Plant Improvement


Testing grapefruit trees expressing an anti-NodT antibody for resistance to HLB

Report Date: 06/17/2022   Project: 18-016   Year: 2022

Testing grapefruit trees expressing an anti-NodT antibody for resistance to HLB

Report Date: 06/17/2022
Project: 18-016   Year: 2022
Percentage Completion: 0.98
Category: Plant Improvement
Author: Tim McNellis
Sponsor: Citrus Research and Development Foundation

This is the final report for project 18-016.   A final comprehensive report is being submitted along with this summary document.  The final comprehensive report contains additional project details and relates the project to previous funding by CRDF. 1. Please state project objectives and what work was done this quarter to address them:  The project objective is to test transgenic ‘Duncan’ grapefruit trees for resistance to citrus greening.  Infections were done by grafting onto infected rough lemon rootstocks, caged infected psyllid feeding, and natural infection outdoors.  Tree symptoms were visually assessed and ‘Candidatus Liberibacter asiaticus’ bacterial titers were determined by quantitative PCR (qPCR) in collaboration with Southern Gardens.  All the tests proposed were performed and have now been completed.  This quarter, we performed final data analysis and began to prepare a manuscript describing the tree citrus greening disease data.  The manuscript is still at an early stage, but figures are nearing completion, which is the first major step.  The text will be adapted from a M.S. student Chad Vosburg’s thesis document. 2. Please state what work is anticipated for next quarter:  This is the final report.  However, we are still working on a manuscript to publish the tree test data. 3. Please state budget status (underspend or overspend, and why):  The project remains underspent in part due to support for graduate stipends and tuition waviers from the Plant Pathology & Environmental Microbiology Department of Penn State and the Penn State Graudate School.   We are reporting the project as 98% completed because we still need to submit the manuscript to a peer-reviewed journal and get it accepted for publication.



Performance of newly released grapefruit cultivars and rootstocks in the Indian River Citrus District

Report Date: 06/14/2022   Project: 18-037C   Year: 2022

Performance of newly released grapefruit cultivars and rootstocks in the Indian River Citrus District

Report Date: 06/14/2022
Project: 18-037C   Year: 2022
Percentage Completion: 1
Category: Plant Improvement
Author: Ronald Cave
Sponsor: Citrus Research and Development Foundation

     A large-scale trial of HLB-tolerant citrus cultivars addresses the need of Indian River growers to identify the best rootstocks and scions for growing fresh citrus fruit. This project had two objectives: (i) Assess the performance of new grapefruit cultivars with selected rootstocks; and (ii) Evaluate the influence of UFR and other rootstocks on grapefruit scion development in comparison to legacy/standard rootstocks. Trial 1 consisted of 18 grapefruit cultivars on three rootstocks (Sour orange, US-942, and X-639). Trial 2 assessed 31 rootstocks with `Ray Ruby’ grapefruit as the scion. The total number of trees with grapefruit scions is 2,741. Control-release polycoated fertilizer was applied appropriately in May 2022. All trees were treated as needed with appropriate agrochemicals to manage canker, Asian citrus psyllids, mites, and citrus leafminers. No trees died due to the subzero temperatures experienced in February 2022.     Trunk diameter and canopy volume were measured on the three middle trees in each experimental plot in October 2021 to assess tree size. At the time, there were significant differences among scion/rootstock combinations. In Trial 1, `Pummelette UF-5-1-99-2′ grapefruit on US-942 was 6X larger (395.5 ft3) than `US 1-83-179′ grapefruit hybrid on US-942 (70.6 ft3). In Trial 2, grapefruit on UFR-15 was 3X larger (314.3 ft3) than on UFR-17 (123.6 ft3) for trees planted September 2019. Measurements were gathered from trees planted in June 2021 for the first time in May 2022. Phenology data collected monthly recorded flush initiation, flower initiation, full flowering, fruit set, fruit development, fruit color, and fruit maturity per tree. No fruit data were collected because a tree’s first crop is not indicative of future production. Nearly all trees in Trials 1 and 2 have set fruit in May 2022. Fruit data will be collected in January 2023. Long-term evaluation of fruit yield and quality is needed to identify the most promising scions and rootstocks to determine their profitability and capability of meeting grower and market needs.     HLB is widespread in the study grove. Visual blotchy mottle symptoms and twig dieback canopy are present in a few treatments but not widespread. Leaf samples for quantifying CLas titer were collected in May 2022 and sent to Southern Gardens for analysis. Trees that are CLas-free (ct values >38) and CLas-infected and symptomatic (ct values of 26-32) can be found in the same plots, but many symptomatic trees are developing vigorous canopies.      The incidences of Asian citrus psyllid and citrus leafminer were frequent during flush periods. The incidences of aphids, root weevils, and orange dogs were sporadic. Canker damage was noticeable but not uniform; it was especially significant on `Ray Ruby’ grapefruit trees. Tree growth has not been significantly affected by these pests due the timely applications of pesticides.     Results of the study were presented at the annual Florida Citrus Show in January 2022 in Fort Pierce, FL by graduate student Martin Zapien and in his Zapien’s MSc thesis presented to the Graduate School in April 2022. The thesis and data collected to date are available upon request.  



Development of SuperSour and other outstanding rootstocks with tolerance to HLB

Report Date: 05/30/2022   Project: 18-004   Year: 2022

Development of SuperSour and other outstanding rootstocks with tolerance to HLB

Report Date: 05/30/2022
Project: 18-004   Year: 2022
Percentage Completion: 1
Category: Plant Improvement
Author: Kim Bowman
Sponsor: Citrus Research and Development Foundation

Create new candidate hybrids.  Crosses were made this spring flowering season, based on parental combinations yielding the best progeny in previous trials.  Hybrids from previous crosses were selected and propagated this quarter.  New seed source trees for advanced selections were field planting in Spring 2022.Propagate and plant new field trials.  One new Stage 2 field trial with Hamlin with selected released rootstocks and the best of the next generation hybrids was field planted this quarter.  Nursery trees for one new Valencia Stage 1 trial with 60 new rootstocks that is planned for field planting in 2022 with a cooperator was delayed to finish the required MTA, and will be planted next quarter. Budwood increase trees were grown in preparation for budding trees for new rootstock trials.  Trees with Valencia scion and HLB-tolerant microcitrus interstocks were planted into a field trial as a preliminary analysis of interstock feasibility.Collect data from field trials.  Measurements of fruit crop and fruit quality data were collected from 9 rootstock trials with Valencia scions.   The USDA researcher assisting with the analysis of fruit quality from USDA rootstock trials retired in December, so responsibility for this aspect of the work shifted entirely onto the Bowman program.  Because of the Bowman program emphasis on rootstock trials, it is anticipated that this change will allow for an increase in the pace of fruit quality analysis associated with the rootstock trials.  Evaluate effectiveness for seed propagation of new rootstocks and develop seed sources.  As the best rootstocks are identified through field trials, seed sources are established. Studies continued to evaluate seed propagation for the most promising SuperSour hybrid rootstocks, using morphological and SSR analyses of seedling progeny for trueness-to-type.  Additional effort was initiated on evaluation of seed propagation for the SuperSour rootstocks as a UF graduate student project.   Field trial results for grower access.  The USDA rootstock trials produce large amounts of information that is useful to help growers make informed decisions about rootstock choice for new plantings.  A manuscript has been prepared that has a detailed comparison of field performance for Valencia on 50 SuperSour rootstocks and other commercial rootstocks, and this has been provided for review to selected citrus growers.  It is expected that this manuscript will be submitted for publication in the coming quarter, and key findings distributed broadly to citrus growers.  During this quarter, updated trial summaries from the 2021-2 season were prepared for uploading to the USDA citrus rootstock program website https://www.citrusrootstocks.org/, and information was provided to individual growers and groups, as requested.Release of superior new rootstocks for commercial use.  Release of new USDA rootstocks is based on robust data from multiple trees in replicated field trials over multiple years.  Several of the SuperSour rootstock hybrids in the Valencia field trial being prepared for publication have exhibited superior performance in comparison with commercial standard rootstocks and have supporting superior performance from one or more other trials.  Performance data continues to be evaluated, and is being used to critically compare the new hybrids with each other and existing rootstocks.  It is anticipated that 2-3 of the most outstanding of the new rootstocks from this set of SuperSour hybrids will be proposed for release in late 2022. 



Upgrading Citrus Genome Sequence Resources: Providing the Most Complete Tools Necessary for Genome Editing Strategies to Create HLB Resistant Cultivars

Report Date: 05/28/2022   Project: 18-010   Year: 2022

Upgrading Citrus Genome Sequence Resources: Providing the Most Complete Tools Necessary for Genome Editing Strategies to Create HLB Resistant Cultivars

Report Date: 05/28/2022
Project: 18-010   Year: 2022
Percentage Completion: 0.65
Category: Plant Improvement
Author: Fred Gmitter
Sponsor: Citrus Research and Development Foundation

We generated raw sequence data for Valencia orange (S, sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, tolerant), and Lisbon lemon (T) and preliminary assemblies and analyses were carried out. Because of reduced sequencing costs, we were able to enter additional important genomes into the pipeline beyond those originally proposed, including Carrizo citrange, sour orange, and Shekwasha (an important breeding parent for HLB tolerance); these also have now been sequenced and preliminarily assembled. Original plans for transcriptome sequencing, necessary to annotate the genome assembles produced, were to rely on Illumina short reads; but we decided to also include long PacBio Sequel II3 reads to capture full length transcripts as well. The first transcriptome data for two target genomes, were found to be inadequate. So, we identified a new vendor for this service. New samples have been collected for RNA preparation.We have focused on the first two genomes for which we had PacBio long read assemblies coupled with Hi-C sequencing using Hi-Rise software for the best quality chromosome scale assemblies. For these two citrus types, we also have access to a collection of resequenced genomes of related mutants and closely related accessions which is enabling us to explore additional potential HLB tolerant or resistant rootstocks. Because these assemblies have much improved contiguity (i.e., completeness), we have been able to better characterize the MITE sequence diversity (MITEs are a type of mobile DNA that inserts into different locations and contributes to genetic and phenotypic diversity) at the locus that controls nucellar embryony, a very important and widespread trait in commercial citrus cultivars. And, as these two genomes contain contributions for several biological citrus species, we can now begin to look at species specific genes. The new Hi-C assemblies have now been completed by Dovetail Genomics, and they have been transferred to us. Next, we can begin the long process of analyzing the assembly output for possible Type 2 errors, attempting to anchor several still unanchored sequence contigs, etc. to polish and produce the most complete and accurate assemblies.   



Part A - The UF/CREC Core Citrus Improvement Program (Complementary to Part B - The UF/CREC Citrus Improvement Program's Field Trial Evaluations)

Report Date: 05/28/2022   Project: 18-011   Year: 2022

Part A - The UF/CREC Core Citrus Improvement Program (Complementary to Part B - The UF/CREC Citrus Improvement Program's Field Trial Evaluations)

Report Date: 05/28/2022
Project: 18-011   Year: 2022
Percentage Completion: 1
Category: Plant Improvement
Author: Fred Gmitter
Sponsor: Citrus Research and Development Foundation

1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Seed from a first group of rootstock crosses was harvested and planted in the calcareous/Phytophthora soil as the first step in the gauntlet screen; parents included several previously selected but unreleased HLB-tolerant rootstocks, as well as some of the UFRs, HLB-tolerant pummelos, and US-897 and US-942.  Fifty seedlings exhibiting tolerance of the poor soil challenge were selected, potted up, and rooted cuttings were produced for further gauntlet steps. Seed from a second group of crosses, using LB8-9 Sugar Belle® and other mandarins as eed parents with pollen from various hybrids of Poncirus trifoliata with citrus accessions, Citrus ichangensis (Ci), different Cleopatra mandarin x Ci hybrids, a Palestine sweet lime x Ci hybrid, and two C. latipes hybrids, were planted in a second round of gauntlet screening, to be completed spring 2022. In collaboration with researchers at IFAPA in Spain, new information has been generated regarding performance of selected UFRs and other unreleased rootstock hybrids from our program in response to drought and flooding, Phytophthora, boron and salinity; UFR-6 has demonstrated good tolerance to all conditions except flooding and salinity, and 9 unreleased rootstock hybrids exhibiting tree size control and HLB tolerance in Florida likewise showed good tolerance of Phytophthora, with one of these showing best performance under all stress conditions. HLB ratings, fruit quality measures, or yield data were collected from 8 different rootstock field trials by our team during the reporting period.2. Develop new, HLB-tolerant scion cultivars from sweet orange germplasm, as well as other important fruit types such as grapefruit, mandarins, and acid fruit. Following extensive phenotyping of a replicated planting of hybrids between Monreal Clementine and an accession of Citrus latipes (perhaps the most HLB-tolerant citrus), we have found at least two hybrids that remain PCR-negative after 6 years under high pressure in the field, produced large fruit somewhat resembling sweet orange. Hundreds of seeds were collected from crosses made using their pollen onto low-acid breeding parents in our program and planted. Seed from new early-maturing (first week of December) Vernia sweet orange clones, which had higher soluble solids than the other selected early-maturing Vernia clones, were collected and planted.  Thirty new grapefruit hybrids generated using HLB and canker tolerant breeding parents were moved from embryo rescue into pots for subsequent field planting. One hundred twenty new EV protoclones, grafted on UFR-15 and US-802, were planted, in efforts to select a more robust early maturing Valencia clone. Three hundred ten new grapefruit hybrids or cybrids were planted at the CREC and in the IR area. Protoplast fusion experiments using W. Murcott suspension protoplasts with various leaf parents were caried out to create new tetraploid breeding parents that can be used for orange and mandarin improvement. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have more than 70 5-year-old-trees of `Marathon’ mandarin on sour orange, that set large crops this last season. Although all trees have HLB, there are few to no obvious disease symptoms in fruit, leaves, or canopy, demonstrating a high degree of tolerance thus far. We have followed closely their performance, and individual trees yielded more than 300 pounds of fruit in 2 harevsts in September and October. Fruit size distributions were determined, and post-harvest behavior and fruit quality data were collected and are now under analysis. We made several selections of apparently HLB-tolerant seedlings from breeding populations of oranges and orange-like hybrids, grapefruit and mandarins, and presented fruit and juice samples at displays in the CREC. 4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. Several new genetic constructs have been developed using newly identified citrus specific promoters (phloem and root tissue), and new putative disease resistance genes, or downstream genes. Transgenic plants have been produced with some of these constructs, and additional transformation experiments are underway with several sweet oranges, grapefruit, and rootstocks. We assessed a diverse population of 459 hybrids from 30 crosses and 53 accessions for HLB tolerance using different morphophysiological traits and compared the relationship of these traits with a visual HLB severity score. We found a significant genetic effect on HLB tolerance which indicates opportunities for genetic improvement of HLB tolerance. Leaf area index (LAI) was the trait most highly correlated with HLB score. suggesting that LAI is a rapid, cost-effective, and reliable method in comparison to other existing HLB phenotyping measurements, and can avoid cognitive bias in phenotyping trees for HLB tolerance.      



Upgrading Citrus Genome Sequence Resources: Providing the Most Complete Tools Necessary for Genome Editing Strategies to Create HLB Resistant Cultivars

Report Date: 05/28/2022   Project: 18-010   Year: 2022

Upgrading Citrus Genome Sequence Resources: Providing the Most Complete Tools Necessary for Genome Editing Strategies to Create HLB Resistant Cultivars

Report Date: 05/28/2022
Project: 18-010   Year: 2022
Percentage Completion: .7
Category: Plant Improvement
Author: Fred Gmitter
Sponsor: Citrus Research and Development Foundation

We are assessing the current inventory of sequence resources we have available, to identify additional needs to successfully complete all 9 genome sequence assemblies to a chromosome scale and with the greatest accuracy and contiguity technically possible. We still are lacking sufficient PacBio long read coverage for Ruby Red grapefruit and Shiikuwasha. New HMW DNA preparations have been made and are pending sequencing. We also need to expand our collection of RNA transcripts, both with Illumina short reads and PacBio long reads, and with broader collections of tissue types to maximize the number of expressed genes we can find for annotation of the assemblies. We have collected samples of tender flush, mature leaves, flowers, young and nearly mature fruit, bark, and for some accessions leaf tissue with and without symptoms of CLas infection and citrus canker. RNA processing and sequencing are pending.Seven of the nine genomes have been assembled using both the PacBio and Hi-C sequencing and assembly using Hi-Rise. Quality control and assessment is underway, to properly phase chromosomes, to identify and anchor unanchored sequence contigs, to find haplotype swaps via 2 different approaches, to define centromeres, and to polish telomeres and resolve highly repetitive sequences in these regions. Phasing of the LB8-9 Sugar Belle® mandarin hybrid genome has been a challenge because of several runs of homozygosity (ROH) throughout the genome; we are trying to address this by using sequence information already available from Dancy tangerine and Duncan grapefruit but using Minneola (the pollen parent) would be better so plans are in place to resequenced it. Comparing the Dovetail assemblies, we found that the highest quality coming from Carrizo citrange, not surprising given that the parents represent the greatest possible genetic diversity.   



Citrus row middle management to improve soil and root health

Report Date: 05/27/2022   Project: 18-059C   Year: 2022

Citrus row middle management to improve soil and root health

Report Date: 05/27/2022
Project: 18-059C   Year: 2022
Percentage Completion: 1
Category: Plant Improvement
Author: Sarah Strauss
Sponsor: Citrus Research and Development Foundation

The overall goal of the project was to provide a detailed assessment of cover crop impacts on soil nutrient cycling and microbial communities, weed growth, tree performance, and economic considerations. Treatments in this trial included two different mixtures of cover crops planted in the row middles: legumes + non-legumes (LG + NL) and non-legumes only (NL). These were compared to a grower standard control (GSC) that primarily consisted of weeds. Cover crops were planted twice during the year: June and late October/early November to correspond with the rainy season. Trials were conducted in two different locations in Southwest Florida (labeled North and South groves).
We found cover crops had a significant impact on components of soil health for this project. Both cover crop mixtures increased soil organic matter, though this increase was only found at the North grove location. The increase in soil organic matter was linked with increased nitrogen availability in the row middle soils where cover crops were planted and significant changes to the soil microbial community. The mixtures of cover crops planted (LG + NL compared to NL only) also significantly impacted the abundance and types of microbes in the soil, and their roles in soil nitrogen availability. For example, there were more bacteria capable of fixing nitrogen in soils under LG + NL, as well as completely different types of bacteria involved in other aspects of the nitrogen cycle. While these changes in soil organic matter, microbial communities, and nitrogen availability in the row middles did not translate to changes in root or tree growth or production, they do indicate changes to soil health are possible. The trees in both locations of this study were 25+ years old, and therefore more time may be necessary to observe changes in production. Based on a survey of citrus growers about the use of cover crops conducted as part of this project, growers were on average willing to wait 4.4 years for cover crops to benefit citrus production. It is possible that changes in production due improved soil health from cover crops may also be evident more quickly in younger trees.
In addition to changes to soil parameters, cover crops also significantly suppressed weed growth. In particular, the cover crop mixtures in this study substantially suppressed the germination and growth of grasses and sedges. Based on the economic analysis conducted in this project, there is an estimated short-term savings of $75.47 per acre due to not frequently mowing the row middles where cover crops are planted. The overall net cost of adopting cover crops was estimated to be $144.32 per acre, on average.
Based on the results from this project, cover crops can significantly impact components of soil health, reduce costs related to weed suppression, and have the potential to provide greater impacts to soil nutrient availability. There have been nine publications from this project to date, with at least five more in preparation. Publications include both articles for the Citrus Industry magazine and peer-reviewed scientific journals. In addition, PIs on this project have presented results at multiple grower meetings. A grower/extension seminar with presentations of the results of this project by all PIs is scheduled to be held at the UF/IFAS SWFREC on June 23.



Comparison of field performance of citrus trees on rootstocks propagated by seedlings, cuttings, and tissue culture

Report Date: 04/15/2022   Project: 18-028C   Year: 2022

Comparison of field performance of citrus trees on rootstocks propagated by seedlings, cuttings, and tissue culture

Report Date: 04/15/2022
Project: 18-028C   Year: 2022
Percentage Completion: 0.9
Category: Plant Improvement
Author: Ute Albrecht
Sponsor: Citrus Research and Development Foundation

 1. Please state project objectives and what work was done this quarter to address them: Objective 1. Investigate effects of rootstock propagation method and the interaction with rootstock on root structure, root growth, and tree performance during the first 3 years of growth in the field.Fruit yield was determined in both commercial trials, but fruit production on these young trees was still low. Fruit samples were collected from each experimental unit and fruit quality was determined. Because of the earlier than usual harvesting time (determined by the grower collaborators) due to the freeze events in January/February, fruit quality was generally poor. Tree ratings (HLB symptoms, canopy color, canopy density) were conducted. Due to the freezes there was considerable defoliation (especially in the SW FL trial) and new flush had died off. Trees at the central ridge location were not as affected by the freezes. Objective 2. Investigate if trees on rootstocks propagated by tissue culture or cuttings differ in susceptibility to Phytophthora-induced decline or wind-induced blow-over compared with trees on rootstocks propagated by seed. We continued our root crowns analyses (measurements and photographic documentation) on the trees that were uprooted during the previous quarter. An in-person/virtual seminar with research results from this project and a hands-on demonstration of root crowns from the uprooting trials was held at SWFREC on March 23rd. Other information from this project was presented at the Florida Citrus Growers’ Institute in Avon Park on April 5th.   3. Please state budget status (underspend or overspend, and why): This project is being continued under an NCE because it was underspent for reasons outlined in the NCE agreement.  



Test of SAMP efficacy in the field and greenhouse on important citrus varieties in Florida

Report Date: 04/15/2022  

Test of SAMP efficacy in the field and greenhouse on important citrus varieties in Florida

Report Date: 04/15/2022

Percentage Completion: 100%
Category: Plant Improvement
Author: Hailing Jin
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them:
This project aims to test SAMP efficacy tests in the field and greenhouse on important citrus varieties in Florida and California. Dr. Megan Dewdney is in charge of all the field trials in FL. A large scale field test on 240 young Hamlin trees with 3 different treatments and 1 control set was laid out in a completely randomized design. A second field test on infected 4-year old ‘Hamlin’ sweet orange bearing trees was also initiated. In addition, we also performed new greenhouse tests on important citrus varieties in collaboration with Dr. Kris Godfrey (UCD). Fruthermore, we also have surprising findings that SAMP may also suppress CLas in ACP in collaboration with entomologists Dr. Kris Godfrey and Dr. Kerry Mauck (UCR). Thus, SAMP is the only antimicrobial peptide that we know of that has three biological functions: 1) bactericidal activity, 2) inducing host immunity activity and 3) inhibiting ACP activity.

2. Please state what work is anticipated for next quarter:
Test I: Field Test I- SAMP efficacy test on 240 newly planted ‘Hamlin’ sweet orange trees
Dr. Megan Dewdney started a field trial with a total 240 young Hamlin’ sweet orange trees to determine the SAMP efficacy on newly planted trees against HLB. The trial will last for at least 2-3 years. The experiment was laid out in a completely randomized design with three treatments and sixty trees per treatment. The treatments are 1. Untreated control, 2. Treatment in the nursery 1 week prior to planting and field applications (10 µM in 158 ml/tree) every 2 months, 3. No nursery treatment with field applications every 2 months starting 10 days post-planting, 4. The injection treatment with Invaio simple injection device. It is expected that the treatment volume will increase as the trees grow over the next two years (Figure 1). We will monitor the tree growth, disease rating, and CLas titer in trees and ACP after this summer.
Test II: Field Test II- SAMP efficacy test on 125 infected 4-year old ‘Hamlin’ sweet orange trees.
The second field trial was set up to test if SAMP can remediate an HLB-affected young citrus grove. This experiment is ongoing in the Ridge region of Florida on the deep sandy soils. We have located a four-year-old commercial ‘Hamlin’ sweet orange grove in the Lake Wales region. The trial involves total of 125 bearing trees and was laid out as a randomized complete block design with five blocks and five replicates per block. The treatments in this trial were an untreated control and bimonthly foliar application. Three additional treatments from an additional funding source are to look at the peptide as various injection treatments. These treatments are 2 injections in spring and summer, 3 injections in spring, summer, and fall, and 3 injections per year starting at the same time as the foliar spray (Figure 2). The treatment started from August 2021. We expect to monitor the trial at least for 2 years. We will monitor the tree growth, disease rating, and CLas titer in trees and ACP.
Test III: Three greenhouse trials with important varieties of California which were conducted in BSL3-UC Davis :
A. Tests of SAMP treatment using trunk injection and foliar spray on the early infected tree with ‘Washington Navel’ orange and ‘Tengo’ Mandarin trees (Table 1, Figure 3 and 4).Total of 30 Navel trees and 22 Tengo trees
B. Test of using SAMP foliar spray to protect trees from CLas infection with 16 ‘Washington Navel’ orange.
SAMP has clear disease control and plant protection effect on all the three trials.

3. Please state budget status (underspend or overspend, and why): The budget was spent in full to purchase 100g SAMP.



Test of SAMP efficacy in the field and greenhouse on important citrus varieties in Florida

Report Date: 04/15/2022  

Test of SAMP efficacy in the field and greenhouse on important citrus varieties in Florida

Report Date: 04/15/2022

Percentage Completion: 100%
Category: Plant Improvement
Author: Hailing Jin
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them:
This project aims to test SAMP efficacy tests in the field and greenhouse on important citrus varieties in Florida and California. Dr. Megan Dewdney is in charge of all the field trials in FL. A large scale field test on 240 young Hamlin trees with 3 different treatments and 1 control set was laid out in a completely randomized design. A second field test on infected 4-year old ‘Hamlin’ sweet orange bearing trees was also initiated. In addition, we also performed new greenhouse tests on important citrus varieties in collaboration with Dr. Kris Godfrey (UCD). Fruthermore, we also have surprising findings that SAMP may also suppress CLas in ACP in collaboration with entomologists Dr. Kris Godfrey and Dr. Kerry Mauck (UCR). Thus, SAMP is the only antimicrobial peptide that we know of that has three biological functions: 1) bactericidal activity, 2) inducing host immunity activity and 3) inhibiting ACP activity.

2. Please state what work is anticipated for next quarter:
Test I: Field Test I- SAMP efficacy test on 240 newly planted ‘Hamlin’ sweet orange trees
Dr. Megan Dewdney started a field trial with a total 240 young Hamlin’ sweet orange trees to determine the SAMP efficacy on newly planted trees against HLB. The trial will last for at least 2-3 years. The experiment was laid out in a completely randomized design with three treatments and sixty trees per treatment. The treatments are 1. Untreated control, 2. Treatment in the nursery 1 week prior to planting and field applications (10 µM in 158 ml/tree) every 2 months, 3. No nursery treatment with field applications every 2 months starting 10 days post-planting, 4. The injection treatment with Invaio simple injection device. It is expected that the treatment volume will increase as the trees grow over the next two years (Figure 1). We will monitor the tree growth, disease rating, and CLas titer in trees and ACP after this summer.
Test II: Field Test II- SAMP efficacy test on 125 infected 4-year old ‘Hamlin’ sweet orange trees.
The second field trial was set up to test if SAMP can remediate an HLB-affected young citrus grove. This experiment is ongoing in the Ridge region of Florida on the deep sandy soils. We have located a four-year-old commercial ‘Hamlin’ sweet orange grove in the Lake Wales region. The trial involves total of 125 bearing trees and was laid out as a randomized complete block design with five blocks and five replicates per block. The treatments in this trial were an untreated control and bimonthly foliar application. Three additional treatments from an additional funding source are to look at the peptide as various injection treatments. These treatments are 2 injections in spring and summer, 3 injections in spring, summer, and fall, and 3 injections per year starting at the same time as the foliar spray (Figure 2). The treatment started from August 2021. We expect to monitor the trial at least for 2 years. We will monitor the tree growth, disease rating, and CLas titer in trees and ACP.
Test III: Three greenhouse trials with important varieties of California which were conducted in BSL3-UC Davis :
A. Tests of SAMP treatment using trunk injection and foliar spray on the early infected tree with ‘Washington Navel’ orange and ‘Tengo’ Mandarin trees (Table 1, Figure 3 and 4).Total of 30 Navel trees and 22 Tengo trees
B. Test of using SAMP foliar spray to protect trees from CLas infection with 16 ‘Washington Navel’ orange.
SAMP has clear disease control and plant protection effect on all the three trials.

3. Please state budget status (underspend or overspend, and why): The budget was spent in full to purchase 100g SAMP.



Fort Pierce Field Test Site for Validating HLB and/or ACP Resistance

Report Date: 04/12/2022   Project: 18-058C   Year: 2022

Fort Pierce Field Test Site for Validating HLB and/or ACP Resistance

Report Date: 04/12/2022
Project: 18-058C   Year: 2022
Percentage Completion: .97
Category: Plant Improvement
Author: Randall Niedz
Sponsor: Citrus Research and Development Foundation

Updates for this quarter:Site management and field trials are progressing well.  The site remains available for access to all researchers and all regulatory protocols for the care and disposal of transgenic material are being observed. The trees have been hedged and topped to promote growth, open canopies and access to nutritional sprays.  The foliar spray program, applied every two weeks, includes: 435 horticultural oil, Magna-Bon CS2005 copper, 20-10-20 and/or 15-5-15 CA-MG, N-Sure 28-0-0, Keyplex 1400 DP, and Ocean Organics 0.2-0-6.  Wedgeworth’s 12-13-15, 2Mg slow release nitrogen and potassium with greening guard was also applied above the root zone in March.   Discussions have begun with APHIS-BRS to set conditions for new or expanded transgenic release permits to allow field trials of a novel system using engineered tissue to delivery therapeutic peptides to non-transgenic trees. This effort is in support of NIFA project 2020-70029-33176, Therapeutic Molecule Evaluation and Field Delivery Pipeline for Solutions to HLB, with field trials expected to begin later this year once all regulatory requirements are met. The testing site is also being actively utilized this breeding season. Crosses have been made with transgenic pollen to help elucidate if sexual embryos can be rescued from polyembryonic females, making use of the transgenic markers to determine if sexual hybridization is successful. This could greatly impact both transgenic and conventional breeding efforts by allowing the use of polyembryonic citrus accessions as females.  A third year of crossings has also been made with the early flowering (FT) transgenics, continuing the work described below.  Recent quarters:A significant USDA-funded infrastructure project has been completed, fully renovating the water management systems and significantly improving storm and flood protection. USDA has also acquired a topper hedger to facilitate canopy management and reflect the best practices of commercial farms.  An additional BRS transgenic release permit was approved (AUTH – 0000043620) for material with confidential business information (CBI) for a project led by R. Shatters. The primary BRS permit has also been renewed and amended to include a new construct from UF (Now AUTH  – 0000206702).  The annual site review from APHIS/BRS has been conducted successfully.  Four new plantings from UF expressing resistance genes and two new plantings from USDA-CRADA partners expressing antimicrobial peptides and anti-CLas plantibodies have been made. With recent plantings, the transgenic site is operating at full capacity. Fall 2021 assessments were completed for USDA plantings as described below for trials #8, #9, #10, #11 and #15. Fruit have been harvested from the second year of controlled crosses using pollen from early flowering (FT) transgenics on traditional varieties maintained in the testing site.  Seeds from these fruit and those of future crossings will be used to assess inheritability of the phenotype and for CRISPR gene stacking to combine genome editing with accelerated breeding traits. The UCRiverside-led trifoliate and trifoliate hybrid trial has concluded, a manuscript regarding identified HLB-tolerance is in preparation; and these trees can be removed as needed to make space available for future plantings. Dr. Stover analyzed data on canker incidence for this trial and a manuscript detailing these results has been published in HortScience DOI: 10.21273/HORTSCI15684-20. Previously established at the site:A number of trials are underway at the CRDF funded Picos Test Site.  A detailed current status is outlined below this paragraph. We continue investigation of potential pollen flow from transgenic trees to assess the possibility of reducing the isolation distances.  Availability of the test site for planting continues to be announced to researchers.   Supplemental: Full details on trial plantings.1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics.  These are on an independent permit, while all other transgenics on the site are under the USDA permits.2) Under the Stover permit, a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted.  These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project.5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars.  Genotyping, HLB phenotyping and growth data have been collected and analysis will continue to be conducted under a new NIFA grant.6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).7) In a project led by Fred Gmitter, there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo.10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls.11) Multiple plantings with grafted trees of l Hamlin, Valencia and grapefruit scions on transgenic rootstock expressing antimicrobial citrus-thionin and bacterial recognition domain fusion proteins (219 trees with controls) as a collaboration between USDA and the New Mexico Consortium.12) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type.13) Planting from Zhonglin Mou of UF that includes transgenic grapefruit (31 plants) and sweet orange (60 plants) scions expressing two different resistance genes and grafted on WT swingle rootstocks; as well as non-transgenic controls. 14) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State15)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.   



Testing grapefruit trees expressing an anti-NodT antibody for resistance to HLB

Report Date: 04/08/2022   Project: 18-016   Year: 2022

Testing grapefruit trees expressing an anti-NodT antibody for resistance to HLB

Report Date: 04/08/2022
Project: 18-016   Year: 2022
Percentage Completion: .95
Category: Plant Improvement
Author: Tim McNellis
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them: This report covers the period of December 1, 2021 – February 28, 2022.  The objective of this project is to test transgenic ‘Ducan’ grapefruit trees expressing an anti-HLB antibody fused to the FT (Flowering Locus T) protein (FT-scFv protein).  Chad Vosburg graduated in December, 2021 with his M.S. degree in Plant Pathology and his M.S. Thesis was approved.  The PI reviewed the M.S. thesis elements related the HLB resistance tests that we completed in the greenhouse and an ongoing test in the field at Fort Pierce.  An initial draft of a manuscript describing these results was prepared by the PI. 2. Please state what work is anticipated for next quarter:In the next quarter, the PI will finalized the manuscript in collaboration with Chad Vosburg and other co-workers.  The manuscript will be submitted by the PI to a peer-reviewed journal for review in the next quarter.  We anticipate requesting another no-cost extension to allow us to use grant funds to pay publication costs for the manuscript.  Review, acceptance and publication could take several additional months, at least. 3. Please state budget status (underspend or overspend, and why):The project remains underspent in part due to support for graduate stipends and tuition waviers from the Plant Pathology & Environmental Microbiology Department of Penn State and the Penn State Graudate School.  We have sufficient funds remaining for publication costs for the upcoming manuscript.



Optimization of the CRISPR technology for citrus genome editing

Report Date: 03/31/2022   Project: 18-025   Year: 2022

Optimization of the CRISPR technology for citrus genome editing

Report Date: 03/31/2022
Project: 18-025   Year: 2022
Percentage Completion: 0.98
Category: Plant Improvement
Author: Nian Wang
Sponsor: Citrus Research and Development Foundation

The purpose of this project is to optimize the CRISPR technology for citrus genome editing. This study is related to the CRDF RMC-18 Research Priorities 4AB. Objective 1. Expanding the toolbox of citrus genome editing. In this study, we will adapt StCas9, NmCas9, AsCpf1 (from Acidaminococcus), FnCpf1 (from Francisella novicida) and LbCpf1 (from Lachnospiraceae) on genome modification of citrus. Lately, we have shown CRISPR-Cpf1 can be readily used as a powerful tool for citrus genome editing. In our recent study, we employed CRISPR-LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time.  Our study showed that CRISPR-LbCas12a can readily be used as a powerful tool for citrus genome editing. One manuscript entitled CRISPR-LbCas12a-mediated modification of citrus has been published on Plant Biotechnol J. We are currently further optimizing LbCas12a-crRNA-mediated genome editing to make homologous biallelic mutations. We are also testing AsCpf1 and FnCpf1 for their application in citrus genome editing and generating homologous biallelic mutations.  We have successfully generated both homozygous and biallelic mutations in the EBE region of LOB1 gene in pumlo. This work has been submitted for publication. We are in the process of generating homozygous and biallelic lines of other citrus varieties.Recently, we have developed multiplex genome editing toolkits for citrus including a PEG mediated protoplast transformation, a GFP reporter system that allows rapid assessment of the CRISPR constructs, citrus U6 promoters with improved efficacy, tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we have successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues.  We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, indicating that the CRISPR-mediated citrus genome editing technology is mature and could be implemented in citrus genetic improvement as a viable approach. In addition, our study lay the foundation for non-transgenic genome editing of citrus. One manuscript entitled Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations has been published on Plant Molecular Biology.We have successfully developed base editing tools for citrus genome editing. This method has been succefully used to generate non-transgenic biallelic mutants of sweet orange.  Objective 2. Optimization of the CRISPR-Cas mediated genome editing of citrus. In this study, we are testing different promoters including INCURVATA2 promoter, the cell division-specific YAO promoter, and the germ-line-specific SPOROCYTELESS promoter, and ubiquitin promoter in driving the expression of Cas9 and Cpf1 orthologs. To optimize the expression of sgRNA and crRNA, we have identified multiple citrus U6 promoters and two of the citrus U6 promoters showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We have further increased the mutation efficacy to 50%. We have further optimized the CRISPR/Cas9 system. Now, the biallelic mutation rate reaches 89% for Carrizo citrange and 79% for Hamlin sweet orange. We have generated one homozygous line in the promoter region of canker susceptibility genes of Hamlin. We have successfully generated one biallelic mutant of grapefruit that is canker resistant. We also successfully generated multiple biallelic and homozygous mutant lines of sweet orange that are canker resistant. Objective 3. Optimization of the CRISPR technology to generate foreign DNA free genome editing in citrus. We have conducted transient expression of Cas9/sgRNA plasmid and Cas9 protein/sgRNA ribonucleoprotein complex in citrus protoplast. We are also conducting citrus genome editing using Cpf1/crRNA plasmids and ribonucleoprotein complex in citrus protoplast. The plasmid-transformed protoplast has 1.7% editing efficiency, and the RNP-transformed samples have approximately 3.4% efficiency. The genome modified protoplast cells are undergoing regeneration. We aim to increase the efficacy to over 20% and eventually generate non-transgenic genome modified citrus. One patent has been filed on the CRISPR-Cas mediated genome editing of citrus. We have lately optimized the citrus protoplast isolation and manipulation, our data showed that more than 98% of the isolated protoplasts were alive. We regularly obtained a transfection efficiency of approximately 66% or above. ErCas12a has been succes for non-transgenic gene editing of embryogenic Hamlin sweet orange protoplast cells. We are editing 6 putative HLB susceptibility genes for sweet orange. One biallelic mutant line has been generated for ACD2.  We have successfully adapted the adenine base editors (ABE) to modify the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit and Hamlin sweet orange. Inoculation of the TATA-edited plants with the canker pathogen Xanthomonas citri subsp. Citri (Xcc) demonstrated that the TATA-edited plants were resistant to Xcc. In addition, cytosine base editors (CBE) was successfully used to edit the acetolactate synthase (ALS) gene of Carrizo citrange, a hybrid of Citrus sinensis `Washington’ sweet orange X Poncirus trifoliata. Editing the ALS genes conferred resistance of Carrizo to the herbicide chlorsulfuron. Two ALS-edited Carrizo plants did not show green florescence although the starting construct for transformation contains a GFP expression cassette. We performed PCR amplification for Cas9 gene in the mutant plants and found that Cas9 gene was undetectable in the herbicide resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression. This work has been been published by Frontiers in genome editing. In addition, we are further developing non-transgenic genome editing technology using ALS gene as a selection marker.   



Part B - The UF/CREC-Citrus Improvement Program's Field Trial Evaluations (Complementary to Part A- the UF/CREC Core Citrus Improvement Program)

Report Date: 03/17/2022   Project: 18-039-C   Year: 2022

Part B - The UF/CREC-Citrus Improvement Program's Field Trial Evaluations (Complementary to Part A- the UF/CREC Core Citrus Improvement Program)

Report Date: 03/17/2022
Project: 18-039-C   Year: 2022
Percentage Completion: 95
Category: Plant Improvement
Author: Jude Grosser
Sponsor: Citrus Research and Development Foundation

True sweet oranges: This was a very exciting quarter for identifying true sweet orange candidates to replace Hamlin.  Multiple Vernia somaclone-derived seedling clones repeated for early maturity (first week of December) for the 3rd consecutive year.  The best of these was MB-R25-T7, which had a 16.17 ratio and the highest brix (11.16), and significantly higher soluble solids than the Hamlin control.  The Vernia somaclone-seedling population, planted in the Mathew Block at the Orie Lee Alligator Grove, has never had psyllid control.  We began supplementing trees with CRF about 2 years ago to prevent decline.  Trees were commercially harvested last week, and there was still no fruit drop observed.  At this time, the brix in the MB-R25-T7 was over 13.  This new Vernia clone was entered into the PTP in January. We also discovered a new early-maturing OLL clone in the somaclone-derived seedling block of OLLs (located in the Lee Family Home Grove in St. Cloud).  Clone FB-R7-T35 had a 14.12 ratio in mid-January with 12 brix, 5.7 lbs. solids/box (exceptionally high for a juvenile tree) with a 37.3 juice color score.  This clone had an extended juvenility and did not have fruit last year.  This new OLL clone will be entered into the PTP the first week of April, when new slots become available. Both of these clones are being propagated at the CREC to expedite further evaluation (with permission from DPI). Promising orange-like hybrid: Triploid hybrid C7-11-7 [Sugar Belle x (Murcott + Succari sweet orange)] produces fruit morphologically indistinguishable from a true sweet orange, but with better external color. The original tree is exceptionally productive and appears to have HLB tolerance slightly better than standard sweet oranges.  Juice from fruit of the original tree (with HLB for many years) had 13.87 brix, 15.1 ratio, 39.3 color score and 8.31 lbs. solids per box (double the state average for Hamlin!) in mid-January.  This selection will also be entered into the PTP in early April.  Potential HLB tolerance/resistance from ‘gauntlet’ rootstock candidates:  Rootstock sprouts recovered from 12 superior gauntlet trees that had the tops cut off to induce sprouting for rootstock recovery, all successfully grafted, were analyzed for CLas by PCR.  Most interestingly, multiple grafted trees from two of these rootstock candidates had ct values above 36, indicating no CLas bacteria replication; and both hybrids were from the same cross (A+HBPxOrange 3-12-12 and A+HBPxOrange 3-12-10). Both hybrids appear to be more vigorous than either parent. Orange 3 is UFR-1, and both tetraploid parents in this cross produce fruit with high soluble solids (good rootstock pedigree).  Further experiments are being designed to determine the value of strong rootstocks that do not support CLas replication in the root systems. Since recovered lines of these two rootstock candidates are pathogen free, TC propagation was initiated. A 3rd hybrid from this cross looks excpetional in the ‘gauntlet’ this spring, and it will also be cut to recover the rootstock.  CREC Trailer Park Trial:  A good candidate for juice blending was identified: 18A-10-11.  This selection is a low-seeded cybrid of ‘Furr’ that produces a large firm fruit, with exceptional quality juice (12.5 brix, 19.84 ratio and 46.1 color in mid-January). We used this selection in juice blends at both the CREC display and Gator Day at the State Capitol, and all were very well received.  The exceptional juice flavor holds up well to pasteurization.   Rootstock candidate identified from Strang/Gapway trial.  We were able to recover the x639 mutant (apparent deletion mutant) showing the exceptional HLB tolerance.  One successfully grafted tree and 12 rooted cuttings have been obtained.  PCR analysis showed a ct value of 40, indicating no CLas bacteria in the recovered rootstock.  Additional propagation is underway as needed for a more robust field trial.  (Also note that the GapWay property has been sold, so the trial has been lost; however, the rootstock tree was dug up and successfully moved back to the CREC).   Field Trial Data Collection, etc.:  various data (tree health, fruit quality, yield, etc.) was collected from the following trials: Tom Hammond, Greene River Citrus, IMG, Peace River Growers, Bryan Paul Citrus, Lee Family Groves, and Duda.  Data analysis and entry onto the Rootstock Data Website: new data posted on website included:  Jackson Citrus, Lykes, GFC Citrus, Lee Family Groves, Banack Citrus and Duda.  Trial data worked on, not yet added to website: Greene River Citrus, Peace River Growers, IMG, Bryan Paul Doe Hill, Citra trial, Cutrale, and the Serenoa trial. The Access Data Plan was also implemented on the website. 



Evaluation of the tolerance of newly developed citrus cultivars, on different rootstocks, to Huanglongbing

Report Date: 03/05/2022   Project: 19-015   Year: 2022

Evaluation of the tolerance of newly developed citrus cultivars, on different rootstocks, to Huanglongbing

Report Date: 03/05/2022
Project: 19-015   Year: 2022
Percentage Completion: .95
Category: Plant Improvement
Author: Nabil Killiny
Sponsor: Citrus Research and Development Foundation

In the current project we are establishing the volatile and non-volatile polar metabolite profiles of new scions and rootstocks and evaluating them for their tolerance to HLB by challenges with psyllids and HLB.Progress on Objectives: This reporting period we focused on data from the study involving a new mandarin hybrid we call “Lucky” (Sugar Belle x Nova×Osceola) and the two parental varieties by challenging with ACP and taking leaf samples after infestation.Objective 1.1.         To understand the mechanism behind the tolerance of new varieties toward HLB. The comparison between the varietal responses will allow us to determine the response to CLas-infected ACP infestation.  Subsequent PCR testing will help determine susceptibility to HLB.a.         For the hybrid “Lucky” (its parents are Sugar Belle and Nova × Osceola), we successfully repeated the challenge with CLas-infected ACPs ending in September. Leaf samples were taken initially (before infestation with ACP) and then 3 days, 1 month, 2 months and 3 months after ACPs were introduced into the cages. We will determine the biochemical response of “Lucky” to ACP infestation and its relative tolerance to CLas over time, with PCR confirmation of HLB status scheduled for Mar 2022 (9 months). The chromatograms have now been integrated and a summary is provided here:b.         VOC profile summary: At this time we have finished the preliminary integration and have a general idea of the VOC profile of each of the three varieties. Overall, we detected 54 volatile compounds among the leaf extracts of the three varieties. `Lucky’ and Nova produced sabinene and beta-ocimene as their major monoterpenes, whereas in Sugar Belle (SB) the major monoterpenes are beta-ocimene and gamma-terpinene. Linalool appears at similar levels in all three, and limonene is relatively low in all three (compared to sweet orange varieties, where it is the dominant monoterpene). The dominant sesquiterpenes are caryophyllene and beta- and gamma-elemene.c.         The majority of the compounds detected were in common between the three varieties, but a few were unique to each variety. As reported previously, the characteristic VOCs for SB included thymol and thymol methyl ester. Neither `Lucky’ nor Nova produced thymol, but Nova produced traces of the thymol methyl ester. In addition, we detected the monoterpenes para-cymenene and para-methatriene in SB, which was not found in the hybrid `Lucky’ offspring or Nova. Overall, the VOC profile of `Lucky’ showed contributions from both parents but did not express all of the possible compounds (44/54). There are many that are found uniquely or in two of the three varieties. These include para-cymene, para-menthatriene, and thymol (in SB only), thymol methyl ester (SB, Nova), germacrene C (Nova only), beta- and gamma-elemene (SB, Nova), 7-epi-thujene (Nova, Lucky), beta-cubebene (Lucky), 4-epicubedol (new, in Nova), and beta-sesquiphellandrene (Nova, Lucky).d.         We will continue the data analysis to determine the effect of the infestation on the VOC profile of the three varieties over time. We expect to see changes in the VOC profile with increasing time after the infestation. e.         Most importantly, survival of ACPs on the three varieties was not good again, as was found previously in our January 2021 experiment, which was attributed to cold weather. The second biology experiment was carried out in a protected growth room between July and August, 2021 and showed that ACP survives and reproduces poorly on the two parents. The ACP count was the highest on the hybrid “Lucky”. Therefore, we can suggest a correlation may exist between the lack of inheriting thymol and several other compounds from its parents and the higher ACP counts on “Lucky”, so maybe it is not very “Lucky” after all.Objective 2.To support objective 2 (understanding the role of rootstocks in citrus tolerance to HLB), we have two remaining experiments. 1.         USDA rootstocks. We collected leaves from all of the USDA rootstocks grown from seeds since last year (US-802, 812, 897, 942, 1283, 1284, 1516) for volatile metabolite profiling.  These samples are in the freezer and await processing and analysis. In addition, we have scheduled challenges with ACP and HLB graft-inoculations in the early spring of 2022 to determine if any of these are tolerant toward HLB.2.         Grapefruit varieties on different rootstocks from the CUPS. The volatile data was reported in the previous reporting period. However, the data for the non-volatile metabolites are still being analyzed at this time.