CAP: COMBINING CULTURAL AND GENETIC APPROACHES FOR GROVE SUCCESS TO UNRAVEL AND ENHANCE RESISTANCE/TOLERANCE TO HUANGLONGBING

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1024604
• Grant No.: 2020-70029-33202
• Cumulative Award Amt.: $10,000,000.00
• Proposal No.: 2020-08471
• Project Start Date: Sep 1, 2020
• Project End Date: Aug 31, 2026
• Grant Year: 2021
• Program Code: [ECDRE]- Emergency Citrus Disease Research and Extension Program

Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521

Performing Department
College of Nat & Agr Sciences

Non Technical Summary
The research objective of this proposal is to investigate the root collapse associated with Huanglongbing (HLB)-impacted trees and ways to mitigate it by promoting root health. Our previous work demonstrates that as HLB severity increases, the root microbiome becomes enriched in soil-borne pathogens. We will conduct experiments to empirically determine if these pathogens exacerbate the HLB-associated root and canopy decline. We will integrate field studies that test HLB resistant/tolerant rootstocks and use of soil amendments that promote root health to determine if they suppress pathogens in the roots and prolong tree longevity/productivity under HLB pressure. These field studies will include newly established groves and mature groves. To support our field trials and decipher the genes/gene pathways that dictate how plants respond to HLB, we will determine how rootstocks and scions respond toCandidatusLiberibacter asiaticus using citrus varieties that are either highly HLB-susceptible or HLB-resistant using a combination of disease phenotyping and gene expression analyses. We will integrate this research with a robust extension and outreach program in combination with an economic cost-benefit analysis structured around adoption of treatments that enhance root health into commercial citriculture.

Animal Health Component

50%

Research Effort Categories

Basic

40%

Applied

50%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	0999	1160	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1160 - Pathology;

Keywords

crispr/cas9

candidatus liberibacter asiaticus

huanglongbing (hlb)

Goals / Objectives
HLB is a major threat to US citrus production. It is highly destructive and lethal to commercial citrus cultivars making it the most serious citrus disease. In the US,CandidatusLiberibacter asiaticus (CLas), is the primary bacterial species associated with HLB and the primary vector is the Asian Citrus Psyllid (ACP),Diaphorina citri. Despite years of research, there is no cure for HLB, and management practices focus on vector management/exclusion and maintaining tree health through nutrition regimes. Root collapse is an issue in HLB-impacted trees andCLas colonization is linked to fibrous root decline prior to manifestation of above ground symptoms.The research objective of this project is to investigate the root collapse associated with HLB-impacted trees and ways to mitigate it by promoting root health. This research will fill a gap in knowledge and provide immediate solutions for growers to cope with HLB.Our research group has demonstrated that high vigor HLB tolerant rootstocks had significantly less fibrous root growth reduction than HLB susceptible rootstocks when infected withCLas in greenhouse studies. Results from large-scale field trials in FL further also suggest that rootstock vigor may contribute to HLB tolerance. In addition, our group has demonstrated that significant tissue-specific microbial shifts occur within the citrus microbiome as trees get sicker.The changes in the root compartment were particularly striking.Specifically, as disease progressed, there were depletions of putative beneficial species in roots and enrichments of parasitic and saprophytic microorganisms as trees got sicker with HLB. We hypothesize that these root pathogens are major contributors to fibrous root decline associated with HLB and, in turn, exacerbate above ground HLB symptoms. In the proposed project we will integrate rootstock testing as a factor in maintaining root health and also monitor the influence of rootstock on the root/rhizosphere soil community to determine if choice of rootstock imparts resistance to the pathogenic microbial enrichments observed in trees in HLB-impacted groves.Mitigating root decline is vital for prolonging a grove's lifespan under high HLB pressure. Hence, FL growers are now using different strategies to enhance root health by improving the soil environment with soil amendments such as compost and humic acids. Preliminary results from our research team indicate that soil amendments also impact microbial communities in the citrus rhizosphere. In the proposed work, we will further refine this research to test how different sectors of the root microbiome contribute to or lessen fibrous root loss and if soil amendments and HLB resistant/tolerant rootstocks can be used to mitigate root loss associated with HLB. To support our field trials and decipher the genes and gene pathways that dictate resistance or susceptibility to HLB from the plant side, we will also determine how rootstocks and scion varieties respond toCLas using the same trifoliate orange (Poncirus trifoliata) andP. trifoliatahybrids that we will be testing in our field and greenhouse trials.The short-term goal of the proposed project is to determine if secondary root pathogens exacerbate overall tree (root and canopy) decline under HLB pressure. The mid-term goal is to provide growers with near-term actionable guidelines in areas of HLB pressure (FL) and areas where HLB remains an imminent threat (CA) that are based on rootstock performance integrated with soil amendments that focus on promoting root health.The long-term goal is to leverage data from these experiments to provide a mechanistic understanding to what dictates HLB susceptibility or resistance in the context of the HLB disease complex.

Project Methods
Objective 1.We will sample mature trees in FL commercial groves that fall into three different HLB severity levels for metagenomic microbiome analysis. We will collect roots and rhizosphere soil from trees for each disease state for metagenomic and metatranscriptomic analysis. DNA libraries will be constructed, sequenced, assembled, and annotated using methods for metagenomic previously employed by Co-PD Stajich. Strand-specific RNA-Seq libraries will be constructed with Nextera kits and sequenced on Illumina Novaseq to a depth of 30 Gb per library. Meta-transcriptomic analysis will follow bioinformatics best practices developed by Co-PDs Stajich and Cantu based on mapping RNA-seq reads. The metagenome/transcriptome data will be processed to compare the dynamics of the taxonomic and functional makeup of root and rhizosphere communities among the different HLB states.PD Roper initiated a curated citrus microbial repository that contains over 1,600 bacterial and fungal isolates derived from citrus groves in FL and CA. We will continue to build this repository by culturing from samples obtained in Objs.1,2,3. This repository will serve as the primary resource for testing pathogen enrichments and their contribution to root decline observed inCLas-infected trees. Our repository contains threeFusariumroot isolates collected from FL groves. Initially, HLB-susceptibleCitrus sinensiscv. Valencia grafted onto Swingle rootstock will be inoculated withCLas using aCLas-positive psyllid colony. Control plants not exposed to psyllids will be similarly inoculated. We will then testPhytophthoracollected from FL using the same experimental design. We will continue inputting putatively pathogenic microbes into this pipeline as our model predicts and as our culture repository expands. In addition to testing single isolates, we will test isolates in combination with one another. Plants will be visually examined weekly for HLB symptom development over a period of 6 months and rated on an arbitrary HLB rating scale of 0 to 5.In addition,CLas titer will be quantified using qPCR. Root decline will be quantified by measuring root biomass and fibrous root length. We will link to the other objectivesand integrate the HLB-resistant/tolerant rootstocks that are being evaluated for field performance into the same experimental design described above to determine if rootstock choice confers resistance/tolerance to soil-borne pathogens. This will include an in-depth evaluation of the transcriptional response not only in the rootstock but also theinteraction of the same tolerant/resistant rootstock varieties grafted with a common scion ('Valencia') to explore the molecular basis of the response of a susceptible scion toCLas when grafted onto rootstocks with varying HLB resistance/tolerance. We will continue to build our culture repository by isolating microorganisms from samples collected in Obj. 1a and Objs. 2, 3. Whole genome sequences will be obtained for microbes of interest.Objective 2.A field trial will be conducted in a recently replanted commercial citrus grove in SW Florida. Trees in this planting are Valencia (C. sinensis) grafted on HLB tolerant US-802 (C. maxima×P. trifoliata) rootstock. Organic amendments will include: 1) certified compost; 2) a combination of humic acids and fulvic acids in granular form; and 3) a control without organic amendment. We will quantify glyphosate half-life in soil among the different treatments as well as glyphosate impact on fruit drop. A second field trial will be conducted in the same commercial citrus grove. Trees in this planting are Valencia grafted on US-802 rootstock and on 3 mandarin (C. reticulata) × trifoliate (P. trifoliata) hybrid rootstocks: US-812, US-897, and X-639. The main plot consists of 200 trees treated with: 1) compost or 2) no compost; and the subplots consist of the 4 different rootstocks arranged in rows of 50 trees. Cover crops or no cover crops will be established as sub-subplot factors in row middles by dividing rootstock plots into two sub-sub plots of 25 trees each. A third field trial will be conducted on an experimental plot at the Lindcove Research and Extension Center (REC) using the CA industry standard mandarin Tango scion (C. reticulata) on six rootstocks planted in 15 rows of 30 trees on berms. Organic amendments (main plots) will include: 1) manure (standard for CA farms); 2) a combination of humic acids and fulvic acids in granular form used in FL; and 3) a control treatment. A random subset of trees from each experimental unit will be analyzed for tree height, canopy volume, and scion and rootstock trunk diameters. In addition, trees in FL will be rated for canopy density, canopy color, and foliar HLB symptoms according to standardized guidelines. To assess the water status of trees, stomatal conductance will be measured seasonally.CLas titer will be quantified by qPCR. Anin-situroot imaging system will be used to non-destructively visualize fibrous roots and measure length, diameter, and seasonal growth. Root cores will be collected annually separated into fibrous and non-fibrous roots, and root lengths and biomasses will be determined. Following all soil amendments described above, glyphosate half-life in the soil, effects of glyphosate on specific root length and exacerbation of HLB-related fruit drop will be quantified as previously described. Yield will be determined in collaboration with a commercial harvesting crew. For the microbiome analyses, rhizosphere, root, and leaf samples will be collected and processed each yearon an Illumina MiSeq platform. Data will be analyzed using published protocols and standard statistical analyses.Objective 3.The soil cultural practices that are being evaluated in Obj. 2 on young orchards will also be tested on established mature orchards in Obj. 3. A field trial will be established in 2021 in a commercial mature grove. Trees are Valencia on US-942 (C. reticulata×P. trifoliata) rootstock, known for its tolerance to HLB and currently the most propagated rootstock in FL. Trees are planted in single rows without furrows. The design will be a split plot design with cover crops or no cover crops as main plot factors and weed management (glyphosate or no-glyphosate) as subplot factors using 6 replications for a total area of 10-12 acres. Cover crops will be planted 3 times a year and mixes will vary by season. A second field trial will be conducted in CA on an existing experimental citrus grove at the Lindcove REC. We will use Tango (C. reticulata) on Carrizo rootstock (C. sinensisxP. trifoliata) planted in 2011. The design will be a split plot design with weed management as the main plot and organic amendment as the subplot. Main plots will consist of rows arranged in 5 biological replicates of 24 trees, and subplots will consist of trees in a block of 12 trees. The block will be split in half (12 trees per row) so that one side of the block will receive humic acid (Humic DG) in the irrigation whereas the other one will receive standard irrigation water as a control treatment. We will measure the impact of weed management strategies on root growth, fruit drop and fruit quality. Weed management treatments will include glyphosate and a no glyphosate control.Objective 4.To identify socioeconomic factors and risk perceptions that affect grower decisions to adopt new cultural practices that reduce the threat of HLB, we will survey growers and pest control advisors and analyze the profitability of adopting root/tree health cultural practices. We will then construct representative citrus production systems to simulate systems in CA and FL and evaluate the economic gains or losses associated with adopting the identified root/tree health cultural practices. Following these analyses, we will draft manuscripts for peer-reviewed journals to share with the scientific community and extension resources to share with targeted end-users.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Our target audiences are grower stakeholders, scientists in academia, government and industry, undergraduate and graduate students and the general public. To reach these target audiences, we presented our work at national and international professional science meetings that are attended by scientists, citrus growers and agricultural industry representatives. These include participation in the 15th International Conference on Plant Pathogenic Bacteria and the 5th International Symposium of Biological Control of Bacterial Plant Diseases, International Research Conference on Huanglongbing, United States Department of Food and Agriculture, National Institute of Food and Agriculture, Specialty Crop Research Initiative, Emergency Citrus Disease Research and Extension Project Directors meeting, UF/IFAS Extension Seminar Series, Soil Science Society of America Annual Meeting, California Citrus Nursery Society Conference, Soil Ecology Society meeting.Our Extension Specialists have disseminated information to target stakeholder audiences through our project website, presentations at stakeholder meetings and through semi-technical stakeholder publications that are listed in the Products (Publications) section of this report. We have also disseminated information about Huanglongbing (HLB), its implications to the citrus industry and specifics of this project through our project website, educational aids and curricula developed by the PDs and PIs of this project. Changes/Problems:Field trials in Florida had to be terminated sooner than expected because of one of the grower collaborator's decision to end all citrus production. Despite the unexpected termination, the FL team collected a large set of data and several publications from these trials are being prepared. We have initiated a new trial to compensate for the shorter than planned other field trials. However, since these trees have been newly planted, it will take time before we can anticipate any meaningful differences. Aside from hurricane Ian in 2022, which compromised all trials in FL, hurricane Helene and Milton occurred in 2024. Though our trials have been spared the worst impact from this year's hurricanes, trees have experienced leaf drop, some fruit drop, and general stress from the wind, salt burn, and heavy rain. To facilitate research on the response of tolerant and susceptible rootstocks to HLB, we obtained the appropriate permits and training to perform research in a biosafety level 3 (BSL-3) facility. We initiated research associated with this grant at the California Citrus Research Foundation (CCRF) BSL-3 facility in March 2022. Issues with building infrastructure have affected containment barriers, which raised concerns about the potential for insect ingress and/or escape from containment. Due to these concerns UCR's Institutional Biosafety Committee suspended research using the insect vector in October 2023. The insects were used to inoculate citrus plants with the pathogenic bacteria. Infrastructure issues were not corrected by CCRF and in May 2023 the USDA-APHIS suspended the permits for all research activities due to inadequate containment. Permits are still suspended and it is unclear when corrective measures will be completed. After permit suspension, all experimental plants were moved to the Contained Research Facility at UC Davis. What opportunities for training and professional development has the project provided?The PDs and PIs of this project have trained members of the project in citrus propagation, DNA extractions from field collected tissues, microbial isolations from plants and soils, natural product isolations, Fusarium disease bioassays and microbial inhibition assays. We have also trained participants in the project on how to construct Illumina libraries and perform the subsequent bioinformatic analysis. Members of the project are also being trained on sequencing, single organism genome assembly and metagenome assembly, metagenome binning, gene prediction and annotation. The members are gaining skills in metagenome comparisons to identify organisms, genes and pathways which vary in composition between samples. The members are also training on amplicon metabarcoding library construction and bioinformatics analyses to support molecular ecology and contrasting of taxonomic distributions in samples with a focus on both archaea and bacteria using the 16S locus and fungi using the ITS locus. In addition, members of the team are being trained in the analysis of RNA-sequencing data, including quantification of gene expression, identification of differentially expressed genes between treatments and/or genotypes, and an introduction to unsupervised statistical learning methods to facilitate exploratory data analysis. Several Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society, Plant and Animal Genome Conference, the American Society of Horticultural Sciences, International Plant Protection Congress, the 15th International Conference on Plant Pathogenic Bacteria and the 5th International Symposium of Biological Control of Bacterial Plant Disease, International Research Conference on Huanglongbing, Genomics-assisted breeding for crop and livestock improvement, Soil Ecology Society Meeting, and the California Citrus Nursery Society Conference. How have the results been disseminated to communities of interest?The Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society, Plant and Animal Genome Conference, the American Society of Horticultural Sciences, International Plant Protection Congress, the 15th International Conference on Plant Pathogenic Bacteria and the 5th International Symposium of Biological Control of Bacterial Plant Disease, International Research Conference on Huanglongbing, the Soil Ecology Society Meeting, and the California Citrus Nursery Society Conference. We have also presented at grower stakeholder meetings through virtual extension presentations and in-person trade show/educational events (UCR Citrus Day, FL Citrus Show, Citrus Expo, FL Citrus Institute and UC ANR). Extension specialists in Florida and California are communicating regularly with growers and grove managers regarding ongoing field trials through emails, phone calls, and in-person meetings. PD Roper has integrated lectures on HLB and citrus microbiome research in the graduate class Bacterial and Viral Disease of Plants (PLPA207) and the undergraduate course, Introductory Microbiology (BIOL/MCBL121, MCBL131). Co-PD Albrecht and Co-PD Strauss have presented seminars at various grower events that included information and research updates from the Florida field trials, and published in trade journal articles. An Open House event at the SW Florida REC was held where Co-PDs Albrecht, Kanissery, and Strauss presented their research projects to the public). Co-PDs Rolshausen and el-Kareamy have presented seminars at various grower events in California that included information and research updates from the California field trials and have published various extension articles.Co-PD Kaplan has presented research findings through online research notes. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Transcriptomic analysis will be completed for tolerance and susceptible rootstocks that were mock-and CLas-inoculated and moved from the containment facility in Riverside to UC Davis. Metagenome analysis of citrus roots and leaves collected in FL from trees exhibiting a range of HLB symptoms will be completed.The metatranscriptomics portion of this work will be initiated on the same samples we used for the metagenomic analysis. This data will fold into the metagenomics data to construct a functional model of the citrus microbiome under HLB pressure. Microbial isolations from CA and FL will continue and isolates will be archived in the Citrus Isolate repository in the Roper lab. Microbial consortia inoculations will be conducted in conjunction with Fusarium, Phytophthora and CLas inoculations. Objective 2. Field trials in FL will continue as described above with biannual applications of soil amendments/cover crops, weed management, biometric measurements, tree health- and root health assessments, leaf and soil nutrient analyses, etc. Pre-harvest fruit drop will be assessed as well as yield and fruit quality in the upcoming harvest season. We will continue to sample the new young plantings in CA to assess the interactions of rootstocks and soil amendments (compost, humic acids). We will compare the performance of HLB-tolerant rootstocks from FL with CA standard rootstocks. The field trials will consist of biannual applications of soil amendments, weed management, biometric measurements, tree health- and root health assessments, leaf and soil nutrient analyses, etc. Microbiome-based data will be analyzed for the different rootstock and soil amendment combinations incorporated into our experimental design. Objective 3. The field trial in FL will continue as outlined in the proposal with additional cover crop plantings (seeding), weed management, biometric measurements and leaf, soil, and root sample collections for physiological, soil and microbiome analyses as described above. Pre-harvest fruit drop will be assessed and fruit yield and fruit quality will be determined in the upcoming harvest season. The field trial in CA will continue as outlined in the proposal with leaf, soil, and root sample collections for physiological and microbiome analyses as described above. Microbiome-based data will be analyzed for the different rootstock and soil amendment combinations incorporated into our experimental design. Objective 4. The economic team will continue to conduct outreach efforts in California and expand them to Florida. We will also complete our development of the epidemiological-agronomic-economic simulation model and tour analysis of identified practices.

Impacts
What was accomplished under these goals? Objective 1. We are now in our fourth year of collecting leaf and root samples from trees with low and high levels of HLB from groves under high disease pressure in FL. Microbiome-enriched samples were processed using two different methods to generate scaffold and shotgun libraries. Libraries from years 0, 1 and 2 have been sequenced and the subsequent data, following computational deconvolution, used for bacterial and fungal community construction. Sequencing and data analysis are ongoing. We established a workflow for growing citrus rootstocks from seeds in a BSL-3P containment facility and inoculating rootstocks with CLas infected psyllids. Ten rootstocks genotypes, including tolerant and susceptible types, have been mock- and CLas-inoculated. Infrastructure failures at the BSL-3P located in Riverside resulted in transfer of experimental plants to a contained research facility at UC Davis in June 2024. A subset of genotypes (3) were sampled prior to plant movement for RNA-sequencing (3 genotypes, 2 treatments, 4 timepoints). RNA-sequencing data for this subset has been analyzed for genotype-specific response to CLas. . Genotypes were selected to include rootstocks in field trials from Obj 2 and 3. We are also determining if root/rhizosphere microorganisms contribute to root collapse under HLB pressure. We have completed Koch's postulates with several HLB-associated Fusarium isolates we obtained from HLB-impacted citrus roots in Florida. We have determined that a subset of these are pathogenic on common citrus rootstocks. We have published whole genome sequences for 18 of these new Fusarium isolates. We have also initiated disease bioassays using two root-associated Phytophthora isolates and have expanded that work to new HLB-tolerant rootstocks. Our previous work indicated that these fungal/oomycete taxa increase in relative abundance in the roots of trees suffering from mid-late stages of HLB. These are known pathogens of citrus and our hypothesis is that their increase in relative abundance contributes to and exacerbates whole tree decline in trees with HLB. This objective is 60% completed. During this reporting period, we have published three technical journal articles and one semi-technical journal article for this objective. Objective 2. The Valencia/US-802 field trial in Southwest FL (trees planted in 2019) had to be terminated because the grower collaborator stopped all citrus production. A manuscript is in preparation (nearly ready for submission) reporting the results for two production seasons. We have processed, archived and extracted all DNAs from these samples. Another manuscript is in preparation assessing the impact of the humic acid treatments on the soil microbiome. The Southwest FL Valencia field trial with different rootstocks also had to be terminated for the same reason. A manuscript is in preparation focusing on soil, fibrous root, and rootstock-related changes in response to the treatments. We have processed, archived and extracted all DNAs from these samples for three sampling periods. We have completed the analysis of the microbiome data. Florida: We started a replacement trial with newly planted Valencia trees on US-942 rootstock at our center in southwest Florida to integrate cover crops with compost and use of individual protective covers in summer 2023. We started sampling the rhizosphere in 2024. California: All six rootstocks that are being evaluated in CA field trials were planted starting in 2022. Tree biometric measurements and tree health assessments were conducted and sample collections for nutrient and microbiome analysis were initiated in 2022. Organic amendment applications (humic acid and mulch) were applied and application of herbicide treatments were performed as outlined in the proposal. We initiated our annual sampling of leaves, roots, rhizosphere and bulk soil of these field trials to monitor changes in microbiome composition when using these amendments. We have processed, archived and extracted all DNAs from these samples for one sampling periods. We have initiated the analysis of the microbiome data. This objective is 60% completed. There have been three extension articles published for this objective. Objective 3.Florida: We continued the field trial in central FL with Valencia scion on US-942 rootstock in accordance with the experimental design outlined in the proposal. Tree biometric measurements and tree health ratings were continued and soil and leaf nutrients and herbicide residues were determined regularly. Rhizosphere microbiome analysis continued. Root health assessments (fibrous root respiration, density, and root size/length distribution) were continued seasonally. New seeds for cover crops were sown seasonally. Cover crop and weed biomass assessments were conducted seasonally. Fruit were harvested in March 2022, 2023, and 2024 and fruit/juice quality was analyzed.The grower has started to inject all trees with Oxytetracycline as part of his management program. California: We established the field trial at the Lindcove Research and Extension Center with Tango on C35 rootstock according to the experimental design outlined in the proposal. Rhizosphere, root and leaf samples were collected from all treatments and stored at -80C prior to processing and analyses. Treatments include control, mulch, glyphosate and humic acid. We continue our annual sampling of leaves, roots, rhizosphere and bulk soil of these field trials to monitor changes in microbiome composition when using these amendments. We have processed, archived and extracted all DNAs from these samples for three sampling periods. We have initiated microbiome analysis for this sub-objective. This objective is 60% completed. There has been one journal article and three extension articles published for this objective. Objective 4. The goal of this objective is to communicate our research to the stakeholder community, the greater academic community and the general public. During this report period, we participated in the Extension events listed in the Other Products (Activities and Events). Further advances have been made on our epidemiological-agronomic-economic model, which has and will be used to conduct simulated economic experiments. During this period we also made progress incorporating the functionality of practices into our simulation model. We now have data from the field trials to complete the analysis. This objective is 75% completed. There have been four extension articles published for this objective.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Lombardo Monia F. , Zhang Yunzeng , Xu Jin , Trivedi Pankaj , Zhang Pengfan , Riera Nadia , Li Lei , Wang Yayu , Liu Xin , Fan Guangyi , Tang Jiliang , Coletta-Filho Helv�cio D. , Cubero Jaime , Deng Xiaoling , Ancona Veronica , Lu Zhanjun , Zhong Balian , Roper M. Caroline , Capote Nieves , Catara Vittoria , Pietersen Gerhard , Al-Sadi Abdullah M. , Xu Xun , Wang Jian , Yang Huanming , Jin Tao , Cirvilleri Gabriella , Wang Nian. 2024. Global citrus root microbiota unravels assembly cues and core members. Frontiers in Microbiology, 15. https://doi.org/10.3389/fmicb.2024.1405751
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: A. Aksenov, A., Blacutt, A., Ginnan, N., Rolshausen, P. E., V. Melnik, A., Lotfi, A., C. Gentry, E., Ramasamy, M., Zuniga, C., Zengler, K., Mandadi, K. K., Dorrestein, P. C., & Roper, M. C. (2024). Spatial chemistry of citrus reveals molecules bactericidal to Candidatus Liberibacter asiaticus. Scientific Reports, 14(1). https://doi.org/10.1038/s41598-024-70499-z
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Campos Vieira, F., Blacutt, A., Drozd, C., Viravathana, P., Ginnan, N., & Roper, M. C. (2024). Thirteen draft genome assemblies of Bacillus spp. isolated from HLB-impacted citrus trees. Microbiology Resource Announcements. https://doi.org/10.1128/mra.00602-24
• Type: Peer Reviewed Journal Articles Status: Under Review Year Published: 2024 Citation: iaz, I., Ostovar, T., Chen, J., Saddoris, S., Schmitz, R.J., Wessler, S.R., Stajich, J., Seymour, D.K. Haplotype phased genome of Fairchild mandarin highlights influences of local chromatin state on gene expression. bioRxiv 2024.01.20.575729; doi:https://doi.org/10.1101/2024.01.20.575729
• Type: Other Journal Articles Status: Published Year Published: 2024 Citation: Campos Vieira, F., Drozd, C and M. Caroline Roper. 2024. New Microbial Tools to Combat HLB-associated citrus pathogens. Citrograph, Spring 2024.
• Type: Other Journal Articles Status: Published Year Published: 2024 Citation: Jewell, K., Yelshetty, N., Singh, A.S., and J. Kaplan "Preliminary results on the potential long-term cost-effectiveness of spraying insecticides for Asian citrus psyllids and rogueing huanglongbing-infected citrus trees in California," Research Note 2024-3, August 2024. https://www.csus.edu/faculty/k/kaplanj/researchnotes/rogue_spray_rn_v2.pdf.
• Type: Other Journal Articles Status: Published Year Published: 2024 Citation: Jewell, K., Yelshetty, N., Singh, A.S., and J. Kaplan"The Potential Long-term Cost-effectiveness of ACP Insecticide Spraying of HLB-Infected Citrus Trees in California: Preliminary Results," Research Note, 2024-2, May 2024. https://www.csus.edu/faculty/k/kaplanj/researchnotes/spraying_rn.pdf.
• Type: Other Journal Articles Status: Published Year Published: 2024 Citation: Jewell, K., Yelshetty, N., Singh, A.S., and J. Kaplan"The Potential Long-term Cost-effectiveness of Rogueing HLB-Infected Citrus Trees in California: Preliminary Results," Research Note 2024-1, February 2024. https://www.csus.edu/faculty/k/kaplanj/researchnotes/rogueing_rn.pdf.
• Type: Other Journal Articles Status: Published Year Published: 2023 Citation: Johnston, E., Kaplan, J., and A. Singh. Potential economic consequences from Huanglongbing (aka citrus greening disease) in California commercial citrus: results for Valencia orange production.  Research Note 2023-4, September 2023. https://www.csus.edu/faculty/k/kaplanj/researchnotes/2023-04-valencias_rn.pdf
• Type: Other Journal Articles Status: Published Year Published: 2024 Citation: Strauss, SL (2024) Covering the basics for summer cover crops Citrus Industry Tip of the Week for April 29, 2024.
• Type: Other Journal Articles Status: Published Year Published: 2023 Citation: Rolshausen P.E. 2023. Challenges and prospects for biopesticide discovery. Progressive Crop Consultant. September/October.

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Our target audiences are grower stakeholders, academic and industry scientists, undergraduate and graduate students and the general public. We have presented our work at national and international professional science meetings that are attended by scientists, citrus growers and agricultural industry representatives. These include participation in UCR Citrus Day, UF/IFAS SWFREC Extension Seminar Series,Polk County Master Gardeners, ASHS Annual Conference, Citrus Expo, Tampa, FL, UF/IFAS Extension In-Service-Training, Soil Science Society of America Annual Meeting, Annual Meeting Florida State Horticultural Society, Pomegranate and Citrus HLB Extension Meeting, University of California Davis, French National Research Institute for Agriculture, Food and Environment, Bordeaux France and UC ANR. Our Extension Specialists have disseminated information to target stakeholder audiences through our project website, presentations at stakeholder meetings and through semi-technical stakeholder publications that are listed in the Products (Publications) section of this report. In addition,our Economist has conducted grower surveys. We have also disseminated information about Huanglongbing (HLB), its implications to the citrus industry and specifics of this project through our project website, educational aids and curricula developed by the PDs and PIs of this project. Changes/Problems:The Southwest FL Valencia trial with different rootstocks has been terminated since the grower collaborator stopped all citrus production and the trees will not receive any management anymore. We started a replacement trial with newly planted Valencia trees on US-942 rootstock at our center in southwest Florida to integrate cover crops with compost and use of individual protective covers in summer 2023. What opportunities for training and professional development has the project provided?Training activities: The PDs and PIs of this project have trained members of the project in citrus propagation, microbial isolations from plants and soils, natural product isolations and microbial inhibition assays. We have also trained participants in the project on how to construct Illumina libraries and perform the subsequent bioinformatic analysis. Members of the project are also being trained on sequencing, single organism genome assembly and metagenome assembly, metagenome binning, gene prediction and annotation. The members are gaining skills in metagenome comparisons to identify organisms, genes and pathways which vary in composition between samples. The members are also training on amplicon metabarcoding library construction and bioinformatics analyses to support molecular ecology and contrasting of taxonomic distributions in samples with a focus on both archaea and bacteria using the 16S locus and fungi using the ITS locus. In addition, members of the team are being trained in the analysis of RNA-sequencing data, including quantification of gene expression, identification of differentially expressed genes between treatments and/or genotypes, and an introduction to unsupervised statistical learning methods to facilitate exploratory data analysis. Professional Development: Several Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society, Plant and Animal Genome Conference, the American Society of Horticultural Sciences, International Society for Citrus Huanglongbing and Phloem-colonizing bacteria, and the Florida State Horticultural Society. How have the results been disseminated to communities of interest?The Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society, the American Society of Horticultural Sciences, Plant and Animal Genome Conference, the American Society of Horticultural Sciences, International Society for Citrus Huanglongbing and Phloem-colonizing bacteria and the Florida State Horticultural Society. We have also presented at grower stakeholder meetings through virtual extension presentations and in-person trade show/educational events (UCR Citrus Day, FL Citrus Show, Citrus Expo, FL Citrus Institute and UC ANR among others). Extension specialists in Florida and California are communicating regularly with growers and grove managers regarding ongoing field trials through emails, phone calls, and in-person meetings. PD Roper has integrated lectures on HLB and citrus microbiome research in the graduate class Bacterial and Viral Disease of Plants (PLPA207) and the undergraduate course, Introductory Microbiology (BIOL/MCBL121). Co-PD Albrecht and Co-PD Strauss have presented seminars at various grower events that included information and research updates from the Florida field trials, and published in trade journal articles. An Open House event at the SW Florida REC was held where Co-PDs Albrecht, Kanissery, and Strauss presented their research projects to the public). Co-PDs Rolshausen and el-Kareamy have presented seminars at various grower events in California that included information and research updates from the California field trials and have published various extension articles.Co-PD Kaplan has presented research findings at seminars and extension meetings as well as published online research notes. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. A set of tolerant and susceptible rootstocks that were mock- and CLas-inoculated will be sampled for transcriptomic analysis and monitored for the progression of HLB symptoms. RNA-sequencing libraries have been produced and sequenced and will be analyzed. Metagenome analysis of citrus roots and leaves collected in FL from trees exhibiting a range of HLB symptoms will be completed.The metatranscriptomics portion of this work will be initiated on the same samples we used for the metagenomic analysis. This data will fold into the metagenomics data to construct a functional model of the citrus microbiome under HLB pressure. Microbial isolations from CA and FL will continue and isolates will be archived in the Citrus Isolate repository in the Roper lab. Microbial consortia inoculations will be conducted in conjunction with Fusarium, Phytophthora and CLas inoculations. Objective 2.Field trials in FL will continue as described above with biannual applications of soil amendments, weed management, biometric measurements, tree health- and root health assessments, leaf and soil nutrient analyses, etc. Pre-harvest fruit drop will be assessed as well as yield and fruit quality in the upcoming harvest season. We will establish the new young plantings in CA to assess the interactions of rootstocks and soil amendments (compost, humic acids). We will compare the performance of HLB-tolerant rootstocks from FL with CA standard rootstocks. The field trials will consist of biannual applications of soil amendments, weed management, biometric measurements, tree health- and root health assessments, leaf and soil nutrient analyses, etc. Microbiome-based data will be analyzed for the different rootstock and soil amendment combinations incorporated into our experimental design.Objective 3.The field trial in FL will continue as outlined in the proposal with additional cover crop plantings (seeding), weed management, biometric measurements and leaf, soil, and root sample collections for physiological, soil and microbiome analyses as described above. Pre-harvest fruit drop will be assessed and fruit yield and fruit quality will be determined in the upcoming harvest season. The field trial in CA will continue as outlined in the proposal with leaf, soil, and root sample collections for physiological and microbiome analyses as described above. Microbiome-based data will be analyzed for the different rootstock and soil amendment combinations incorporated into our experimental design. Objective 4. The economic team will continue to conduct outreach efforts in California and expand them to Florida to gain information from growers, advisors, and others involved in citrus production that we can use to consider more varied scenarios for consideration with our simulated economic experiments. We will also make further strides on preparing the epidemiological-economic simulation model for use once details on cultural practices are available.

Impacts
What was accomplished under these goals? Objective 1. We collected leaf and root samples from trees with low and high levels of HLB from groves under high disease pressure in FL. Microbiome-enriched samples were processed using two different methods to generate scaffold and shotgun libraries. These libraries have been sequenced and the subsequent data, following computational deconvolution, used for metagenome-assembled genome construction. Sequencing and data analysis are ongoing. We established a workflow for growing citrus rootstocks from seeds in the UCR BSL-3P containment facility and inoculating rootstocks with CLas infected psyllids. Ten rootstocks genotypes, including tolerant and susceptible types, have been mock- and CLas-inoculated and sampling and transcriptomic analysis has begun. Genotypes were selected to include rootstocks in field trials from Obj 2 and 3. We are also determining if root/rhizosphere microorganisms contribute to root collapse under HLB pressure. We have completed Koch's postulates with several HLB-associated Fusarium isolates we obtained from HLB-impacted citrus roots in Florida. We have determined that a subset of these are pathogenic on common citrus rootstocks. We have published whole genome sequences for 18 of these new Fusarium isolates. We have also initiated disease bioassays using two root-associated Phytophthora isolates and have expanded that work to new HLB-tolerant rootstocks. Our previous work indicated that these fungal/oomycete taxa increase in relative abundance in the roots of trees suffering from mid-late stages of HLB. These are known pathogens of citrus and our hypothesis is that their increase in relative abundance contributes to and exacerbates whole tree decline in trees with HLB. This objective is 50% completed. During this reporting period, we have published two technical journal articles for this objective. Objective 2. The Valencia/US-802 field trial in Southwest FL (trees planted in 2019) was continued in accordance with the experimental design outlined in the proposal. Trees were harvested in February 2022 and 2023 and fruit quality determined. Tree biometric measurements and tree health assessments were conducted and sample collections for nutrient, herbicide residue, and rhizosphere microbiome analysis continued. Organic amendment applications (compost and humic acids) were continued biannually and application of herbicide treatments were performed as outlined in the proposal. Weed biomass assessments were conducted as needed. Root health indicators (fibrous root respiration, root size/length distribution, root density) were measured seasonally. Leaf and soil nutrient analysis were conducted and stomatal conductance and soil moisture measurements have been performed regularly. Fruit drop due to hurricane Ian was assessed in 2022. We continue our annual sampling of leaves, roots, rhizosphere and bulk soil of these field trials to monitor changes in microbiome composition when using these amendments. We have processed, archived and extracted all DNAs from these samples. The Southwest FL Valencia field trial with different rootstocks was continued with biannual applications of compost, tree biometric measurements, tree health assessments, soil and leaf nutrient analyses, and fibrous root health (respiration, root length, root density) assessments as described under objective 2a. Trees were harvested in February 2022 and 2023 and fruit quality was determined. Minirhizotron images were captured seasonally for root growth analyses. Soil moisture monthly, flush ratings, stomatal conductance, weed biomass assessment were conducted regularly. Fruit drop due to hurricane Ian was assessed in 2022.We continue our annual sampling of leaves, roots, rhizosphere and bulk soil of these field trials to monitor changes in microbiome composition when using these amendments. We have processed, archived and extracted all DNAs from these samples for three sampling periods. We have initiated the analysis of the microbiome data.Florida: The Southwest FL Valencia trial with different rootstocks has been terminated since the grower collaborator stopped all citrus production and the trees will not receive any management anymore. We started a replacement trial with newly planted Valencia trees on US-942 rootstock at our center in southwest Florida to integrate cover crops with compost and use of individual protective covers in summer 2023. California: All six rootstocks that are being evaluated in CA field trials were planted starting in 2022. Tree biometric measurements and tree health assessments were conducted and sample collections for nutrient and microbiome analysis were initiated in 2022. Organic amendment applications (humic acid and mulch) were applied and application of herbicide treatments were performed as outlined in the proposal. We initiated our annual sampling of leaves, roots, rhizosphere and bulk soil of these field trials to monitor changes in microbiome composition when using these amendments. We have processed, archived and extracted all DNAs from these samples for one sampling periods. We have initiated the analysis of the microbiome data.This objective is 50% completed. There have been two journal articles and four extension articles published for this objective. Objective 3.Florida: We continued the field trial in central FL with Valencia scion on US-942 rootstock in accordance with the experimental design outlined in the proposal. Tree biometric measurements and tree health ratings were continued and soil and leaf nutrients and herbicide residues were determined regularly. Rhizosphere microbiome analysis continued. Root health assessments (fibrous root respiration, density, and root size/length distribution) were continued seasonally. New seeds for cover crops were sown seasonally. Cover crop and weed biomass assessments were conducted seasonally. Fruit were harvested in March 2022 and 2023 and fruit quality was analyzed. Fruit drop due to hurricane Ian was assessed in 2022.California:We established the field trial at the Lindcove Research and Extension Center with Tango on C35 rootstock according to the experimental design outlined in the proposal. Rhizosphere, root and leaf samples were collected from all treatments and stored at -80C prior to processing and analyses. Treatments include control, mulch, glyphosate and humic acid. We continue our annual sampling of leaves, roots, rhizosphere and bulk soil of these field trials to monitor changes in microbiome composition when using these amendments. We have processed, archived and extracted all DNAs from these samples for three sampling periods. We have initiated microbiome analysis for this sub-objective. This objective is 50% completed. There have been two journal articles and four extension articles published for this objective. Objective 4. The goal of this objective is to communicate our research to the stakeholder community, the greater academic community and the general public. During this report period, we participated in the Extension events listed in the Other Products (Activities and Events). Plans to conduct semi-structured interviews and develop a survey questionnaire to distribute to Florida and California growers, advisors, and others involved in citrus production to assess hurdles to adopting root/tree health cultural practices that may reduce the threat of HLB. We conducted outreach efforts in California with growers, PCAs, grower liaisons and California Department of Food and Agriculture staff. The information we gather is to be used to parameterize an epidemiological-economic model, which will be used to conduct simulated economic experiments. During this period we also made progress incorporating the functionality of practices that promote root/tree health through the microbiome into our simulation model. This objective is 50% completed. There have been five extension articles published for this objective.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Kurbessoian, T., Heimlich-Villalta, G., Ginnan, N., Vieira, F. C., Rolshausen, P. E., Roper, M. C., & Stajich, J. E. (2023). Genome Sequence and Assembly of 18 Fusarium Isolates from Florida Citrus under High Huanglongbing Disease Pressure and California Citrus under Low Huanglongbing Disease Pressure. Microbiology Resource Announcements. https://doi.org/10.1128/mra.00101-23
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Xi, M., Deyett, E., Stajich, J. E., El-Kereamy, A., Roper, M. C., & Rolshausen, P. E. (2023). Microbiome diversity, composition and assembly in a California citrus orchard. Frontiers in Microbiology, 14. https://doi.org/10.3389/fmicb.2023.1100590
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Castellano-Hinojosa A, Albrecht U, Strauss SL. (2023). Interactions between rootstocks and compost impact the active rhizosphere bacterial communities in citrus. Microbiome. doi:10.1186/s40168-023-01524-y
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Castellano-Hinojosa A, Kanissery R, Strauss SL (2023) Cover crops in citrus orchards impact soil nutrient cycling and the soil microbiome after three years but effects are site-specific. Biology and Fertility of Soils. Doi: 10.1007/s00374-023-01729-1
• Type: Other Status: Published Year Published: 2023 Citation: Semi-technical: Albrecht U, Pokhrel A, Strauss SL, Kanissery R (2023) Integrating use of organic soil amendments and weed management. Citrus Industry 104 (6): 15-17
• Type: Other Status: Published Year Published: 2023 Citation: Strauss SL, Dawson E, Karlsen-Ayala E (2023) Using cover crops to improve soil health. Citrus Industry 104 (6): 18-20
• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Flowers A, Kaplan J, and A Singh. Belief in Neighbor Behavior and Confidence in Scientific Information as Barriers to Cooperative Disease Control.
• Type: Other Status: Published Year Published: 2023 Citation: Johnston E, Kaplan J, and A Singh (2023) "Potential economic consequences from Huanglongbing (aka citrus greening disease) in California commercial citrus: Results for Valencia orange production", Research Note 2023-4, September 2023.
• Type: Other Status: Published Year Published: 2023 Citation: Johnston E, Kaplan J, and A Singh (2023) "Potential economic consequences from Huanglongbing (aka citrus greening disease) in California commercial citrus: Results for Navel orange production", Research Note 2023-3, August 2023.
• Type: Other Status: Published Year Published: 2023 Citation: Johnston E, Kaplan J, and A Singh (2023) "Potential economic consequences from Huanglongbing (aka citrus greening disease) in California commercial citrus: Results for tangerine & mandarin production",Research Note 2023-2, August 2023.
• Type: Other Status: Published Year Published: 2023 Citation: Strauss SL (2023) Preparing for summer cover crops. Citrus Industry Tip of the Week for May 2, 2023
• Type: Other Status: Published Year Published: 2023 Citation: Albrecht U, Pokhrel A, R, Strauss S, Kanissery (2023). Integrating use of organic soil amendments and weed management. Citrus Industry 104(6): 15-17
• Type: Other Status: Published Year Published: 2023 Citation: Johnston E, Kaplan J, and A Singh (2023)"Potential economic consequences from Huanglongbing (aka citrus greening disease) in California commercial citrus: Results for lemon production", Research Note 2023-1, July 2023
• Type: Other Status: Published Year Published: 2023 Citation: Rolshausen, P. (2023) Challenges and Prospects for Biopesticide Discovery. Progressive Crop Consultant

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Our target audiences are grower stakeholders, academic and industry scientists, undergraduate and graduate students and the general public. To reach these audiences, we have presented our work at national and international professional science meetings that are attended by scientists, citrus growers and agricultural industry representatives. We have input the specific locations and titles of these meetings in the Other Products (Activities and Events) section in the report. Our Extension Specialists have disseminated information to target stakeholder audiences through our project website, presentations at stakeholder meetings and through semi-technical stakeholder publications that are listed in the Other Products (Events and Publications) section of this report. In addition,our Economist has conducted grower surveys. We have also disseminated information about Huanglongbing (HLB), its implications to the citrus industry and specifics of this project through the Citrus Clonal Protection Program (CCPP) budwood distribution system (over 6,000 users), our project website, educational aids and curricula developed by the PDs and PIs of this project. Our public in-person outreach has increased this year as COVID-19 restrictions regarding in-person events and travel have eased. Changes/Problems:The economic outreach component of this project continued to face challenges reaching growers, advisors and others in the citrus industry through in-person meetings due to the COVID-19 pandemic. The economic team will nonetheless continue their outreach efforts with those within the California and Florida citrus industries. What opportunities for training and professional development has the project provided?Training activities: The PDs and PIs of this project have trained members of the project in citrus propagation, microbial isolations from plants and soils, natural product isolations and microbial inhibition assays. We have also trained participants in the project on how to construct Illumina libraries and perform the subsequent bioinformatic analysis. Members of the project are also being trained on sequencing, single organism genome assembly and metagenome assembly, metagenome binning, gene prediction and annotation. The members are gaining skills in metagenome comparisons to identify organisms, genes and pathways which vary in composition between samples. The members are also training on amplicon metabarcoding library construction and bioinformatics analyses to support molecular ecology and contrasting of taxonomic distributions in samples with a focus on both archaea and bacteria using the 16S locus and fungi using the ITS locus. In addition, members of the team are being trained in the analysis of RNA-sequencing data, including quantification of gene expression, identification of differentially expressed genes between treatments and/or genotypes, and an introduction to unsupervised statistical learning methods to facilitate exploratory data analysis. Professional Development: Several Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society, the American Society of Horticultural Sciences and the Florida State Horticultural Society (listed in Other Products). How have the results been disseminated to communities of interest?The Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society, the American Society of Horticultural Sciences, the Soil Science Society of America, and the Florida State Horticultural Society. We have also presented at grower stakeholder meetings through virtual extension presentations and in-person trade show/educational events (UCR Citrus Day, FL Citrus Show, Citrus Expo, FL Citrus Institute). Extension specialists in Florida and California are communicating regularly with growers and grove managers regarding ongoing field trials through emails, phone calls, and in-person meetings. PD Roper has integrated lectures on HLB and citrus microbiome research in the graduate class Bacterial and Viral Disease of Plants (PLPA207) and the undergraduate course, Introductory Microbiology (BIOL/MCBL121). Co-PD Albrecht and Co-PD Strauss have presented seminars at various grower events that included information and research updates from the Florida field trials, and published in trade journal articles. An Open House event at the SW Florida REC was held where Co-PDs Albrecht, Kanissery, and Strauss presented their research projects to the public). Co-PDs Rolshausen and el-Kareamy have presented seminars at various grower events in California that included information and research updates from the California field trials and have published various extension articles. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: A set of tolerant and susceptible rootstocks that were mock- and CLas-inoculated will be sampled for transcriptomic analysis and monitored for the progression of HLB symptoms. RNA-sequencing libraries will be produced and sequenced. Metagenome analysis of citrus roots and leaves collected in FL from trees exhibiting a range of HLB symptoms will be completed. The metatranscriptomics portion of this work will be initiated on the same samples we used for the metagenomic analysis. This data will fold into the metagenomics data to construct a functional model of the citrus microbiome under HLB pressure. Microbial isolations from CA and FL will continue and isolates will be archived in the Citrus Isolate repository in the Roper lab. Microbial consortia inoculations will conducted in conjunction with CLas inoculations. Objective 2. Field trials in FL will continue as described above with biannual applications of soil amendments, weed management, biometric measurements, tree health- and root health assessments, leaf and soil nutrient analyses, etc. Pre-harvest fruit drop will be assessed as well as yield and fruit quality in the upcoming harvest season. We will establish the new young plantings in CA to assess the interactions of rootstocks and soil amendments (compost, humic acids). We will compare the performance of HLB-tolerant rootstocks from FL with CA standard rootstocks. The field trials will consist of biannual applications of soil amendments, weed management, biometric measurements, tree health- and root health assessments, leaf and soil nutrient analyses, etc. Objective 3. The field trial in FL will continue as outlined in the proposal with additional cover crop plantings (seeding), weed management, biometric measurements and leaf, soil, and root sample collections for physiological, soil and microbiome analyses as described above. Pre-harvest fruit drop will be assessed and fruit yield and fruit quality will be determined in the upcoming harvest season. The field trial in CA will continue as outlined in the proposal with leaf, soil, and root sample collections for physiological and microbiome analyses as described above. Objective 4. The economic team will continue to conduct outreach efforts in California and expand them to Florida to gain information from growers, advisors, and others involved in citrus production that we can use to consider more varied scenarios for consideration with our simulated economic experiments. We will also make further strides on preparing the epidemiological-economic simulation model for use once details on cultural practices are available.

Impacts
What was accomplished under these goals? Objective 1. A systems-level approach to unraveling the HLB disease complex and its link to root decline. Obj. 1a. We have collected leaf and root samples from trees with low and high levels of HLB from groves under high disease pressure in FL. We developed to enrich these samples for microbiome content to preserve microbial diversity. Microbiome-enriched samples were split and processed using two different methods to generate scaffold and shotgun libraries. These libraries have been sequenced and the subsequent data used for metagenome-assembled genome construction. Within these data are reconstructed genomes belonging to CLas, known plant-associated bacteria and some that have yet to be cultured or characterized. We have established a workflow for growing citrus rootstocks from seeds in the UCR BSL-3P containment facility and inoculating rootstocks with CLas infected psyllids. Ten rootstocks genotypes, including tolerant and susceptible types, have been mock- and CLas-inoculated and sampling for transcriptomic analysis will begin in January. Genotypes were selected to include rootstocks in field trials from Obj 2 and 3.Objective 1b. The purpose of this sub-objective is to determine if root/rhizosphere microorganisms contribute to root collapse under HLB pressure.Using the large citrus microbial repository in the Roper laboratory, we have initiated disease bioassays with three Fusarium isolates we obtained from HLB-impacted citrus roots in Florida. We have also initiated disease bioassays using two root-associated Phytophthora isolates. Our previous work indicated that these fungal/oomycete taxa increase in relative abundance in the roots of trees suffering from mid-late stages of HLB. Using the large citrus microbial repository established in the Roper laboratory, we have begun disease bioassays with three Fusarium isolates we obtained from HLB-impacted citrus roots in Florida as well as two root-associated Phytophthora isolates. Preliminary data from stem inoculation assays indicate that all aforementioned isolates were able to cause significant lesions on S-1 citron, Swingle citrumelo, and Carrizo citron seedlings in comparison to controls. We have obtained whole genome sequences for all Fusarium isolates we have obtained from citrus in CA and FL. We have developed a medium throughput pipeline for screening compounds for prophylactic and curative effects in citrus plants in the UC Riverside BSL3 facility. We are now propagating citrus plants under BSL3 containment that are infected with Candidatus Liberibacter asiaticus (CLas). We have also established an Asian Citrus Psyllid colony under BSL3 high containment at UC Riverside that we are using as an additional method to inoculate citrus plants with CLas. Infected citrus plants will serve as a source of CLas for inoculation of tolerant and susceptible citrus rootstocks and subsequent gene expression analysis. This objective is 30% completed. We have published two technical journal articles and one that is under review for this objective. We have published one extension article for this objective. Objective 2. Integrating a formula of successful grove practices associated with root health in newly planted HLB-impacted groves. Objective 2a. Florida:The Valencia/US-802 field trial in Southwest FL (trees planted in 2019) was continued in accordance with the experimental design outlined in the proposal. Trees were harvested in February 2022 and fruit quality determined. Tree biometric measurements and tree health assessments were conducted and sample collections for nutrient, herbicide residue, and rhizosphere microbiome analysis continued. Organic amendment applications (compost and humic acids) were continued biannually and application of herbicide treatments were performed as outlined in the proposal. Weed biomass assessments were conducted as needed. Root health indicators were measured seasonally. Leaf and soil nutrient analysis were conducted and stomatal conductance and soil moisture measurements have been performed regularly. Fruit drop due to hurricane Ian was assessed. We continue our annual sampling of leaves, roots, rhizosphere and bulk soil of these field trials to monitor changes in microbiome composition when using these amendments. We have processed, archived and extracted all DNAs from these samples.Objective 2b. Florida: The Southwest FL Valencia field trial with different rootstocks was continued with biannual applications of compost, tree biometric measurements, tree health assessments, soil and leaf nutrient analyses, and fibrous root health (respiration, root length, root density) assessments as described under objective 2a. Trees were harvested in February 2022 and fruit quality was determined. Minirhizotron images were captured seasonally for root growth analyses. Soil moisture monthly, flush ratings, stomatal conductance , weed biomass assessment were conducted regularly. Fruit drop due to hurricane Ian was assessed. We continue our annual sampling of leaves, roots, rhizosphere and bulk soil of these field trials to monitor changes in microbiome composition when using these amendments. We have processed, archived and extracted all DNAs from these samples.Objective 2c.California: We are propagating all six rootstocks that will be evaluated in CA field trials starting in 2022.This objective is 30% completed. There have been five extension articles published for this objective. Objective 3.Integrating a formula of successful grove practices associated with tree health in existing HLB-impacted groves: A focus on grove rehabilitation.Objective 3a. We continued the field trial in central FL with Valencia scion on US-942 rootstock in accordance with the experimental design outlined in the proposal. Tree biometric measurements and tree health ratings were continued and soil and leaf nutrients and herbicide residues were determined regularly. Rhizosphere microbiome analysis continued. Root health assessments (fibrous root respiration, density, and root size/length distribution) were continued seasonally. New seeds for cover crops were sown seasonally. Cover crop and weed biomass assessments were conducted seasonally. Fruit were harvested in March 2022 and fruit quality was analyzed. Fruit drop due to hurricane Ian was assessed.Objective 3b.We established the field trial at the Lindcove Research and Extension Center in California with Tango on C35 rootstock according to the experimental design outlined in the proposal. Rhizosphere, root and leaf samples were collected from all treatments and stored at -80C prior to processing and analyses. Treatments include control, mulch, glyphosate and humic acid.We continue our annual sampling of leaves, roots, rhizosphere and bulk soil of these field trials to monitor changes in microbiome composition when using these amendments. We have processed, archived and extracted all DNAs from these samples. This objective is 30% completed. There have been five extension articles published for this objective. Objective 4. Integration of Research and Extension. During this report period, we have accomplished this by participating in the Extension events listed in the Other Products (Activities and Events). Plans to conduct semi-structured interviews and develop a survey questionnaire to distribute to Florida and California growers, advisors, and others involved in citrus production to assess hurdles to adopting root/tree health cultural practices that may reduce the threat of HLB are back underway in California withgrowers, PCAs, grower liaisons and California Department of Food and Agriculture staff. The information we gather is to be used to parameterize an epidemiological-economic model, which will be used to conduct simulated economic experiments. During this period we also made progress incorporating the functionality of practices that promote root/tree health through the microbiome into our simulation model. This objective is 30% completed.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Ginnan, N. A., De Anda, N. I., Campos Freitas Vieira, F., Rolshausen, P. E., & Roper, M. C. (2022). Microbial Turnover and Dispersal Events Occur in Synchrony with Plant Phenology in the Perennial Evergreen Tree Crop Citrus sinensis. MBio, 13(3). https://doi.org/10.1128/mbio.00343-22
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Xi M, Deyett E, Ginnan NA, Ashworth V, Dang T, Bodaghi S, Vidalakis G, Roper MC, Glassman S, Rolshausen P. 2022. Geographic location, management strategy and Huanglongbing disease affect arbuscular mycorrhizal fungal communities across US citrus orchards. Phytobiomes Journal, 6 (4). https://doi.org/10.1094/PBIOMES-03-22-0014-R
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Castellano-Hinojosa A, Albrecht U, Strauss S. (2022). Interactions between rootstocks and compost impact the active rhizosphere bacterial communities in citrus. Microbiome. Under review.
• Type: Other Status: Published Year Published: 2022 Citation: Albrecht U, Pugina G, Castellano-Hinojosa A, Strauss S. (2022). Does the use of compost improve young tree growth? Citrus Industry 103 (5):10-13.
• Type: Other Status: Published Year Published: 2022 Citation: Strauss S, Castellano-Hinojosa A, Kadyampakeni D, Kanissery R, Wade T. (2022) Experimenting with cover crops: results from three years of trials. Citrus Industry 103 (5): 18-21
• Type: Other Status: Published Year Published: 2022 Citation: Kanissery R (2022) Managing weeds in row middles. Citrus Industry Tip of the Week for April 19, 2022
• Type: Other Status: Published Year Published: 2022 Citation: Strauss S (2022) Getting read for cover crops. Citrus Industry Tip of the Week for May 3, 2022
• Type: Other Status: Published Year Published: 2022 Citation: Kanissery R and Bashyal M (2023) Effects of composting and organic amendment on citrus weed control. Citrus Industry Magazine, Feb 2023 (Under review).
• Type: Other Status: Published Year Published: 2022 Citation: P.E. Rolshausen. (2022). Mycorrhizae: An Underground Support Network for Trees. Topics in Subtropics. Volume 22, Fall Issue.

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Our target audiences are grower stakeholders, academic and industry scientists, undergraduate and graduate students and the general public. To reach these audiences, we have presented our work at national and international professional science meetings that are attended by scientists, citrus growers and agricultural industry representatives. We have input the specific locations and titles of these meetings in the Other Products (Activities and Events) section in the report. Our Extension Specialists have disseminated information to target stakeholder audiences through our project website, presentations at stakeholder meetings and through semi-technical stakeholder publications that are listed in the Other Products (Events and Publications) section of this report. In addition, our Economist has conducted grower surveys. We have also disseminated information about Huanglongbing (HLB), its implications to the citrus industry and specifics of this project through the Citrus Clonal Protection Program (CCPP) budwood distribution system (over 6,000 users), our project website, educational aids and curricula developed by the PDs and PIs of this project. Our public in-person outreach has been hampered this year due to COVID-19 restrictions regarding in-person events. Changes/Problems:The laboratories of the PDs and PIs were closed for a period of time due to COVID-19 mandates from our institutions and research ceased for a period of time. We were then placed in a restricted research ramp up phase that included limits on the number of personnel in the lab at a given time. Thus, research was slower than originally planned. We are now able to work in labs with safety precautions, such as masking and frequent hand washing, but without social distancing requirements in place. The economic outreach component of this project originally relied on in-person meetings to gather data for the economic analysis and subsequent outreach and extension efforts. As an alternative strategy, the team met with growers, advisors, and researchers from Florida and California over ZOOM. With this information and COVID-19 travel restrictions easing, the team has begun to design in-person surveys of growers, advisors, and others involved in citrus production and will begin to solicit responses at public meetings in Florida and California in the coming year. What opportunities for training and professional development has the project provided?The PDs and PIs of this project have trained members of the project in citrus propagation, microbial isolations from plants and soils, natural product isolations and microbial inhibition assays. We have also trained participants in the project on how to construct Illumina libraries and perform the subsequent bioinformatic analysis. Members of the project are also being trained on sequencing, single organism genome assembly and metagenome assembly, metagenome binning, gene prediction and annotation. The members are gaining skills in metagenome comparisons to identify organisms, genes and pathways which vary in composition between samples. The members are also training on amplicon metabarcoding library construction and bioinformatics analyses to support molecular ecology and contrasting of taxonomic distributions in samples with a focus on both archaea and bacteria using the 16S locus and fungi using the ITS locus. In addition, members of the team are being trained in the analysis of RNA-sequencing data, including quantification of gene expression, identification of differentially expressed genes between treatments and/or genotypes, and an introduction to unsupervised statistical learning methods to facilitate exploratory data analysis. Several Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society, the American Society of Horticultural Sciences and the Florida State Horticultural Society (listed in Other Products). How have the results been disseminated to communities of interest?The Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society Plant Health online, the American Society of Horticultural Sciences and the Florida State Horticultural Society. We have also presented at grower stakeholder meetings through virtual extension presentations and in-person trade show/educational events (FL Citrus Show, Citrus Expo). Extension specialists in Florida are communicating regularly with growers and grove managers regarding ongoing field trials through emails, phone calls, and in-person meetings. PI Roper has integrated lectures on HLB and citrus microbiome research in the graduate class Bacterial and Viral Disease of Plants (PLPA207) and the undergraduate course, Introductory Microbiology (BIOL/MCBL121). Co-PD Albrecht has presented guest lectures in the undergraduate course Citrus Culture and Production (FRC 3212) and graduate courses Advanced Citriculture (HOS 6545) and Citrus Pathology (PLP 5115C). Due to COVID-19 restrictions that started during this reporting period, much of our interactions with the communities of interest have been virtual. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: A set of tolerant and susceptible rootstock seedlings will be introduced to the high containment BSL3 plant facility at UC Riverside. These seedlings will be mock- and CLas-inoculated at 9 months of age and monitored for the progression of HLB symptoms. Metagenome analysis of citrus roots and leaves collected in FL from trees exhibiting a range of HLB symptoms will be completed. The metatranscriptomics portion of this work will be initiated on the same samples we used for the metagenomic analysis. This data will fold into the metagenomics data to construct a functional model of the citrus microbiome under HLB pressure. Microbial isolations from CA and FL will continue and isolates will be archived in the Citrus Isolate repository in the Roper lab. Objective 2. Field trials in FL will continue as outlined in the proposal with biannual applications of soil amendments, weed management, biometric measurements, and leaf, soil, and root sample collections for physiological and microbiome analyses. Cover crops will be established in trial 2. Yield and fruit quality will be assessed as trees become productive. Objective 3. The field trial in FL will continue as outlined in the proposal with additional cover crop plantings (seeding), weed management, biometric measurements and leaf, soil, and root sample collections for physiological and microbiome analyses. Yield and fruit quality will also be assessed. Objective 4. The economic team will travel to Florida and throughout California to survey growers, advisors, and others involved in citrus production. With the responses to the surveys, hurdles to adoption of tree/root health strategies will be assessed as well as the role risk perception may play in those decisions.

Impacts
What was accomplished under these goals? Objective 1. A systems-level approach to unraveling the HLB disease complex and its link to root decline. Hypothesis: Disrupted root microbiomes influence the rate of HLB development and consequently hasten tree decline. Obj. 1a. The purpose of this sub-objective is to determine the functional landscape of the citrus root and rhizosphere microbiomes under different levels of HLB severity. We have collected leaf and root samples from trees with low and high levels of HLB from groves under high disease pressure in FL. We have extracted the DNA and built the metagenome libraries and these have been sent for sequencing. Once we receive the sequences back we will begin the analyses. We have also sequenced the genome of the citrus host, Citrus reticulata (Fairchild mandarin) that will fold into the transcriptomics portion of this objective that will aid in understanding how different citrus genotypes (HLB susceptible and HLB tolerant) respond to andidatus Liberibacter asiaticus (CLas) infection. Objective 1b. The purpose of this sub-objective is to determine if root/rhizosphere microorganisms contribute to root collapse under HLB pressure. Using the large citrus microbial repository established in the Roper laboratory, we have initiated disease bioassays with three Fusarium isolates we obtained from HLB-impacted citrus roots in Florida. We have also initiated disease bioassays using two root-associated Phytophthora isolates. Our previous work indicated that these fungal/oomycete taxa increase in relative abundance in the roots of trees suffering from mid-late stages of HLB. These are known pathogens of citrus and our hypothesis is that their increase in relative abundance contributes to and exacerbates whole tree decline in trees with HLB. We have, thus far, set up growth chamber and greenhouse experiments using Citron D1 grown from seed. We have inoculated these with individual isolates of the three Fusarium and two Phytophthora strains we are testing. We are now preparing to inoculate the common rootstocks, Carrizo and Swingle. We have developed a medium throughput pipeline for screening compounds for prophylactic and curative effects in citrus plants in the UC Riverside BSL3 facility. We are now propagating citrus plants under BSL3 containment that are infected with CLas. We have also established a Asian Citrus Psyllid colony under BSL3 high containment at UC Riverside that we will use as an additional method to inoculate citrus plants with CLas. Infected citrus plants will serve as a source of CLas for inoculation of tolerant and susceptible citrus rootstocks and subsequent gene expression analysis. Objective 1 is 15% completed. There has been one microbiome review article and one extension article published for this objective. Objective 2. Integrating a formula of successful grove practices associated with root health in newly planted HLB-impacted groves: A focus on replanted groves. Hypothesis: Promoting root health by using soil amendments will support robust establishment of trees in young grove replants in HLB-impacted areas (FL) and areas where HLB is an imminent threat (CA). Objective 2a. The purpose of this sub-objective is to integrate of organic soil amendments with weed management in young replanted FL grove trials and assess the impact on root health and root/rhizosphere microbiome health. Florida: We have established a Valencia/US-802 field trial in Southwest FL (trees planted in 2019) was established in accordance with the experimental design outlined in the proposal. Field plots were marked and trees were randomly selected in each plot for biometric measurements and sampling. Baseline measurements (tree biometrics) and baseline sample collections (soil, leaves, roots) for nutrient, herbicide, and rhizosphere microbiome analysis were conducted. The first round of organic amendments (compost and humic acids) was applied. Objective 2b. The purpose of this sib-objective is to integrate compost, cover crops, and HLB-resistant rootstocks in young replanted FL grove trials and assess the impact on root health and root/rhizosphere microbiome health. Florida: The Southwest FL Valencia field trial with different rootstocks was continued with biannual applications of compost, tree biometric measurements, soil and leaf nutrient analyses, and root health assessments. Minirhizotrons were placed for root growth analyses. Objective 2c. The purpose of this sub-objective is to integrate organic soil amendments in young replanted CA grove trials and assess the impact on root health and root/rhizosphere microbiome health. California: We are propagating all six rootstocks that will be evaluated in CA field trials starting in 2022. Objective 2 is 15% completed. There have been two extension articles published for this objective. Objective 3.Integrating a formula of successful grove practices associated with tree health in existing HLB-impacted groves: A focus on grove rehabilitation. Hypothesis: Integration of cultural practices that promote root health will aid in rehabilitation of HLB-affected groves and increase productivity of groves. Objective 3a. The purpose of this sub-objective is to integrate of cover crops in existing groves of older trees (6-10 years old) in FL. We established the field trial in central FL with Valencia scion on US-942 rootstock was established in accordance with the experimental design outlined in the proposal. Field plots were marked and trees were randomly selected in each plot for biometric measurements and sampling. Baseline measurements (tree biometrics) and baseline sample collections (soil, leaves, roots) for nutrient, herbicide, and rhizosphere microbiome analysis were conducted. Seeds for cover crops were sown and cover crops are growing well. Objective 3b. The purpose of this sub-objective is to integrate soil amendments in existing groves of older trees in CA. We established the field trial at the Lindcove Research and Extension Center in California with Tango on C35 rootstock according to the experimental design outlined in the proposal. Rhizosphere, root and leaf samples were collected from all treatments and stored at -80C prior to processing and analyses. Treatments include control, mulch, glyphosate and humic acid. Objective 3 is 15% completed. There have been two extension articles published for this objective. Objective 4. Integration of Research and Extension. The goal of this objective is to communicate our research to the stakeholder community, the greater academic community and the general public. During this report period, we have accomplished this by participating in the Extension events listed in the Other Products (Activities and Events). In addition, we included HLB brochures in 1,404 budwood shipments to 1,050 Citrus Clonal Protection Program Budwood users. Plans to conduct semi-structured interviews and develop a survey questionnaire to distribute to Florida and California growers, advisors, and others involved in citrus production to assess hurdles to adopting root/tree health cultural practices that may reduce the threat of HLB were altered to comply with COVID-19 restrictions. Our original plan to conduct in-person semi-structured interviews and clicker surveys at various citrus events were changed to include interviews conducted on ZOOM and a delay in survey growers, advisors, and others until now. A survey instrument has been developed and responses will be solicited at in-person events in Florida and California in the coming year.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhang, Y.,Trivedi, P., Xu, J., Roper, M.C. and Nian Wang, 2021. The Citrus Microbiome: From Structure and Function to Microbiome Engineering and Beyond, Phytobiomes Journal, https://doi.org/10.1094/PBIOMES-11-20-0084-RVW
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Rolshausen P.E. 2021. Healthy Roots, Healthy Trees. Topics in Subtropics 19 (Winter issue Issue):8-9.
• Type: Other Status: Published Year Published: 2021 Citation: Tip of the Week in Citrus Industry Magazine on planting cover crops : What to consider before planting cover crops published May 4, 2021
• Type: Websites Status: Published Year Published: 2021 Citation: PD Rolshausen is maintaining and updating our project website on a regular basis, https://ucanr.edu/sites/Citrus@UCR/.