The fall cover crop mix planted in early November at both locations had some of the best growth we have seen so far, and crops from the planting in November continue to grow. This mix included daikon radish, coker, Wrens grain rye, and dove millet. Sunnhemp, alyce clover, sesbania, dixie crimson clover, and yellow sweet blossom clover were included in the mixes for the nitrogen-fixation treatments (1/2 of the rows). Germination counts and biomass samples have been collected and are currently being analyzed.
Yield and juice quality data were collected for the North grove location in mid-March. Unfortunately, due to COVID-19, we were not able to collect yield and juice quality data for the South grove location. However, preliminary analysis of the North grove yield data found trends of increased yield under the Cover Crop Mix#1 with eco-mowing.
After 1-year treatment in the North grove, the content of soil organic matter significantly increased in the row middles treated with cover crops (with values in the range of 3.6-4.2%) compared to the grower-standard control (3.3%). Under the tree canopy, we observed a greater, but not significant, increase in soil organic matter in treatments under eco-mowing (with values in the range of 4.1-4.8%) compared to those without in both cover crop mixes (values in the range of 3.8-4.3%) and the grower-standard control (4.1%). Canopy size measurements in all treatments continue every six months though significant differences and trends have not been detected. However, changes in these parameters could take up to 2 to 3 years in mature citrus trees following changes in practices. Preliminary measurements and analysis of changes in soil organic matter for the South grove are in progress. Measurements are in progress to determine the abundance of nitrogen-fixing genes (important with legumes since they are related to changes in the content of ammonium in soils) and denitrification genes (often linked to changes in soil organic carbon, organic matter, and nitrogen losses from the soils). Multivariate statistical analyses are also in progress to evaluate the influence of abiotic (soil properties) on biotic (microbial gene abundance) variables in treatments with and without cover crops.
Dataloggers and soil moisture probes continue to record soil moisture every hour. Root growth measurements using the mini-rhizotron tubes installed in both groves were performed in March and July 2019 and Feb 2020. Data on these measurements are currently being analyzed and will continue in Fall 2020. Additional nutrient measurements are still in progress.
The next measurements of weed density, soil and tree nutrients, and microbial communities will occur in August 2020.
The next set of cover crops will be planted in both groves are being planted on May 15 and May 19, 2020. The mixes will include sunn hemp, cow pea, several millet species, and buckwheat.
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. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc-facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBEP thA4 -CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBEP thA4 -CsLOBP. A total of seven 35S-LbCas12a-transformed Duncan plants were generated, and they were designated as #D35 s1 to #D35 s7, and ten Yao-LbCas12a-transformed Duncan plants were created and designated as #Dyao 1 to #Dyao 10. LbCas12a-directed EBEP thA4 -CsLOBP modifications were observed in three 35S-LbCas12a-transformed Duncan plants (#D35 s1, #D35 s4 and #D35 s7). However, no LbCas12a-mediated indels were observed in the Yao-LbCas12a-transformed plants. Notably, transgenic line #D35 s4, which contains the highest mutation rate, alleviates Xcc.pthA4:dCsLOB1.4 infection. Finally, no potential off-targets were observed. 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. 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 are further characterizing the citrus U6 promoters and testing their efficacy in driving sgRNA and crRNA in genome editing of citrus. We have significantly increased the transient expression efficiency. 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. One manuscript is in preparation for publication.
Citrus huanglongbing (HLB) is presently the most devastating citrus disease worldwide, and the Florida citrus industry is fighting for its survival against HLB. However, high disease pressure and natural selections of citrus genetic variants provide a golden opportunity for selecting seedlings and/or budsports with greater HLB resistance/tolerance, along with advantage of less time course and no regulatory constraints. This project was built upon a proof of concept on the screening of large numbers of Duncan seedlings via graft inoculation, and the performance observation of 15-25 years old Red Ruby volunteer seedlings (VS) in HLB endemics fields. Twenty of the 300 VS trees were selected, and propagated on sour orange rootstocks in greenhouse. These propagates were evaluated for HLB resistance/tolerance via graft inoculation twice (six months apart) with two different Candidatus Liberibacter asaticus (Las) isolates. Four out of the 20 lines were further selected and propagated on three different rootstocks (sour oranges and US 942) with over 1000 trees. These selections were then planted either in USHLR research farm, Pico’s Farm or the Scott Groves where the VS trees were identified. After almost three years’ evaluation in Pico’s Farm with an extremely high psyllid and HLB disease pressure, the obvious and significant results have been perceived by stakeholders during their field trips. In details, some of the selections showed much lower infection rate (less than 20%) than the control (40%) and poor performers (40-50%) after 26 months evaluation with periodic qPCR assays. It is worth to point out that among the four selections, the best one displayed the lowest disease rate (13.0%) and better growth canopy. Meanwhile, the new plantings (750 trees) in Scott Groves are less than one year old, but they grow well as expected and will be further evaluated. The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected VS trees showed no significant difference from their neighbor maternal trees and commercial grapefruit trees as control both in the original VS trees and their progenies in the new plantings. In other words, these selected VS trees inherited the genetics for good fruit quality when they are propagated on good rootstocks. One such a new selection of rootstocks not only shares the general feature of sour orange, but also yield less size of canopy, which makes it possible for high density of planting. It is well known that it takes at least 5-6 years to evaluate a citrus cultivar for its HLB resistance/tolerance and other major horticulture features in fields. Although the grant terminated in the middle of the project, the selectee and the methods developed for rapid selection and evaluation of citrus variants may serve as bases for developing a new approach to obtain HLB resistant/tolerant cultivar(s) for future citrus industry.
Create new candidate hybrids. Sexual hybridization is completed between selected elite parents during spring flowering and seed collected in the fall. During this quarter, seed were planted from crosses of elite rootstock hybrids US-942 and US-897 with pummelos. Selected hybrids will be grown-out for propagation, testing, and establishment of seed trees. Emphasis of hybridization in the USDA rootstock program is among parents with superior tolerance to HLB, CTV, and Phytophthora, along with showing favorable effects on grafted tree yield, fruit quality, and tree size. Propagate and plant new field trials. Replicated multi-year field trials with commercial scions are essential to evaluate performance of rootstocks, both to determine whether each new rootstock should be released for commercial use, and to develop comparative performance information among new and existing rootstocks for a diversity of scions, soils, and management conditions. Most of the rootstock field trials are planted with a single scion representing a common commercial type on each of 40-60 different rootstocks. Adequate replication is considered a critical factor in the USDA rootstock trials, with 6-7 replications the minimum and 12 replications the optimum to provide an acceptable level of reliability for results. Plans for planting new field trials this quarter were delayed because of institutional Coronavirus shutdown. Nursery trees for four rootstock trials are being maintained in the greenhouse for planting sometime later in 2020.Collect data from field trials. Information on tree performance is collected from established field trials, and includes measurement of tree size, fruit crop, fruit quality, and pathogen titer, HLB symptoms, and assessments of tree health. Measurements related to cropping are on an annual cycle based on the scion, while measurements of health and tree size are on a schedule determined by the specific conditions and goals of the trial. Cropping data was collected from nine trials with Valencia scions during this quarter. Assessments of tree health and measurements of tree size were completed on 13 trials during this quarter.Evaluate effectiveness for seed propagation of new rootstocks and develop seed sources. Some of the newest hybrid rootstocks can be uniformly propagated by seed, but others cannot. As the best rootstocks are identified through testing, seed sources are established and used to determine trueness-to-type from seed. Studies were continued this quarter to evaluate seed propagation for 25 of the most promising SuperSour hybrid rootstocks, including SSR analysis of progeny.Posting field trial results for grower access. The USDA rootstock trials produce large amounts of information that is useful to identify the most promising of the new hybrids, as well as comparative information on the relative performance of many commercially available rootstocks. During this quarter, the website https://citrusrootstocks.org/ came online, and will be updated regularly to share current summaries of performance information from the USDA rootstock trials. Release of superior new rootstocks for commercial use. Several of the 300 advanced Supersour rootstock hybrids in field trials are exhibiting outstanding performance in comparison with the commercial standard rootstocks. Perforance data continues to be collected, but it is anticipated that 2-3 of the most outstanding of these will be officially released in 2021-22.
In the first quarter of 2020, Juvenile Tissue Citrus Transformation Facility (JTCTF) accepted four new orders. One client decided to request more plants from previously placed orders and that counted as two new orders. We also received request to work on two additional binary vectors from another client. All these orders were for production of transgenic Duncan grapefruit plants. In accord with the decision of University of Florida leadership, JTCTF stopped working in the middle of March because of the COVID19 epidemic.Between January and April of 2020, JTCTF produced 60 plants. Among the produced plants there were: 47 Duncan grapefruit plants, 12 Valencia orange plants, and one Murraya koenigii (Indian curry leaf) plant. Duncan grapefruit plants were product of experiments with seven different Agrobacterium strains carrying following plasmids: ZM15-2, ZM16, ZM17, JJ8, HGJ87, HGJ88, and TAC1. Valencia orange plants were produced from experiments with bacteria carrying BB3 and BB4 plasmids. Finally, Indian curry leaf plant was produced from experiment with bacteria carrying BB4 plasmid.The JTCTF is undergoing transition to EBA unit in accordance with demands presented to me from both UF and CREC leadership. In the last three weeks, I have been working with the different people in UF and CREC administration to complete the EBA form that will determine the future price list for JTCTF services. In my future reports, I will share the progress of this process. Currently, there are six employees in the facility. Once we return to work from the leave caused by COVID19, I will have clearer picture about the future labor force of JTCTF.
Update for this quarter: No additional trees were planted this quarter, but data collection continues for ongoing projects on previously established trees. Stover analyzed data on canker incidence in a block of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside, from data collected 8/17 and 9/19. Most notably: Almost all accessions with lower ACC lesion incidence were hybrids vs. pure trifoliate, though a few pure Poncirus had lower ACC than most. Based on chloroplast genome data from 57K Affymetrix SNP chip, provided by M. Roose, 11 of 33 reported seed parentage for hybrids was inaccurate, convention of female first was not followed. Of 34 hybrids validated, similar numbers had Poncirus, grapefruit, and sweet orange chloroplasts. Chloroplast type did not affect ACC incidence, but in each year accessions with grapefruit chloroplasts had small but statistically higher ACC severity than those with Poncirus chloroplasts. Hybrids of Citrus with Poncirus have markedly reduced ACC sensitivity compared to Poncirus, indicating that this trait is readily overcome in breeding. Seed from fruit harvested for transgenic gene flow experiment coninue to be processed for PCR. Previously established at the site: A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. Renewal and approval for BRS permit effective 9/1/19 through 8/31/20. 4) Continuation of an experiment on pollen flow from transgenic trees. FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. Early-flowering transgenic Carrizo (flowered ex-vitro within five months of seed sowing, and used at 12 months) was used to pollinate some of the same FF-5-51-2 What should be the final samples from the C. Ramadugu-led Poncirus trial (#3 below) completed preparation and were shipped in ethanol to UC Riverside. Availability of the test site for planting continues to be announced to researchers. 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 Stover permit. 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. Likely 2019 will be the last year for data collection. 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 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) 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. 12) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State13)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
Project rationale and focus: The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Most recent quarter:A partial shut-down of USHRL was initiated 3/20/2020, as a response to the Covid-19 pandemic. ACP colonies are Stover lab:5460 ACP used for inoculating 390 detached leaves, 78 no-choice small trees ,and seven homogenate assays of peptides. Bowman lab: Prepared a group of grafted plants and planned to ACP-inoculate in March, but this experiment was disrupted by the Covid-19 slowdown. These ill be inoculated when personnel are allowed more extensive time at USHRL Other users:· 180 for Robert Shatters · 500 for Yongping Duan
The first evaluations have been completed with citrus plants we already had on hand which were received from Dr. Grosser in 2018-19. We evaluated new citrus hybrids for their potential use as windbreaks and trap plants. These hybrids were produced from the cross of C2-5-12 Pummelo [Citrus maxima (L.) Osbeck] x pollen from Citrus latipes. Seeds were germinated in calcareous alkaline (pH 8) clay soil supplemented with Phytophthora nicotiana and P. palmivora as a preliminary screen. Three vigorously growing putative hybrid seedlings were selected based on leaf morphology and seedling vigor. Hybrids were verified by Simple Sequence Repeat (SSR) markers, and exhibited a compact and upright tree architecture as seen in the pollen parent (C. latipes). Volatiles extracts showed that hybrid II contained higher amounts of volatiles compared with other hybrids, and was more attractive to D. citri than the other hybrids and its parents. Likewise, the levels of most of released volatiles in hybrid II were higher than those released from the other hybrids. The leaf morphology, seedling potency, and compact and upright architecture of hybrid II suggest that it could be a successful windbreak, whereas its attractiveness to D. citri indicated that it might also act as a trap plant. Planting hybrid II as a windbreak at the edges of citrus orchards could help attract D. citri, and allow focused insecticide applications, thus killing D. citri before reaching the commercial citrus trees. Further experiments have been established in our greenhouse: 1) Cuttings of Lucky (Sugar Belle x Nava Osceola) are well established and were ready to begin sampling of the Spring flush, but has been delayed due to Covid-19 CREC closure.2) Seeds of seven new rootstocks (UFR 1, 2, 4, 5, 6, 15, and 17) were planted in our greenhouse, germinated, and are now about three months old. These were received from Drs. Grosser and Gmitter. 3) Scion grafts – we side-grafted five new scions onto UFR-2 in November and mid-December 2019. The six scions include C2-2-1, OLL-8, N-40-6-3, RBB-7-34, Grapefruit 914, and 46 x 20. Some of these need to be re-grafted with new budwood. When the new rootstocks/scions are well established, we can begin challenging the new varieties with D. citri to evaluate their attraction/tolerance to psyllids and HLB. Following the psyllid biology experiments, leaf samples can be taken for metabolite and VOC analyses, perhaps in the next 6-12 months.
The objectives of this project are to produce mature citrus transgenics that will flower & fruit naturally using Agrobacterium as a service for customers, increase transformation efficiency, & conduct research to further biolistic transformation, so that it will also become an efficient service requiring less deregulation. The number of transgenics produced, successfully micrografted, & secondary grafted for this quarter are listed in Table 1 below: Table 1. The number of transgenics produced for customersCustomers Vectors Cultivars # Transgenics Zale A Kuharski 5 Dutt B B770 1 Dutt C Kuharski 2 Mou D Hamlin 5 Mou E Kuharski 10 Mou E Swingle 1 Mou E X639 1 Dutt F US942 3 Dutt G Kuharski 11 Dutt H Valquarius 1 Total 40 Five transgenics must still be verified with PCR because the transgene rearranges in 30% of events. In addition, not all transgenics have been transferred to customers because they were too small at the time of UF closure because of the global pandemic, so we are maintaining them in cone-tainers. Five transgenics out of ~25 with the new plant selectable marker were micrografted for the Zale lab & have been transferred to the growth room to encourage growth for molecular analyses.
Root growth of trees at trials 2 and 3 in Hendry County and in Polk County, respectively, continued to be monitored bimonthly using rhizotrons and an imaging system. Trees in both trials were evaluated for foliar HLB symptoms and canopy health. Leaves were collected from trees and are currently being processed for CLas titer determination. Root growth measurements commenced in trial 4 which was established in November in Indian River County using the same rhizotron/imaging system that is used in the other trials. The analysis of the large data set from trial 1 at SWFREC which was terminated (whole tree excavations) during the last quarter has been completed. Data on root architecture, tree growth, and nutrients were presented during the Florida Citrus Show in Fort Pierce in January and during a FNGLA nursery workshop in Lake Alfred in February. Pdf files of presentations are available upon request. The study Influence of Rootstock Propagation Method on Traits of Grafted Sweet Orange Trees by U. Albrecht, S. Bodaghi, B. Meyering, K.D. Bowman was accepted for publication in HortScience and is currently in press (for open access). Two abstracts were submitted for presentation of research data at the FSHS and ASHS annual meetings (both in Florida). The two-year horticultural evaluations of trees in trials 2 and 3 were planned to commence at the end of this quarter but had to be postponed because of UF closure and research halt due to Covid-19.
In preparation for harvest and fruit quality analysis, multiple field trips were conducted to relabel trees with plastic tags where necessary and to reflag trees for easy identification. Due to the unusually high preharvest fruit drop, dropped fruit and remaining fruits on trees were counted for the two sentinel trees in each 8-tree plot to calculate the percentage of fruit drop. Fruit drop ranged from 33% to 74% with some differences among trees on different rootstocks but no consistent trend among the two locations. In January, Hamlin trees were harvested including all 8 trees in each replicated plot. Fruit quality analyses had been conducted in December 2019. Horticultural assessments were performed and included tree height, canopy volume, and trunk diameters. Disease ratings and canopy health ratings were repeated in this quarter, because trees looked more HLB symptomatic than during the last quarter. Significant differences among rootstocks were found for most variables measured. The cumulative yield averaged across both production years and locations was highest in trees on FA-5, UFR-5, C-54, and X-639 (61.3-65.0 lbs/tree) and lowest in trees on Amb+Benton, Changsha Benton, Sorp+Sh-991, and UFR-3 (28.9-34.4 lbs/tree). Tree height and canopy spread was largest in Hamlin trees on X-639, C-54, and C-57 and smallest in trees on Amb+Benton, UFR-3, Green 3, and Changsha+Benton. The highest yield efficiency (lbs fruit per canopy volume) across both seasons and locations was found for trees on Green 3, Amb+Benton, Changsha+Benton, and FA-517. Yield efficiency was lowest for trees on X-639, FA-31, C-146, and FA-13. Due to the UF closure and halt of all research activities because of Covid-19, arrangements were made for a private contractor to conduct the fruit harvest in both Valencia trials and collect fruit for fruit quality analysis. Prior to the research halt, dropped fruit and remaining fruit on trees were counted to assess rootstock effects on preharvest fruit drop and obtain yield estimates in case harvest data collection would be impossible. Horticultural measurements were initiated but were not completed due to the research halt. Data is currently summarized for an upcoming article in Citrus Industry magazine (July issue).Abstracts were submitted for presentation of research data at the FSHS and ASHS annual meetings in Florida.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Seeds of twelve new rootstock crosses made in spring 2019 were harvested, planted into the calcareous soil/ Phytophthora screen, the first stage of the gauntlet screening protocol, and good performers have been identified and moved forward in the Gauntlet process. Seeds harvested from the UFRs have been distributed to interested nurseries. Seeds from various unreleased rootstock candidates, showing good performance (HLB tolerance, good yields and fruit quality, some tree size controlling with more efficient canopies) in several field trials throughout the industry were harvested to be available for future trials with interested industry partners in Florida, as well as for new trials in other citrus production areas. 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. Spring 2019 crosses for this objective were numerous. We harvested fruit from more than 2 dozen interploid crosses, including several designed to produce sweet orange-like fruit, using HLB tolerant parents selected by our program previously. In addition, selected HLB and canker tolerant pummelo breeding parents were crossed with diploid and tetraploid grapefruit, to produce new grapefruit hybrids with enhanced tolerance of HLB. Embryo rescue has been completed and plant production has proceeded. Over 100 new somaclones have been regenerated in vitro from EV1 and EV2, for further selection; these are being grown off for propagation and eventual field planting. Six new grapefruit hybrid selections were made in this autumn 2019 and January 2020 that produce fruit very similar to grapefruit in appearance, color and flavor, but with improved fruit quality attributes and substantially more HLB-tolerant trees; many of these have been entered into the DPI Parent Tree Program for cleanup. We worked together with USDA and citrus industry colleagues to write a manuscript titled: Rationale for reconsidering current regulations restricting the use of hybrids in orange juice; this was a joint effort led by Drs. Stover and Gmitter, and submitted to a major international horticulture journal, Horticulture Research (under the Nature banner). The objective of this manuscript is to work together with the Florida Citrus Processers Association, to lay the groundwork for a revision of the current Standards of Identity for Orange Juice, to enable use of HLB-tolerant sweet orange-like hybrids in OJ. We facilitated the commercial harvest of grove run Sugar Belle® fruit for commercial evaluation by a major juice processor, to assess its ability to be transported in trailers to the processing plant, and the quality of its juice through the processing process. This was done to support the interests of some industry players to consider this most HLB-tolerant variety for blending to improve OJ color, flavor, and overall quality 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have collected extensive data using new approaches to quantify tree responses to HLB, in addition to the previously used subjective approaches. Specifically, we have measured photosynthetic parameters and leaf canopy indexes, to produce repeatable and reliable quantitative data in support of further genetic analyses of tolerant types. Analysis of these data is underway, and we are comparing the results from different methods. We are looking for consistency in the rankings and weighing this against the simplicity and efficiency of collecting data. Continuing through the fruit harvest season, we have evaluated fruit quality of the more tolerant types of sweet orange-like hybrids, as well as mandarins and grapefruit hybrids, and identified some worthy of further evaluation as potential new cultivars. 4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. Using the quantitative data described in 3. above, we began additional GWAS to validate previously identified or to identify new genomic regions associated with HLB tolerance and/or sensitivity; results will be analyzed in the coming months. 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 were carried out.
The present report covers the period of December 1, 2019 through February 29, 2020. During this period, the M.S. student on the project (Mr. Chad Vosburg) made a visit to the USDA United States Horti-cultural Laboratory in Fort Pierce, FL, in December and January. In collaboration with USDA personnel, in particular, Dr. Greg McCollum and Dr. Tim Gottwald and members of their laboratories, Chad prepared for and helped set up psyllid-mediated HLB infections for 20 plants for each of two transgenic anti NodT-FT-scFv lines of ‘Duncan’ grapefruit. Asian Citrus Psyllid (ACP) mediated transfer was done in tents in the greenhouse. The transfer was deemed complete after psyllid feeding and reproduction on the plants was observed. The trees are now in the greenhouse. Time zero DNA samples were taken from the trees, and the samples were analyzed by qPCR for ‘Candidatus Liberibacter asiaticus’ (CLas) DNA. No CLas DNA was detected in the test plants. Control untransformed ‘Duncan’ grapefruit trees were also included in the experiment. All trees used for this experiment are on their own roots (ungrafted). In addition, transgenic FT-scFv buds were grafted onto rough lemon roostocks heavily infected with CLas during this trip by Chad. Populations of two high-expressing FT-scFv lines have been started in a field trial at the Pecos Farm site of the United States Horticultural Research Laboratory at Fort Pierce, FL, and these trees have begun to grow as of the end of February, 22020. This field trial includes non-transgenic ‘Duncan’ trees as well. Tree performance measurements according to standard parameters have been initiated for all trees. The trees for the outdoor trial were also tested for CLas DNA by qPCDR and found to be negative at the time of planting.
Potential breakthrough: The Florida NFC industry would love to have high quality sweet orange cultivars that mature in the December/early January window to replace Hamlin. The EV’s (that mature in November) appear to be too early for this slot. During this quarter, we evaluated fruit quality from 80 new Vernia clones (somaclone-derived seedlings and a few cybrids), that were mostly grown in St. Cloud (Orie Lee Mathew Block) without psyllid control on their own roots. From fruit harvested the first week of December, 7 clones were identified that had ratios higher than 15, indicating full maturity at this time (they are: R24T2; R25T2; R25T12; R25T7; R25T5; R26T10; and R26T4. Two clones with much higher soluble solids were also identified: N40W-V7-9 (also 14.51 ratio) and 18A-V8. These clones are being propagated for further evaluation. These clones have clear potential to fill this slot, and Vernia has shown much better tolerance to HLB than Hamlin.
Rooted cuttings were obtained from approximately 30 new gauntlet rootstock candidates. A population of 60+ vigorous rooted cuttings of the best Picos gauntlet rootstock selection S10xS15-12-25 were moved up to 4×4 citripots to be budded for an advanced field trial; a 2nd set of 80+ cuttings of this selection were successfully rooted, This hybrid is a sour orange-type produced from a cross of HBPummeloxShekwasha mandarin X HBPummeloxCleo mandarin (with both parents previously selected for salinity tolerance). The original gauntlet tree was vigorous yet a precocious heavy bearer.
Potential gauntlet rootstock resistance: Monthly PCR testing was initiated on the top 50 gauntlet rootstock candidates at the USDA Picos Farm. 10 selections were identified that repeated for ct values of 32 or above, indicating no active CLas infection; and their roots were also negative; they were: A+HBPxWhite 1-12-13; A+HBPxWhite 1-12-20; A+HBPxSORP-13-79; A+HBPxSORP-13-60; Orange 15 mutants 25 & 34; A+HBPxCH+50-7-12-8l A+HBJL2xOrange19-09-7; and S10xUS-812-11-2. Several others were negative/questionable both samplings, including A+VolkxOrange 19-11-8 that is producing abundant polyembryonic seed. These results show promise for development of a rootstock that can completely mitigate the disease.
The first gauntlet SugarBelle rootstock (S13xLB8-9-15-16) continues to show no HLB symptoms although the ct value indicated a high CLas titer (but no titer was detected in the roots). The high titer was attributed to heavy psyllid pressure on the tree. Limited cuttings of this selection were put on the mistbed for further evaluation of this promising rootstock.
Seasonal Field Trial Data Collection (tree health status, yield and fruit quality where possible) during the quarter was collected from the following trials: Duda, Smoak, Wheeler Bros., IMG, Bryan Paul, Post Office Block, Peace River Growers, Hidden Golf Course trial, Banack, Hammond, Teaching Block, OrangeCo., CPI and Serenoa. Two new trials were added to the website and four trials were updated.
In this quarter, several transgenic rootstocks expressing the AtNPR1 transgene were inoculated with HLB infected Valencia budsticks (November). Most of these were repeats of ones that had failed from the september graftings. As with pervious attempts, the Ct values of the infected budsticks were evaluated prior to stick grafting. Ct values ranged from 24.1 to 27 in the repeat budded set. The objective behind this study was to understand the effect of the transgenic rootstock on HLB. The budsticks have been slow to flush due to the winter. qPCR was performed on both transgenic and control roots as well as leaves but no HLB has been detected so far. it is estimated that it will be several more months before the inoculum can be detected. Transgenic rootstocks budded with non-transgenic valencia scion have also been prepared for planting in the field since we expect the field trial will provide the most conclusive data.
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