Progress 09/15/23 to 09/14/24
Outputs
Target Audience:Our target audience are the commercial growers (organic and conventional), buyers, distributors, seed companies, grower and marketing organizations and those scientists and extension specialists and agents and others involved in supporting their commercial sweet basil growers. Our consortium includes producers and processors of sweet basil from field and indoor production (e.g., greenhouses and vertical farming), organic and conventional growers, seed companies, scientists and extension researchers that provide technical assistance to their commercial growers and industry. Our stakeholders are our target audience and as such are a focus of our effort during the duration of the project. They have been directly involved in the selection and commercialization of improved sweet basil materials, and will be in testing and trialing our and other available lines by other groups (e.g. the seed industry), and participate in our winter and summer annual meetings and in field trials during which they provide feedback to us which we then use to guide our work. Changes/Problems:The genome sequencing effort was delayed and took longer time to organize. With a clear definition of race structure of P. belbahrii, we will be able to nowinvestigate genetic diversity of this pathogen using comparative genomics in this upcoming year. What opportunities for training and professional development has the project provided?In this second year of this project has provided mentoring, training and professional development for six graduate students, 28 undergraduate students and two post-doctoral research associates. Specialized workshops for these students and others on genetics and plant breeding, new varietal development, including experiential training on cross pollination, plant grow-out, experimental design in greenhouse and field, seed harvesting, seed cleaning, seed storage and then advancing generations were provided in this second year. Additionally, the students and post-docs received training in the chemical characterization of aroma volatiles using GC/MS and in laboratory practices, and disease scouting, identification and control options. Student were also trained in plant pathology, culturing fungal and bacterial colonies. Importantly, many of these students also received hands-on training in DNA extraction, DNA analysis, tissue culture, gene editing and in genomic annotation. Many of the students also were mentored and participated in the preparation of posters and oral presentations at grower/industry and scientific conferences. Unique to our research project, many graduate and undergraduate students were also mentored and trained in science communication, using videos to create science-in-action stories to share with growers, the industry and the public. Many students participated in the making or showing and release of the science documentary, called Fields of Devotion that highlights the story and connection between Rutgers scientists, the agricultural community needs and student training in our development of creating downy mildew resistant sweet basils. Students were also mentored in ways to both listen to growers and the industry and then also in ways to effectively communicate scientific information and recommendations to commercial growers and the public. How have the results been disseminated to communities of interest?The results generated from our current project builds upon our prior USDA funded project as well, therefore enabling us to move ahead with initial dissemination strategies. Information was disseminated using traditional approaches by participating and presenting the research in extension, grower and trade show meetings, field trips, field tours, greenhouse tours and through state winter educational meetings and a remotely held zoom conference. Additionally, results were shared by presenting part of the ongoing research at national and international scientific conferences. A new website (usbasilconsortium.rutgers.edu) was designed and operational to serve multiple functions. It will mirror the approach taken in this grant to promote research, extension and outreach and will be separated into sections for each. The Research pillar of the website will be a place to post about new publications and report new information. It will also serve as a repository for genomic resources. The V0 'RUSB22' sweet basil genome has been available for a year to the scientific community and has been downloaded over 25 times. The Extension pillar will be a central location to find essential news and information for commercial and home growers of basil. It will link to the Basil Ag Pest Monitor website for reporting occurrences of basil downy mildew as well as extension bulletins and fact sheets from Rutgers Pest Advisory and Cornell Vegetable Center (See: https://www.vegetables.cornell.edu/pestmanagement/disease-factsheets/basil-downy-mildew/; and we continued the Rutgers basil social media presence via the instragram, @rutgersbasil which has over 2500 followers). We have also made social media accounts on Facebook, Instagram and Twitter for our US Basil Consortium that we are calling the Basil Advanced Studies and Innovation Laboratory (B.A.S.I.L.). We also contribute to the Rutgers Plant Pest and Advisory Board and HortiDaily with news bulletins and facts sheets. Co-PI Andy Wyenandt was interviewed and quoted in the Washington Post as well. The Outreach pillar will be geared towards communicating scientific results to a broader audience and making the science more accessible. Currently 'Our Favorite Uses of Basil' is a new series we are testing geared towards showcasing our favorite uses of basil. We also have written pages about historical and cultural information about basil. Another website, was built and focused on promoting and dissemination information about 'Fields of Devotion' that highlights our basil research process and accomplishments through https://fieldsofdevotion.rutgers.edu/. The full length documentary is being used in colleges and high school biology, chemistry and plant science classes. Fields of Devotion, an award winning, science-in-action short documentary film featuring basil plant breeding research has been released to the public and viewed in person and online at science film festivals, at agricultural meetings, local gatherings, and in college classrooms. In-person events provided audience members the opportunity to hear directly from scientists and filmmakers during the question-and-answer sessions that followed the film screenings. In August 2023, Fields of Devotion reached wider audiences through New Jersey PBS (August 19, 2023) was and release through a streaming service. The film was featured on the NJPBS website with the trailer available to all viewers (https://www.thirteen.org/programs/nj-pbs-specials/fields-of-devotion-trailer-pqyfzh/ ). Fields of Devotion was released on Kanopy streaming services which allows public access via university and public libraries giving the film the potential for national (and possibly international) reach. A website is kept up-to-date to share current information about the film and future screenings are available (https://fieldsofdevotion.rutgers.edu/). What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Investigating the genetics of the emergent races of P. belbahrii through genome sequencing, RNASeq analyses and a disease differential panel. In parallel, develop new BDM resistant lines to combat these races which have overcome previous resistant varieties. P. belbahrii Genome The isolates that are to be sequences have been selected and preparations have begun to collect enough tissue for successful extractions. Our team will extract high molecular weight DNA from the selected isolates and sequence their whole genomes in next reporting period. Subsequent downstream analysis will be done such as identifying repeat regions and providing gene annotation. RNAseq & Disease Differential Panel Disease differential panels will continue to be grown in various field sites in order to monitor the status and condition of the pathogen race in those areas. The RNAseq study is set to commence at the beginning of the third year of this grant. Sweet basil cultivars and project design is complete. New Resistant Lines Further advanced breeding lines with new sources of BDM resistance will continue development and a new series of DMR basils will be patented this coming grant period. Objective 2: Develop the knowledge and resources to launch a new BLS resistance breeding program for basil and release BLS resistant sweet basil cultivars. P. cichorii Collections Isolates from both New Jersey and Florida have been collected over the last year with a 10+ isolate library constructed at the University of Florida. This adds to our current collection and brings the total isolate count near 20. Isolates will continue to be collected from affected stakeholders and screened for virulence on multiple species of Ocimum. A differential panel comprised of individuals displaying resistance to complete susceptibility is currently being developed to test for differential virulence between isolates. If this panel is a success all isolates that are in frozen culture will be screened against it in the coming grant period. BLS Resistance Screening The final screens of our germplasm collection will be completed in the coming year. Confirmatory replication screens on all putatively resistant individuals will be completed and the results of the entire germplasm screen will be published. Newly collected and developed material will continue to be screened for BLS resistance as the program continues. Screening Mutant Pseudomonas Library & Investigating Type III Secretion System: This work is planned for the last year of the grant period. Potential RNASeq Analysis of BLS Infected and Healthy Sweet Basil: This work is planned for the last year of the grant period. Objective 3: Identify relevant disease and horticultural-related genes in the basil genome via a genome wide association study. The forthcoming year will see the start of the complete GWAS analysis. SNPs have already been identified for use within the pipeline and relevant methodologies and analyses are being identified. Phenotypic data will be cleaned and converted to BLUPs where necessary for analysis. These results will begin to be written up with the aim for publication in the following year. Objective 4: Explore homologous resistance genes and validate putative resistance genes found via de novo annotation using CRISPR/Cas9 knockdowns and de novo annotation of the 'SB22' genome to spur quicker development of new elite basil cultivars. Production of ObOGO- and ObNPR3-edited basil plants: We will continue to transform basil tissues to produce more transgenic plants expressing our Ob2OGO and ObNPR3 CRISPR-gene editing cassettes. We will characterize the integration of the transgenes and the creation of mutations in these host genes. After the confirmation of the mutants, we will test their resistance to P. belbahrii infection and conduct the essential il profiling of these plants. Production of ObIAN9-, ObJAZ-, and ObZAT18-edited basil plants for BLS resistance: After the construction of the CRISPR-editing vectors targeting these host genes, they will be used to transform SB22 and Obsession embryogenic calli and produce gene-edited basil plants. Subsequently, the mutant plants will be tested for P. belbahrii resistance and profiled for the essential oils. Study of P. cichorii virulence: We will search in silico for the DNA sequences encoding the components of the Type Three Secretion System (TTSS) in P. cichorii Boca, such as HopA1, and srfA, srfB, srfC and srfD of the Scrf gene cluster. We will perform CRISPR-gene editing of these virulence factors in P. cichorii Boca in order to determine the contribution of this pathogenic system in the bacterial infection and interaction with basil host plants. Genome Annotation: As mentioned the genome annotate of the SB22 draft genome has been complete and will be published this coming grant period. However as more information if identified via other aspects of this grant (ie MTAs, important SNPs, effector proteins, etc.) this will be added to the annotation and posted as version improvements on our website.
Impacts
What was accomplished under these goals?
Objective 1: Investigating the genetics of the emergent races of P. belbahrii through genome sequencing, RNASeq analyses and a disease differential panel. In parallel, develop new BDM resistant lines to combat these races. P. belbahrii Genome SSR and SNP data has been generated to guide selection of candidates for sequencing. Multiple Race 0 and Race 1 isolates have been identified as potential isolates for sequencing, as well as an avirulent isolate for comparison. RNAseq & Disease Differential Panel We completed the screen for P. belbahrii: finalized the screen panel, defined the race structure of P. belbahrii. We initiated a comparative study of F. oxysporum f. sp. Basilici: Sequenced genomics of two strains, Generated transcriptomics data, Initiated analysis of both data sets. New Resistant Lines We continue breeding new disease resistant lines in NJ and FL. Objective 2: Develop the knowledge and resources to launch a new BLS resistance breeding program for basil and release BLS resistant sweet basil cultivars. P. cichorii Collections Activity 1. A protocol for testing basil plants with isolates of Pseudomonas spp. was established. Experiments were conducted to screen basil lines with new isolates of presumably Pseudomonas spp. the bacterium causing BLS in basil. 16 basil varieties were screened against 2 bacterial isolates per experiment. Inoculated plants were maintained in the laboratory under cooler conditions. and the plants were rated on a 0 (no foliar disease)-5 (100% foliar disease) scale (as used with lettuce BLS). Basil needs to be inoculated and incubated in the lab under lower temperatures Activity 2. Confirmation of isolations of Pseudomonas spp. Additional experiments were conducted using 4 isolates and 4 basil varieties per experiment with the goal of conducting Koch's postulates from the isolates collected throughout the season. 16 of the 17 isolates were recovered using a new Pseudomonas Bacterial Isolation Procedure. We extracted DNA from these 16 isolates recovered from Koch's postulates and their original collections. Sequencing of P. cichorii Boca strain: We isolated the virulent Boca strain from field-grown basil at the Rutgers NJAES Snyder Farm, NJ. Bacterial DNA was purified and sent to Quintara Biosciences for NGS (NextGen Sequencing). From the sequencing data, we identified potential DNA sequence encoding the Avirulence protein E1 (AvrE1), which contributes to plant cell death upon infection by Pseudomonas spp. BLS Resistance Screening Over 300 basil accessions were screened for BLS resistance. Broad patterns emerged that confirm putative resistance profiles across species and types of basil. Thai and lettuce leaved basils exhibited greatest susceptibility, Genovese and sweet basils have moderate to high susceptibility, lemon basils displayed moderate to low susceptibility, with least affected from the more exotics in O. tenuiflorum and O. americanum. Screening Mutant Pseudomonas Library & Investigating Type III Secretion System: Our team sequenced and assembled the genome of the P. cichorii isolate RUPC1 and have identified multiple effector genes for CRISPR knock out. And, putative susceptibility genes were identified in Rutgers SB-22 as targets for CRISPR knock outs. Potential RNASeq Analysis of BLS Infected and Healthy Sweet Basil: Multiple Isolates have been acquired as well as resistant and susceptible sister lines that will be utilized in this study. Objective 3: Identify relevant disease and horticultural-related genes in the basil genome via a genome wide association study. All 5 GWAS field trials have now been completed (3 in NJ, 1 in FL, 1 in Israel) and phenotypic data on a multitude of traits including basil downy mildew resistance were recorded. Our germplasm collection was sent for DArT sequencing which resulted in a newly developed collection of 55,000 raw SNPs prior to post processing and filtering. The raw reads have also been aligned and SNPs have been called from the RUSB-22 genome. Filtering and processing has lead to the development of a 20-25,000 SNP library that will be employed in the GWAS bioinformatics pipeline. Objective 4: Explore homologous resistance genes and validate putative resistance genes found via de novo annotation using CRISPR/Cas9 knockdowns and de novo annotation of the 'SB22' genome to spur quicker development of new elite basil cultivars. Characterizing gene targets for controlling BLS in basil: Applying the CRISPR/Cas9 system for gene editing to and provide further annotation of the Rutgers 'SB22' genome through knock-out studies of genes that condition susceptibility to BDM and BLS; validate putative resistance and susceptibility genes against BDM and BLS. Previously, we identified 2 BDM susceptibility genes in sweet basil, ObHSK encoding homoserine kinase and Ob2OGO encoding a putative 2-oxoglutarate-Fe(II) oxygenase. In 2022, 6 transgene-free, ObHSK-edited basil lines together with the non-edited, wild type (WT) were field-grown with a USDA- APHIS permit to evaluate their resistance to BDM in the field. Some ObHSK-edited plants demonstrated a delayed onset and reduced severity of BSM. Four plants were transferred back to the greenhouse to produce seeds. In 2023, seeds from these plants were transplanted into the field again, and the selections displayed similarly delayed onset and reduced severity of BDM. Production of ObOGO-edited basil plants: We also constructed sgRNA-based, transient CRISPR vector pRD528 to mutate Ob2OGO. We regenerated 18 plantlets from Rutgers Devotion DMR calli bombarded by pRD528 plasmid in combination with a mutated oligonucleotide gRNA to edit the Ob2OGO gene. ICE (Inference of CRISPR Edits) analysis of these 18 T0 plants indicated that 4 of them, carry deletion mutations at the expected Ob2OGO target site. Seeds have been produced and collected from these plants. We constructed the dual tRNA-based, multiplexing, integrating CRISPR vector pRD589 to mutate the Ob2OGO gene. We used Agrobacterium tumefaciens GV3101 containing pRD589 to transform embryogenic calli induced from Rutgers Obsession DMR and the susceptible SB22 basil seeds. Embryogenic calli have also been transformed by gene gun bombardment. We improved our basil TC system, and have a dozen newly regenerated shoots from pRD589 Agrobacterium-transformed Obsession and SB22 embryogenic calli. Production of ObNPR3-edited basil plants for both BDM and BLS resistance: NPR3 (non-expressor of pathogenesis-related 3) gene which, like 2OGO, has been shown to be an immunity suppressor. We used BLAST to query our SB22 transcriptomic and genomic data with the Arabidopsis AtNPR3 gene sequence and identified the basil orthologue ObNPR3 gene from the SB22 genome. We have PCR-cloned partial gDNA sequence of ObNPR3 from SB22 and constructed the dual tRNA-based, multiplexing, transient (pRD630) and integrating (pRD631) CRISPR gene editing vector to knock-out ObNPR3. We have used the gene gun bombardment method to transform SB22 embryogenic calli induced from the mature seeds with pRD631 and regenerated 15 shoots for integration of the transgene cassettes and the mutation of ObNPR3. Production of ObIAN9-, ObJAZ-, and ObZAT18-edited basil plants for BLS resistance: IAN9 is a member of the GTPase family of proteins, shown to suppress immunity against P. syringae by possibly targeting a transcription factor in Arabidopsis. We have used the Arabidopsis genes to query SB22 genome and identified the orthologue ObIAN9, ObJAZ, and ObZAT18 genomic DNAs. We are in the process of constructing the CRISPR-editing vectors to knock-out these genes to investigate their involvement in basil susceptibility to BLS and to produce BLS resistant basil plants. Genome Annotation: Genome Annotate has been accomplished.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Allen, K.S., Barrett, A., Mattera, R., Ben-Naim, Y., DeIulio, G.A., Pyne, R., Cohen, Y., Simon, J.E., Ma L-J (July 2024). Racing against resistance: understanding the genetic and molecular drivers of race development in the basil downy mildew pathogen Peronospora belbahrii. Poster and Flash Talk presented at The Sainsbury Laboratory Summer Conference in Plant-Microbe Interactions for Early Career Researchers, Norwich, U.K. (Grant acknowledged in poster and talk)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Li-Jun Ma (March 2024) Antifungal-resistance from both human health and plant protection perspectives. Keynote at 3rd International Conference on Antimicrobial resistance. Novi Sad, Serbia
- Type:
Websites
Status:
Published
Year Published:
2024
Citation:
Wyenandt, C.A. and J.E. Simon. Preparing for basil downy mildew in the field in 2024. 11 June 2024
https://plant-pest-advisory.rutgers.edu/options-for-controlling-basil-downy-mildew-in-the-field-2-3-2-2/
- Type:
Websites
Status:
Published
Year Published:
2024
Citation:
Wyenandt, C.A. and J.E. Simon. Controlling basil downy mildew in the greenhouse in 2024.12 June 2024
https://plant-pest-advisory.rutgers.edu/controlling-basil-downy-mildew-in-the-greenhouse-2/
- Type:
Other Journal Articles
Status:
Submitted
Year Published:
2024
Citation:
Allen, K.S., DeIulio, G.A., Pyne, R., Maman, J., Guo, L., Lyon, R., Johnson, E.T., Wick, R.L., Gershenson, A., Simon, J.E., Ma L-J. Identification of novel basil downy mildew resistance genes using de novo comparative transcriptomics. MPMI (submitted)
- Type:
Other Journal Articles
Status:
Awaiting Publication
Year Published:
2024
Citation:
Li, G., Newman, M., Yu, H., Rashidzade, M., Mart�nez-Soto, D., Caceido, A., Allen, K.S., Ma, L-J. Fungal effectors: past, present, and future. Current Opinion in Microbiology (in press)
- Type:
Other Journal Articles
Status:
Published
Year Published:
2023
Citation:
Li, G, McWilliams, M, Rodrigues, M, Mearkle, B, Jaafar, N, Golla, V, et al. CUR(E)ating a new approach to study fungal effectors and enhance undergraduate education through authentic research. Biochem Mol Biol Educ.https://doi.org/10.1002/bmb.21783
- Type:
Other Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Mattera III, R., L.J. Brindisi, C.A. Wyenandt and J.E. Simon. Advancing basil genomics: The first pseudo-chromosome level assembly and annotation of sweet basil (Ocimum basilicum). G3 (submitted).
- Type:
Other Journal Articles
Status:
Submitted
Year Published:
2024
Citation:
Ben Naim, Y., Mattera, R., Cohen, Y., & Simon, J. E. Predicting the resistance of basil entries to downy mildew based on their genetics, pathogen race, growth stage, and environmental conditions. Planta: (Under review).
- Type:
Websites
Status:
Published
Year Published:
2024
Citation:
Wyenandt, C.A. Basil downy mildew found in New Jersey. 18 June 2024
https://plant-pest-advisory.rutgers.edu/basil-downy-mildew-confirmed-in-southern-and-central-new-jersey-alert-63016-2-2-2-2-2-2/
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Progress 09/15/22 to 09/14/23
Outputs
Target Audience:Our target audience are the commercial growers (organic and conventional), buyers, distributors, seed companies, grower and marketing organizations and those scientists and extension specialists and agents and others involved in supporting their commercial sweet basil growers. Our consortium includes producers and processors of sweet basil from field and indoor production (e.g., greenhouses and vertical farming), organic and conventional growers, seed companies, scientists and extension researchers that provide technical assistance to their commercial growers and industry. Our stakeholders are our target audience and as such are a focus of our effort during the duration of the project. They have been directly involved in the selection and commercialization of improved sweet basil materials, and will be in testing and trialing our and other available lines by other groups (e.g. the seed industry), and participate in our winter and summer annual meetings and in field trials during which they provide feedback to us which we then use to guide our work. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In this first year of this project has provided mentoring, training and professional development for fivegraduate students, seven undergraduate students and two post-doctoral research associates. Specialized workshops for these students and others on genetics and plant breeding, new varietal development,including experiential training on cross pollination, plant grow-out, experimental design in greenhouse and field, seed harvesting, seed cleaning, seed storage and then advancing generations were provided in this first year. Additionally, the students and post-docs received training in the chemical characterization of aroma volatiles using GC/MS and in laboratory practices, and disease scouting, identification and control options. Student were also trained in plant pathology, culturing fungal and bacterial colonies. Importantly, many of these students also received hands-on training in DNA extraction, DNA analysis, tissue culture, gene editing and in genomic annotation. Many of the students also were mentored and participated in the preparation of posters and oral presentations at grower/industry and scientific conferences. Unique to our research project, many graduate and undergraduate students were also mentored and trained in science communication,using videos to create science-in-action stories to share with growers, the industry and the public. Many students participatedin the making or showing and release of thescience documentary, called Fields of Devotion that highlights the story and connection between Rutgers scientists, the agricultural community needs and student training in our development of creating downy mildew resistant sweet basils. Students were also mentored in ways to both listen to growers and the industry and then also in ways to effectively communicate scientific information and recommendations to commercial growers and the public. How have the results been disseminated to communities of interest?The results generated from our current project builds upon our prior USDA funded project as well, therefore enabling us to move ahead with initial dissemination strategies.Information was disseminated using traditional approaches by participating and presenting the research in extension, grower and trade show meetings, field trips, field tours, greenhouse tours and through state winter educational meetings and a remotely held zoom conference. Additionally, results were shared by presenting part of the ongoing research at national and international scientific conferences. A new website (usbasilconsortium.rutgers.edu)was designed and operational toserve multiple functions. It will mirror the approach taken in this grant to promote research, extension and outreach and will be separated into sections for each. The Research pillar of the website will be a place to post about new publications and report new information. It will also serve as a repository for genomic resources. This year, we postedthe V0 'RUSB22' sweet basil genome to make it available to the scientific community. The Extension pillar will be a central location to find essential news and information for commercial and home growers of basil. It will link to the Basil Ag Pest Monitor website for reporting occurrences of basil downy mildew as well as extension bulletins and fact sheets from Rutgers Pest Advisory and Cornell Vegetable Center (See: https://www.vegetables.cornell.edu/pestmanagement/disease-factsheets/basil-downy-mildew/; and we continued the Rutgers basil social media presence via the instragram, @rutgersbasil). The Outreach pillar will be geared towards communicating scientific results to a broader audience and making the science more accessible. Currently 'Our Favorite Uses of Basil' is a newseries we are testing geared towards showcasing our favorite uses of basil. We also have written pages about historical and cultural information about basil. Another website, was built and focused on promoting and dissemination information about 'Fields of Devotion' that highlights our basil research process and accomplishments through https://fieldsofdevotion.rutgers.edu/. The full length documentary is being used in colleges and high school biology, chemistry and plant science classes.Fields of Devotion, an award winning, science-in-action short documentary film featuring basil plant breeding research has been released to the public andviewed in person and online at science film festivals, at agricultural meetings, local gatherings, and in college classrooms. In-person events provided audience members the opportunity to hear directly from scientists and filmmakers during the question-and-answer sessions that followed the film screenings.In August 2023, Fields of Devotion reached wider audiences through New Jersey PBS (August 19, 2023) was and release through a streaming service. The film was featured on the NJPBS website with the trailer available to all viewers (https://www.thirteen.org/programs/nj-pbs-specials/fields-of-devotion-trailer-pqyfzh/ ). Fields of Devotion was released on Kanopy streaming services which allows public access via university and public libraries giving the film the potential for national (and possibly international) reach. A website is kept up-to-date to share current information about the film and future screenings are available (https://fieldsofdevotion.rutgers.edu/). What do you plan to do during the next reporting period to accomplish the goals?We plan to continue each of the studies initiative in Year 1 as they are multi-year; and to initiate those studies highlighted above in the accomplishment section that had not yet begun and were to begin in Y2. In Year 2, we will also begin as planned winter nurseries in Florida allowing us to advance new genetic lines much faster; we are exchanging genetic materials between the Bar Ilan Univeristy and Rutgers basil breeding programs to better and more effectively streamline improved sources of DMR and BLS resistance. We will complete the genomic anottation of the basil genome; and focus on the the collection and screening of many more DM and BLS isolates using the new differentil panel developed for BDM and the new rapid screening for BLS. Target sites will be identified and CRISPR-editing vectors will be constructed. In the accomplishment section we detailed many experiments including cloning and sequencing that were in process. In Y2 we expect these to be completed. That is, our expected outcomes in Year 2 are to continue to test the new disease differential panel and develop race-specific resistance in basil; and to use the foundation of the new rapid screening method to detect BLS resistance and susceptiblity for the launching of a new BLS resistant basil breeding program. In Y2 we will continue to seek to identify relevant disease and horticultural-related genes in the basil genome via GWAS and de novo annotation of the 'SB22' genome to accelerate our efforts in developing new elite basil cultvars (sweet basil and Thai basils). In Y2, we will continue to explore homologous resistance genese and seek to vailidate putative resistance genese identified via de novo annotation using CRISPR/Cas9 knockdowns. Together, in Y2 and throughout the remainder of this project, we will seek to provide stakeholder with enhanced options for the manafement and control of BDM and BLS. We plan to continue our outreach and educational approaches to disseminate our research findings via social media, video storytelling and in using the documentary film as a focal point to bring different communities together and discuss their needs.
Impacts
What was accomplished under these goals?
1) In-depth investigation into the BDM Genome using (b) Genome sequencing of the different BDM races to provide information on genetic diversity, phylogenetics, and race-specific effectors and molecular markers:Earlier we collected >130 isolates of P. belbahrii from infected basil plants.This year we collected 50 + isolatesincluding 11 new isolates from Israel, Italy, Sicilyand NJ. These isolates arebeing characterized for their pathogenicity with our newly developed BDM race determination panel. A DNA extraction protocol was developed and isolates will be identified by race and genotyped for a genetic diversity analysis. For our BDM genome analyses and phylogenetic analysis 42 representative isolates from the Israeland 23 from the USwere used for DNA extraction and we began to examine the quality of the DNA and the primers that were specially designed for'MIG-seq' genome wide analysis. c) Establish disease differential panels to identify race specific resistance:Previously, we performed P. belbahrii race evaluations over a putative differential system of basil plantsbasedscreening>500 accessions of BIU and RU teams. In 2023, weare using a new differential. This new differential set includes Superbo (Pb0 susceptible control which lacks BDM resistance), Eleonora (resistance unknown) KL-3 (new source of resistance with unknown genetics); Devotion DMR, Obsession DMR, and Passion DMR (MRI) - showing race, non-specific resistance; and Prospera CG and Prospera PS5 (Pb1), D-9-2 (Pb2) and Prospera Active 69 and Prospera Active 55 (Pb1/Pb2) which show race specific resistance. We validated this new tool and identified 2 new races (Pb1 and PB2) of BDM and can now share this differential screening panel system with others. d) Develop new BDM resistant lines to combat those races which have overcome previous resistant varieties:F2 Populations of Rutgers DMR x Prospera resistant lines werescreened for BDM resistance and selections are being made for the individuals to continue to advance. Backcross lines from this population are currently in the F1 stage. F1 interspecific hybrids have been made with O. americanum species and O. tenuiflorum species that show strong and broad based resistance. Newly identified resistant individuals are being shunted into the breeding program to increase genetic diversity and sources of disease resistance. New populations of Rutgers DMR Devotion have been crossed with a range of other sweet basils to alter the DMRs current aroma and taste and morphological profile. Populations of these F4 plants were field tested for the first time in 2023 and promising advanced lines with DMR resistanceidentified. A series of Thai basils were developed for DMR and in 2023, each of these advanced lines were generationally advanced and then screened in the field and results showed these new genetics to be DMR and high quality. 2) Exploring Bacterial leaf spot caused by Pseudomonas chicorri on sweet basil by collecting and identifying isolates from afflicted stakeholders:We have a library of 5 P. chicorri isolates collected from across the US and collecting 10 to 20 additional isolates. These isolates are run through Koch's postulates to test virulence and identified via 16S RNA sequencing. An informative bulletin was developed to spread awareness about the disease as well as to assist in sample collection. b) establishing a high-throughput screening system to identify resistant sweet basils:A novel high-throughput screen was developed to rapidly identify basil germplasm with resistance to Bacterial leaf spot. This protocol allows us to screen up to 32 basil accessions at once within 3-6 days. This methodology is currently being used to screen our entire germplasm collection. More than half of our 500+ germplasm collection wasscreened. Results indicate clear differential resistance between Ocimum species and related cultivars suggesting that multiple putative sweet basil sources of BLS resistance are present and may be useful in breeding for BLS in sweet basil. 3) Genome-Wide Association Study:GWAS will allow for extensive detection of genetic markers associated with critical horticultural traits as well as disease resistance--including new sources of basil downy mildew resistance and bacterial leaf spot resistance, based upon a genome-wide association study of 550 Ocimum spp. accessions. In 2023, GWAS Field Trial 4 was conducted in New Jersey. Our differential panel system definedthe local race at Rutgers Snyder Research Farm as BDM race 0. Multiple horticultural traits were phenotyped. These trials have identified new putative sources of BDM disease resistance. 4) Applying the CRISPR/Cas9 system for gene editing to a and b) provide further annotation of the Rutgers 'SB22' genome through knock-out studies of genes that condition susceptibility to BDM and BLS; validate putative resistance and susceptibility genes against BDM and BLS:Previously,we identified 2 BDM susceptibility genes in sweet basil, ObHSK encoding homoserine kinase and Ob2OGO encoding a putative 2-oxoglutarate-Fe(II) oxygenase. Using our established CRISPR-gene editing platform, we constructed single guide RNA (sgRNA)-based, transient CRISPR vectors pRD321 to mutate ObHSK. Using gene gun transformation of embryogenic calli induced from embryos of the BDM susceptible SB22 cultivar, we regenerated plantlets without antibiotic selection with a regeneration rate of over 75%. ObHSK mutant T0 plants were identified by cloning and sequencing the genomic DNA (gDNA) spanning the gene-editing target site. In 2022, 6transgene-free, ObHSK-edited basil linestogether with the non-edited, wild type (WT) were field grownwith aUSDA-APHIS permit to evaluate their resistance to BDM in the field. Some ObHSK-edited plants demonstrated a delayed onset and reduced severity of BSM. Four plants were transferred back to the greenhouse with a controlled environment to produce seeds. In 2023, seeds from these plants were transplanted into the field again, and these selections displayed similarly delayed onset and reduced severity of BDM. The major aromatic volatile compounds, linalool, eucalyptol and eugenol, in the 9 ObHSK-edited plants were at similar levels as the WT plants. This demonstrates that the gene editing of ObHSK did not alter the biosynthesis of volatiles that define the aroma of sweet basil. Production of ObOGO-edited basil plants: We also constructed sgRNA-based, transient CRISPR vector pRD528 to mutate Ob2OGO. We regenerated 18 plantlets from Devotion (a BDM resistant cultivar) calli bombarded by pRD528 plasmid in combination with a mutated oligonucleotide gRNA to edit the Ob2OGO gene. ICE (Inference of CRISPR Edits) analysis of these 18 T0 plants indicated that 4 of them, carry deletion mutations at the expected Ob2OGO target site. Seeds have been produced and collected from these 4 plants. We also constructed the dual tRNA-based, multiplexing, integrating CRISPR vector pRD589 to mutate the Ob2OGO gene. We used Agrobacterium tumefaciens GV3101 containing pRD589 to transform embryogenic calli induced from Rutgers Obsession DMR basil seeds. Out of 200 seeds as the explant starting material, 50 T0 plants were regenerated with hygromycin selection throughout the callus, shooting and rooting stages. These T0 plants are being analyzed for their Ob2OGO gene mutation status. Production of ObNPR3-editing basil plants for both BDM and BLS resistance: NPR3 (non-expressor of pathogenesis-related 3) gene which, like 2OGO, has been shown to be an immunity suppressor. We used BLAST to query our SB22 transcriptomic and genomic data with the Arabidopsis AtNPR3 gene sequence and identified the basil orthologue ObNPR3 gene. We designed primers and PCR-cloned partial gDNA sequence of ObNPR3 from SB22 and are cloning and sequencing this cloned ObNPR3 gDNA.
Publications
- Type:
Websites
Status:
Published
Year Published:
2023
Citation:
https://fieldsofdevotion.rutgers.edu/
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Brindisi, L.J., S. Mudiyala, R. Mattera, J. Honig and J.E. Simon. Genetic linkage mapping and QTL analysis of sweet basil (Ocimum basilicum L.) to identify genomic regions associated with cold tolerance and major volatiles. PlosOne:
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
2023. Brindisi, L.J. and J.E. Simon. Preharvest and postharvest techniques that optimize the shelf life of fresh basil (Ocimum basilicum L.): a review. Frontiers Plant Science. 14-2023. https://doi.org/10.3389/fpls.2023.1237577
- Type:
Websites
Status:
Published
Year Published:
2023
Citation:
usbasilconsortium.rutgers.edu
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
2023. Barrett, A., Wyendandt, C.A., and J.E. Simon JE.Development of a High-Throughput Screen for Bacterial Leaf Spot caused by Pseudomonas cichorii in Basil (Ocimum spp.) Poster presentation at the American Society of Horticultural Science Annual Conference, Orlando, Florida.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
2023. Brindisi L., Mattera, R., Barrett, A., Styles, T., Amer, I., Karunaratne, K., Dager, E., Bombardiere, J., Wyenandt, C.A., Homa, K., Barney, W.P., OBrien, R., Tepper, B., Di, R., Lawton, M. and J.E. Simon. Updates on basil research in developing disease resistance and improved new varieties. Oral talk at the NJ Agricultural Convention & Trade Show, Atlantic City, New Jersey.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
2023. Mattera, R., Brindisi, L., Honig, J., Wyenandt, C.A., Hartman, D.A., Raid, R., Ben-Naim, Y., Cohen, Y. and J.E. Simon. Identifying QTL in Sweet Basil for Basil Downy Mildew and Other Horticultural Traits Using the MRI x SB22 Recombinant Inbred Line Mapping Population. Oral talk at the American Society of Horticultural Science Annual Conference, Orlando, Florida.
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