treatment. Therefore, from all inoculated citrus fruit, 69.4% produced symptoms, and 30.5% remained asymptomatic. The indications from this one experiment suggest that citrus fruit are susceptible at all stages of their development regardless of their maturation time. A second trial was planned for 2021 to determine the period of fruit susceptibility to P. citricarpa but the trees only recently flowered and the fruitlets are not yet ready for inoculation.  



Novel multi-metal systemic bactericide for HLB control

Report Date: 12/20/2021   Project: 18-020   Year: 2021

Novel multi-metal systemic bactericide for HLB control

Report Date: 12/20/2021
Project: 18-020   Year: 2021
Percentage Completion: 0.95
Category: Other
Author: Swadeshmukul Santra
Sponsor: Citrus Research and Development Foundation

Five multimetal bactericides containing Cu as a minor constiuent: Cu-ZnS, Cu-ZnO, MgCu-gel, TMN 113-MgSOL-Cu, and ZnBoric-Cu were evalauted against 3 respective controls MgSOL, ZnBoric, and Kocide. MgCu-gel was varied with regards to the Mg:Cu ratio in the 2021 canker trial to include four subtreatment groups MgCu 16:4, MgCu 16:3, MgCu 16:2, MgCu 16:1 in order to elucidate any effect(s) on observable canker control and yield.  All treatment groups experienced relatively low canker disease pressure at the trial site in 2021 where the untreated control group reported canker fruit lesion incidences (% of 100 fruit) of 0.1, 0.1, 0.9, 3.3, 4.8 and 5.7 in June, July, August, September, October, and November, respectively. The standard copper control (Kocide 3000) treatment group was observed to have a disease incidence of 2.1 in November (time of highest disease pressure in the trial for all treatment groups). All five mutlimetal treatment groups performed at a statistically similar level as compared to the Kocide copper control.  The MgCu treatment groups reported the lowest magnitude of canker incidence ranging from 0.5-0.7 (% per 100 fruit, november). All Mg:Cu copper ratios included in this years trial performed stiatiscally similar to each other with regards to canker incidence. Cu-ZnS, Cu-ZnO, TMN 113-MgSOL-Cu and ZnBoric-Cu performed statistically similar to the MgCu treatment groups at 0.7, 1.4, 0.8, and 0.8, respectively (% per 100 fruit, november). Although not statiscally different ZnBoric performed at a disease incidence of 1.1 and ZnBoric-Cu at 0.8 suggesting some improvement from the multi-metal combination as documented in prior field trials by Cu-ZnS, Cu-ZnO, etc. All experimental treatments signifcantly 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 statsically different from the kocide control Cu-ZnO, TMN 113-MgSOL-Cu, and ZnBoric-Cu, and ZnBoric reported average yields of  202, 203, 189, and 207 lbs/tree, respectively. Cu-ZnS did outperform all meterials significantly with an observed yield of 229 lbs/tree in this years trial. All Mg:Cu ratios evaluted in the MgCu-gel subtreatment group promoted yields statistically similar to each other and the kocide control.                



Near-term approaches of using alternative HLB-tolerant cultivars for increased production and improved juice quality

Report Date: 11/29/2021   Project: 19-024   Year: 2021

Near-term approaches of using alternative HLB-tolerant cultivars for increased production and improved juice quality

Report Date: 11/29/2021
Project: 19-024   Year: 2021
Percentage Completion: 100
Category: Other
Author: Yu Wang
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them:This project ends at the end of October, 2021, so this is the last report. There are two objectives in this project, and theprogress of each objective is listed here: Objective 1. Evaluation of blended juice using released HLB-tolerant sweetorange/mandarin cultivars via analyses of sensory and consumer acceptance. All the sensory studies have beencompleted to verity the potential of blending Sugar Belle juice with early variety (Hamlin) and late variety (Valencia). Fromthe results obtained, 50/50 Sugar Belle blend was preferred by most panel. Ten percentage of Sugar Belle blended withHamlin could improve the juice appearance and flavor significantly, while 10% of Sugar Belle blended with Valencia didn’timprove the juice quality significantly. In addition, a shelf life study for the Sugar Belle juice has been studied. In a 90-dayshelf-life study, Sugar Belle juice was stored at 4 °C and samples were tested at day 0, 30, 60 and 90. A sensory panelwas used to determine the changes of sweetness, bitterness and overall flavor. For the samples of the first year, we didn’tobserve any significant changes (p=0.05) over time for the storage intervals, while for the second year, samples at day 0were different from other intervals indicating the thawing process may affect the samples at day 0. Objective 2. Identifymore tolerant cultivars resembling the quality of Valencia for the juice market, and identify a chemistry definition ofconsumer accepted orange flavor. In this quarter, our main focus was to put all the data and results into a publication. Apublication entitled “Identification of key flavor compounds in citrus fruits: a flavoromics approach” has been completedand now published in Journal of ACS Food Science & Technology:https://pubs.acs.org/doi/10.1021/acsfoodscitech.1c003042. Please state what work is anticipated for next quarter:This project has been completed.3. Please state budget status (underspend or overspend, and why):The budget has been spent appropriately.



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

Report Date: 10/14/2021   Project: 20-002C   Year: 2021

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

Report Date: 10/14/2021
Project: 20-002C   Year: 2021
Percentage Completion: 0.6
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:

I. Near term field management

(a) Develop methods to time management actions

Working with 6 commercial citrus growers throughout central Florida, we have been developing a robust dataset to describe the seasonal phenology (“life cycle”) of lebbeck mealybug populations. Describing the seasonal life cycle of this pest helps determine key timepoints for management of its population in relation to tree development and/or time of year. In addition to intense sampling to describe the annual seasonal biology of lebbeck mealybug, we are also testing rudimentary pheromone traps to see if we can attract male mealybugs into traps. Reducing the number of males in a mating population can reduce offspring production by females (data shown in a previous report). We are hoping to determine if traps based on pheromones can help detect populations and their relative phenology in the future, which could enable growers to fine tune management actions. Starting in January 2021, we observed zero to very low mealybug population until late March 2021 from all citrus groves sampled in Florida. However, since onset of spring season (April 2021), we have been recording greater mealybug population densities in the citrus groves with mealybug populations peaking in late spring 2021 (June 2021), before a steady decline in population up till July 2021 (Figure 1). Immatures appear to reach a population peak in early Spring (May 2021), while crawler populations appears to reach a peak in late Spring 2021 (June 2021) (Figure 1). We also observed that mealybugs were more likely to be located around the calyx and peduncle of fruit set and developing fruits. They feed on the developing citrus fruit causing premature fruit abortion and/or fruit necrosis in 2 weeks which will result in yield loss (Figure 2).

Figure 2. Abundance of different life stages of lebbeck mealybugs inn central Florida

Figure 2: infested developing fruit over 2 week period

(b) Expand laboratory insecticide and adjuvant screening.

EPF data presented in previous report.

Adjuvant screening: Preliminary trials have been conducted with adjuvants alone to determine their lethality to lebbeck mealybug. A total of 10 adjuvants, including and 4 from Helena Agri Enterprises were mixed with DI water at label rates and sprayed until dripping on Volk lemon leaves with 2nd and 3rd instar mealybugs attached. 8 of the adjuvants resulted in significantly greater mealybug mortality over a 7-day period compared to a DI water control, and were comparable to the morality achieved by the insecticide Delegate (Spinetoram) applied at maximum label rate.

All adjuvants and Delegate were also tested against adults and larvae of Cryptolaemus montrouzieri to determine their potential impact on known predators of lebbeck mealybug. 3 of the adjuvants resulted in significantly higher C. montrouzieri larvae mortality compared to a DI water control, while none of the adjuvants resulted in higher C. montrouzieri adult mortality. The insecticide Delegate resulted in 35% mortality of C. montrouzieri larvae, and 100% mortality of C. montrouzieri adults, both significantly greater than a DI water control.

Next steps involve testing adjuvants against mealybug ovisacs and combining adjuvants with Delegate to determine if the combination of adjuvant and insecticide results in increased mealybug instar and ovisac mortality.

c. Evaluate promising materials in open grove setting

The insecticides Malathion and Assail (Acetamiprid) were tested on an ad hoc basis on heavily infested young citrus trees. Trees were part of a separate experiment and became naturally infested with lebbeck mealybug. Both Malathion and Assail were applied at maximum label rate, and the number of living mealybugs of all life stages at 3, 6, and 15 days post application were compared to the number of living mealybugs pre-application.

Both Malathion and Assail resulted in close to 100% mortality of adults and instars. However, in 35% of ovisacs, at least some eggs survived treatment and crawlers emerged in the following days. These results suggest that Malathion and Assail can be useful to knock down infestations of lebbeck mealybug, but that populations may rebound without additional treatments.

We have additional trials planned pending appropriate infestation levels at 3 locations.

(1d) Ant Management

We are testing a variety of methods to remove red imported fire ants (Solenopsis invicta) from citrus groves. Four experimental treatments were tested and compared to an untreated control: Chlorpyrifos drench followed by a Bifenthrin drench after 3 months, Clinch ant bait (Abamectin) applied every 3 months, Extinguish ant bait (S-methoprene) applied every 3 months, and spot treating ant colonies with hot water.

Fire ant abundance was assessed by counting the number of surviving colonies, and by determining presence/absence of foragers with cookie baits. Natural enemy abundance was assessed by dissecting mealybug clusters for predator larvae. Sampling for each of these methods took place every 2 weeks.

Preliminary data show that both insecticidal baits and hot water treatments significantly reduced fire ant colony abundance initially, while the chlorpyrifos drench had no effect on colony abundance. However, fire ant colony abundance rebounded in the hot water treatments after 3 months and has returned to close to the same levels as control plots. Colony abundance in both bait treatments has remained consistently low. Finally, although Chlorpyrifos had no effect on colony abundance, once bifenthrin was applied, colony abundance dropped and has continued to trend downwards, although this treatment still remains statistically similar to control plots.

Predator populations within mealybug clusters were unaffected by treatment, and numerous predators have been found in mealybug clusters across all treatment types.

Future work includes retreating hot water plots and continuing to monitor colony abundance for a total of 1 year. Ant forager identification and quantification is currently underway.

II. Long term management

a. Assessment of predator- what is currently in the system, can they be enhanced, how to implement use of predators alongside insecticide use for ACP and mealybugs

Predators currently present in the system:

Using molecular gut content analysis, we have identified a broader suite of predatory arthropods, including several species of spider, that consume lebbeck mealybug than anticipated. We are still extracting DNA from the remaining field-collected predators and anticipate having more to share with the regards to this sub area in our next report.

Predators available for purchase or present locally for potential augmentative control:

Predators who directly consume prey:

The commercially available predators Cryptolaemus montrouzieri, Orius insidiosus, Adalia bipuctata, Hippodamia convergens, and Chrysoperla carnea have been screened using no-choice assays to determine if they will feed on 2nd and 3rd instar lebbeck mealybugs, and on mealybug ovisacs. Additionally, several wild-caught and lab reared predators have also been screened, including the coccinellids Diomus austrinus, Coccinella septempunctata, and Olla v-nigrum; larva of the genus Ceraeochrysa (colloquially called trash bugs); and the earwig Euborellia annulipes.

For commercially available predators, only Cryptolaemus montrouzieri consistenty consumed both lebbeck mealybug instars and ovisacs. Chrysoperla carnea consumed larvae at comparable rates to C. montrouzieri, but only infrequently consumed ovisacs. Adalia bipuctata and Hippodamia convergens ate only a few instars, and no ovisacs. Finally, Orius insidiosus consumed virtually no instars, and no ovisacs.

For wild-caught and lab-reared predators, both Ceraeochrysa larvae and Euborellia annulipes consumed instars and ovisacs at high rates, and although not quite as consistently as C. montrouzieri. Diomus austrinus and Coccinella septempunctata consumed a few larvae, and only infrequently consumed ovisacs. Olla v-nigrum ate very few instars and no ovisacs.

From our results, only Cryptolaemus montrouzieri shows promise as a commercially available biological control agent for lebbeck mealybug, although Ceraeochrysa larvae and Euborellia annulipes may act as natural biological control in the grove. Chrysoperla carnea may be useful for controlling mealybug larvae, but not reproductive adult females.

Future work will consist of conducting choice assays with predators that did feed on lebbeck mealybug, as well as testing greater numbers of all predators to increase sample size.

Field trials:

Cryptolaemus montrouzieri were tested in the field against a lebbeck mealybug infestation on an ad hoc basis. A grower experienced a lebbeck mealybug infestation inside a Citrus Under Protective Screening (CUPS) grove and ordered Cryptolaemus montrouzieri to release to control the outbreak. Trees infested with mealybugs were marked and the infestation recorded as low or high. 10,000 C. mountrouzieri were released to cover an area of ~3,150 mature trees, ~100 of which were infested. Marked trees were reassessed 2 weeks after release.

After 2 weeks, most of the infested trees had visibly reduced mealybug abundance, and in many cases, all mealybugs had been consumed. In approximately half of infested trees, Cryptolaemus montrouzieri larvae could be seen feeding on mealybug clusters. The majority of mealybug clusters that remained undamaged either appeared to have died on their own accord or were composed of very few individuals. While preliminary, these results indicate that Cryptolaemus montrouzieri can effectively be used control infestations of lebbeck mealybug in enclosed settings.

Parasitoids:

Limited numbers of Anagyrus pseudococci, a commonly used parasitoid for commonly occurring mealybugs in greenhouses, were tested to determine if they would parasitize lebbeck mealybug adults and ovisacs. A. pseudococci adults were placed in arenas with adult lebbeck mealybugs, or ovisacs and left for 40-48 hrs. Surviving parasitoids were then transferred to adult Citrus mealybugs and given an opportunity to parasitize them, to act as a positive control. Mealybugs were checked for parasitism after 16 days. Most parasitoids died in arenas with lebbeck mealybugs, and none parasitized adults or ovisacs. However, half of those that survived went on to parasitize Citrus mealybug adults. These preliminary results suggest that A. pseudococci will not readily parasitize lebbeck mealybug, however further trials with more individuals and life stages of mealybugs are needed.

b. Determine how to implement mealybug management concurrent with other pest management programs

Ongoing population monitoring shows that, to date, there is no correlation between flush development and population development of lebbeck mealybug. We had anticipated that this pest, because it feeds in a similar manner to ACP, may have populations that were in sync with flush development, however flush phenology does not appear to be directly tied to mealybug population growth. What we do see, however, is a large increase in population concurrent with fruit set and development. Large populations have been documented developing at the calyx junction, often leading to fruit death and abortion of fruit well before physiological fruit drop. This is still an ongoing area of work; however, we have been able to confirm that fruit development is a key period to target management actions, and several growers have been successful in keeping populations of mealybugs down while protecting their fruit from heavy impacts of this pest by targeting appropriate, systemic materials during this time point. We still have several months remaining in this study, but the data generated so far have been crucial to understanding when to time management actions.

c. Determine what insecticide chemistries inhibit feeding

We are continuing to tease apart the feeding biology of lebbeck mealybug, which is the integral step to enable this sub area to be completed.

d. Develop tools to minimize spread

Killing crawlers with isopropyl alcohol

Different concentrations of isopropyl alcohol were tested to determine how lethal they are to 1st instar lebbeck mealybugs. 50%, 70%, and 90% solutions of isopropanol were sprayed onto mealybug crawlers placed on cloth swatches and compared to a DI water control. Both 1 spray at each concentration, and 2 sprays at each concentration were tested. Mortality was assessed after 5, 10, and 15 minutes. The test was repeated, this time assessing mortality at 30 min, 1hr, and 2hrs. For all concentrations of isopropanol, 1 spray resulted in significantly greater mealybug mortality compared to the control. However, several mealybug crawlers remained alive and active after a single spray for all concentrations. 2 sprays of each concentration resulted in almost 100% mortality or incapacitation at all time points.

Killing ovisacs with isopropyl alcohol

50%, and 70% solutions of isopropanol were also tested against lebbeck mealybug ovisacs to assess lethality. Single ovisacs were sprayed both 1 and 2 times with each concentration and compared to a DI water control. Mortality of the adult females was assessed at 0, 3, and 5 days, and ovisacs were held for up to 3 weeks to see if crawlers emerged.

Adult mortality was high by the end of 5 days for both concentrations and spray regimes and was significantly greater than the DI water control. However, most ovisacs remained viable, and crawlers emerged within several days for all treatments. Our results suggest that isopropanol is effective against adults but does not reliably penetrate the ovisac to kill eggs.

Next steps involve testing 90% isopropanol against ovisacs, and more thoroughly soaking ovisacs with each different concentration to determine if any amount of isopropanol will result in reliable egg mortality.

Using steam to kill adult mealybugs and ovisacs

Adult mealybugs and ovisacs may be accidentally transferred from infested groves on tools and equipment. This is a particular concern for Individual Protective Covers (IPCs), where ovisacs can easily be transferred to new young trees if IPCs are reused. Steam treatments may be an effective method of sanitizing equipment and killing both adults and ovisacs.

Using a steam cabinet on-station, we steam treated adults and ovisacs at 100, 120, and 130 degrees Fahrenheit for 5, 10, and 15 minutes to determine mortality. Previous preliminary experiments showed steam treatments at 130 degrees Fahrenheit for 15 and 30 minutes resulted in 100% mortality. We also tested ovisacs inside of a folded IPC to determine if ovisacs within bags would be more insulated from heat and moisture during steam treatments. For ovisacs in IPCs, 120 degrees for 10 and 15 minutes, 130 degrees for 5 and 10 minutes, 135 degrees for 5 minutes, and 140 degrees for 5 minutes were all tested.

Mortality was assessed at 0, 3, and 5 days after treatment, and ovisacs were held for an additional 2 weeks to see if crawlers emerged. For ovisacs and adults without an IPC, adult mealybug and ovisac mortality was not significantly different than the control at 100 degrees F for all time points. Crawlers emerged from all ovisacs at this temperature. At 120 degrees F for 5 minutes, mortality was also functionally 0%, and crawlers emerged. However, mortality rose to 100% at 120 degrees F for 10 and 15 minutes, and no crawlers survived. At 130 degrees F, mortality was 100% for all time points with no crawlers. Inside of an IPC, there were no crawlers and mortality was 100% at 130 degrees for 10 minutes, and at 140 degrees for 5 minutes. All other time points had functionally 0% mortality, and crawlers emerged.

It appears that IPCs insulate ovisacs and adults from steam treatments and require additional time or higher temperatures to sterilize adults and eggs. Currently, exposed ovisacs will be sterilized at 120 degrees for 10 minutes, and at 130 degrees for 5 minutes. Inside of an IPC, ovisacs will be sterilized at 130 degrees for 10 minutes, and 140 degrees for 5 minutes.

A final test will be conducted with ovisacs inside IPCs at 120 degrees to determine how much time is required to achieve 100% mortality at this temperature.

Future work will include sanitation procedures for large equipment and vehicles to develop recommendations for growers and scouts.

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

Many of the updates provided in the previous sections are updates to ongoing research. These projects will continue to make progress over the next quarter.

3. Please state budget status (underspend or overspend, and why):

We are currently on track with our spending for this project.



Novel multi-metal systemic bactericide for HLB control

Report Date: 10/12/2021   Project: 18-020   Year: 2021

Novel multi-metal systemic bactericide for HLB control

Report Date: 10/12/2021
Project: 18-020   Year: 2021
Percentage Completion: 0.9
Category: Other
Author: Swadeshmukul Santra
Sponsor: Citrus Research and Development Foundation

Industry-grade multi-metal formulations containing 2 wt% metallic Cu were manufactured and supplied by our industry collaborator for further evaluation. The industry-grade Cu-Mg was compared with two different versions of lab-grade Cu-Mg, Cu-hydroxide industry standard and Cu sulfate pentahydrate at the same metallic Cu content for differences in particle size, surface charge, pH, and antimicrobial efficacy. The industry-grade formulation presented an average hydrodynamic radius of 207 nm with a broader size distribution (PDI 0.482) as compared to the lab-grade formulation which had a similar radius of 293 nm but smaller size distribution (0.174 PDI). In contrast, the surface charge of both formulations was found to be comparable, indicating that the primary particle for both formulations is stable in suspension after dilution. FTIR analysis was also conducted on the formulations to compare the interactions between particle and adjuvants. Both lab-grade and industry-grade presented almost identical spectra, which indicates that the industry-grade formulation’s components behave similar to that of the lab-grade formulation. The antimicrobial properties of both formulations were compared. The minimal inhibitory concentrations (of Cu) for both materials were the same when assessed on Xanthomonas alfalfae and Pseudomonas syringae at 32 ppm Cu and 64 ppm Cu, respectively. While Clavibacter michinganesis’ minimum inhibitory concentration for the industrial-grade formulation was one stage higher than that of the laboratory synthesized formulation. Both formulations outperformed copper sulfate and the Cu-hydroxide industry standard for all pathogens tested. A more quantitative assessment of the bactericidal potency of these materials will be performed and reported. Additionally, leaf residue of the formulations was imaged through scanning electron microscopy. From our overall assessments, it was observed that both the industry and laboratory synthesized multi-metal formulations were of similar particle size, chemical characteristic, and antimicrobial potency. Preliminary field trial of the industry-grade multi-metal formulation is underway from which efficacy and metal residue data will be reported in the near future. Additionally, we will be reporting on tissue sample characterization including sap to understand the systemic movement of the A.I. 2021 harvest data is tentatively scheduled to be collected for the week of Nov 15th.