Location: Genetic Improvement for Fruits & Vegetables Laboratory
2024 Annual Report
Objectives
Objective 1: Enhance genetic resources for pepper and tomato through development of cultivars and breeding lines optimized for production in controlled environment agriculture and with durable resistance against major diseases.
Sub-objective 1.A: Characterize and develop functional and regulatory mechanisms involved in anthracnose resistance for enhancement of tomato fruit rot resistance.
Sub-objective 1.B: Develop and release tomato breeding lines and genetic stocks with novel traits that enhance adaptation to indoor vertical production CEA environments.
Sub-objective 1.C: Exploit Capsicum germplasm resources to further characterize tospovirus resistance and introgress tomato chlorotic spot virus resistance into adapted culinary pepper breeding lines.
Objective 2. Enhance genetic resources for potato by evaluating germplasm and developing improved varieties for heat tolerance, nitrogen use efficiency, and resistance against important microbial pests.
Sub-objective 2.A: Screen diploid potato germplasm for common scab resistance and developing scab-resistant varieties.
Sub-objective 2.B: Screen potato germplasm and develop varieties with improved agronomic performance under low nitrogen conditions.
Sub-objective 2.C: Identify and characterize the physiological traits associated with internal heat necrosis in potato germplasm.
Objective 3. Develop and utilize molecular biology and biotechnology tools to accelerate trait discovery and germplasm improvement of solanaceous vegetable crops for enhanced biotic and abiotic stress tolerance and adaptation to diverse production systems.
Sub-objective 3.A: Develop TCSV-resistant pepper germplasm with potential broad-spectrum tospovirus resistance via RNAi-mediated silencing.
Sub-objective 3.B: Generate transgenic solanaceous plants to test the role of specific genes in Verticillium wilt resistance in potato.
Sub-objective 3.C: Characterization of molecular mechanisms involved in potato nitrogen use efficiency or nitrogen use efficiency-associated traits.
Sub-objective 3.D: Elucidation of molecular pathways that distinguish susceptibility and tolerance to potato internal heat necrosis.
Objective 4. Expand management options against important pathogens of solanaceous vegetable crops through characterization of pathogen populations, identification of virulence mechanisms, and testing of novel chemical and biological disease management tools.
Sub-objective 4.A: Identify and test natural and synthetic chemical controls of common scab, late blight, and early blight of potato.
Sub-objective 4.B: Characterize the diversity and distribution of pathogens that cause scab diseases of potato in the United States.
Sub-objective 4.C: Identify and functionally characterize pathogen virulence factors in common scab of potato.
Sub-objective 4.D: Identify and test endophytic microbes for control of biotic (common scab, early blight, late blight, and anthracnose) and abiotic (heat) stress.
Approach
Identification, selection, and manipulation of key plant and pathogens genes involved in plant-pathogen interactions or plant physiology will be performed through a variety of experimental pipelines. For tomato-anthracnose interactions, target genes involved in steroidalglycoalkaloid synthesis will be knocked down or overexpressed and impact on tomato disease susceptibility assessed through pathogen infection assays. For TCSV infection of pepper, transgenic pepper plants expressing constructs to silence the broad diversity of TCSV types through RNAi machinery will be transgenically expressed in pepper and tested for viral resistance. Standard traditional breeding approaches for introgressing TCSV resistance into pepper germplasm will be employed in parallel to identify and introgress that resistance into culinary germplasm.
For potato, lines and cultivars with enhanced agronomic response to nitrogen, resistance to internal heat necrosis, and resistance to common scab disease will be identified through screening available potato germplasm resources in field and greenhouse trials. Genetic loci or biochemical pathways responsible for the desired traits will be identified through genome wide association studies or comparative metabolomics and transcriptomics between resistant and susceptible genotypes. The diversity of common scab pathogens in the United States will be examined through broad isolation of pathogens from infected tubers. Genomic analyses will be employed to identify key genes in virulence expression and the role of those genes will be tested through knockdown or overexpression. Chemical common scab disease suppression through low dose application of synthetic auxin analogs will be optimized through field trials with different spray regimens. The presence and effect of endophytic fungi on growth, yield and stress tolerance will be studied through use of fungal isolation and re-inoculation methods, using individual and mixed endophytic isolates. The role of endogenous application of melatonin, along with genetic manipulation of genes in the pathway of melatonin production will be examined to determine effects on biotic and abiotic stresses. The role of allelic diversity of Verticillium resistance genes in Verticillium wilt resistance will be tested through transgenic overexpression.
Progress Report
In support of Objective 1, we demonstrated that VIGS-based transient silencing of the steroidal glycoalkaloid metabolic genes GAME31 and GAME5 in anthracnose-susceptible tomato fruit yielded enhancements to anthracnose resistance. Metabolic profiles of steroidal glycoalkaloids in fruit of gene-silenced plants supported our observations in RIL lines segregating for anthracnose resistance that accumulation of tomatine and several of its derivatives were important for inhibiting fungal fruit rot development. Progress was made in developing stable transformants with silenced GAME31 and GAME5 metabolic genes. We have advanced stable transformants through the T1 generation. Plans are underway to develop analogous CRISPR-based lines to circumvent public resistance over acceptance of traditional GMO plants. Second-year taste panels and data evaluations were also completed and demonstrated that SGA levels in RIL lines were sufficiently low to prevent fruit bitterness but inhibit fungal fruit rot.
In support of Objective 1, we completed an APHIS-approved field trial of tomato lines transformed with a synthetic construct that reduced photorespiration, thus enhancing photosynthetic efficiency. Results demonstrated increased biomass and fruit counts in indeterminate but not in determinate tomato genotypes. Complete data analysis is in progress. The research targets enhanced metabolic efficiency for application in input intensive vertical production environments.
In support of Objective 1 and Objective 3, population development continues to develop segregating populations from crosses between Tomato Chlorotic Spot Virus (TCSV) resistant and TCSV susceptible pepper genotypes. The germplasm will be utilized to evaluate gene action and inheritance of TCSV resistance in pepper. A
complementary approach to develop resistant pepper utilizes transformation with cooperator constructs conferring broad-based tospovirus resistance. Initial screens to identify the most efficacious constructs utilizes transient infiltration into pepper leaves to evaluate efficacy. Preliminary evaluations have focused on transient silencing in pepper leaves using the phytoene desaturase (PDS) gene.
Value-added crops can be very profitable in comparison to conventional forms of the commodity. Analysis of extensive data on Jalapeno pepper fresh-cut quality from the prior Project Plan was completed. That research is summarized as an Accomplishment in this report. Also completed from the prior Project Plan were evaluations for the final release of a new ornamental pepper cultivar ‘Purple Rain’. That research is also summarized as an Accomplishment in this report.
In support of Objective 2, multiple lab protocols were established and automated greenhouse watering and benchtop improvements were installed. Laboratory protocols for tissue culture propagation of potato breeding lines were improved. At ARS research farms in Northern Maine, 25 research trials were planted during May 2024. The size of research plots in these trials range from single plants to 200 plants/plot. In total, thousands of potato breeding lines were planted this spring at these locations. This includes thousands of new breeding lines received from a collaborator and hundreds of historical potato varieties which are not present in any gene bank collection. In addition to being critical towards meeting the goal of potato variety development, these field trials are critical towards meeting the milestones for Sub-objective 2. From the 223 potato breeding lines, 100 will be selected based on the available seed and based on maximizing the genetic diversity. These 100 breeding lines will be evaluated for scab resistance at infected fields at the Aroostook Research Farm in Presque Isle, Maine, and the Penn State Russell E. Larson Agriculture Center in Rock Springs, Pennsylvania, during the 2025 field season.
In collaboration with the University of Maine, a potato monoploid, derived from a diploid inducer line, was developed which will be used in future research to explore the molecular mechanisms for haploid induction in potato.
In support of Objective 3B, Genes in the biosynthetic pathway for melatonin production were cloned from potato, introduced into a constitutive-expression vector and transformed by Agrobacterium into stable transgenic Kennebec and Bintje potato cultivars. Forward and reverse copies of COF were used along with reverse copies of SNAT. Reverse copies (gene silencing) did not produce changes in plant growth or tuber production, while the COF forward copy resulted in small plants with thick leaves. Tubers had numerous secondary growths similar to ginger root. Pathogen resistance screening will proceed in year two.
Nitrogen (N), potassium (K) and phosphorus (P) are crucial elements required for plant growth and development. Nitrogen deficiency causes lethal effects on plants and compromises plant growth with yield penalty. In support of Objective 3C, an attempt was made to develop a nitrogen assay (N) at varying N application rates to see the physiological impact along with agronomic impact toward a broader goal of characterizing nitrogen use efficiency (NUE) and associated traits. The experiment results revealed that potato agronomic indicators such as potato tuber yield and plant biomass depend on N application rates within the same cultivar. NUE was differential across two cultivars of potato with differential physiological and agronomic performances. These results indicate that the window of genetic improvement of NUE traits in potato lies at less than 50% of the current use of N which needs to be further researched.
Internal heat necrosis is a devastating phenomenon impacting tuber yield and quality and screening of resistant and susceptible IHN varieties along with development of IHN tolerant potato varieties is needed. In support of Objective 3D, we developed an assay for IHN to test the impact of different heat conditions on early and late tuber formation and maturity. Growth chamber availability is a limiting factor. Two growth chambers were made available in mid-February 2024. Preliminary studies carried out in the growth chamber indicated that heat stress impacts on tuber formation and development are potato growth stage specific. Shortage of growth chambers only allowed to test one heat stress condition. More experiments are underway to test heat conditions for late tuber maturity to further evaluate IHN incidents in potato tubers.
Streptomyces genomes are notoriously fragmented due to high GC% and numerous repetitive elements. High quality genomes are needed for accurate reference-based assemblies and for analyses of overall genome structure. In support of Objective 4B, finished or nearly finished genomes were generated using hybrid sequencing for 18 species of Streptomyces, including the type strains of all of the species groups known to cause common scab disease of potato. These high-quality genomes will serve as reference genomes for improved assembly of other genomes in the species group. Structural analysis of the finished genomes suggested substantial genome plasticity and few plasmids. Core and pan genome analyses revealed sets of genes specific to the pathogenic lineages.
Also in support of Objective 4B, whole genome sequencing of more than 170 Streptomyces strains revealed substantial diversity of strains. Several new species-level groups were identified, two of which are currently in the process of being formally described. Surprisingly, up to eight pathogenic species were identified in a single field site indicating substantial within-field diversity of the pathogen.
Toward completing Objective 4D, Endophytic fungi were isolated from multiple potato cultivars grown in greenhouse and field conditions. Robust surface sterilization eliminated many superficial fungi, revealing true endophytes. A limited number of fungi were isolated, and there was little overlap between potato varieties. Isolated fungi were often known to be endophytic in other plants. Fungi considered pathogenic on monocot plants were found as endophytes in the dicot potato.
Thiamin is a known elicitor of immune priming for several plants. The ability of Thiamin applications to reduce two diseases of potato, early blight caused by Alternaria solani, and common scab, caused by several species of Streptomyces, was tested. Thiamin treatments significantly attenuated early blight disease. RNA sequencing and metabolomics suggested several pathways involved in the thiamin-mediated resistance. Direct foliar treatments did not attenuate common scab disease. However, soil drenches of thiamin led to reduced disease. Thiamin did not directly impact pathogen growth but did suppress production of the key phytotoxin necessary for manifestation of disease symptoms.
Accomplishments
1. Improved soil detection of common scab pathogens to understand population dynamics and disease risk. A qPCR detection assay for Streptomyces bacteria, the causative agent of potato common scab disease, was implemented in the field and greenhouse to identify pathogen population dynamics in the soil. In both field and greenhouse settings and across multiple species of the pathogen, a spike in the pathogen population occurred mid-growing season indicating that the pathogen abundance increases during initial tuber infection. This time point is therefore the most promising for sampling to ascertain pathogen abundance in analysis of disease trials. ARS researchers in Beltsville, Maryland, also identified for the first time active growth of multiple pathogen lineages during disease infection. The qPCR detection assay was also optimized to identify phytopathogenic Streptomyces in potato tuber skin lesions. These results have delivered an additional diagnostic tool for stakeholders which has been implemented multiple times to provide growers rapid disease diagnostic information and improved disease management.
2. Modified potato tuber production through expression of pathogen proteins. Verticillium wilt remains a major disease problem in potato production, resulting in millions of dollars in annual losses. Understanding how the Verticillium fungus develops a successful infection is critical to developing management strategies. Verticillium produces a limited number of proteins inside the infected plant, and it is believed that they aid the infection. ARS scientists in Beltsville, Maryland, determined that one protein was very similar to a protein already in the potato plant. The potato gene was expressed continuously in transformed potato plants and found to control plant growth, resulting in shorter plants. A striking feature was the production of small tubers along the stem, suggesting a change in plant resource allocation and developmental modification. This discovery provides new avenues for understanding why Verticillium produces certain proteins, also revealing the potential use of a potato gene to develop aerial tubes that can be harvested independent of soil tuber production. This information will benefit scientists in understanding both Verticillium infections as well as potential modifications to potato production which can provide new economic benefits.
3. New pepper cultivar ‘Purple Rain’. Ornamental peppers have the highest per unit value of any pepper product. They have become an innovative way for small farmers to produce a high-value alternative crop. New cultivars that exploit considerable diversity in Capsicum for fruit and leaf shape, size and color, as well as plant habit will help sustain renewed interest in ornamental peppers. A new ornamental pepper cultivar, ‘Purple Rain’, was developed by researchers in Beltsville, Maryland. ‘Purple Rain’ is a true-breeding small-statured ornamental pepper with vibrant green-purple-white variegated foliage with contrasting black immature and red mature fruit colors. ‘Purple Rain’ is easily propagated from seed, has short cropping time, heat and drought tolerance, and excellent keeping quality. This cultivar affords growers a new crop to add to their pot plant and bedding/color/landscape plant assortment. ‘Purple Rain’ is also well-suited as an accent or filler for container arrangements. ’Purple Rain’ is a public release and will benefit growers as well as seed producers and plant propagators who supply seed and plants to the wholesale and retail ornamental markets.
4. Simplified breeding for Jalapeno fresh-cut pepper quality. During the past 40 years, the U.S. fresh-cut market has experienced a consistent increase in demand because consumers prioritize health and convenience. This has led to innovations in breeding, product selection, and packaging. Despite increased popularity of bell pepper and chile pepper, research of fresh-cut jalapeno pepper is limited. ARS researchers in Beltsville, Maryland, characterized fresh-cut attributes in a diverse collection of Jalapeno germplasm to establish measures that could be easily used to assess fruit quality during storage and be readily used in breeding to develop Jalapeno cultivars with superior fresh-cut quality. The research demonstrated that measures of fruit softening and tissue firmness could replace laborious electrolyte leakage assays for fresh-cut quality. For breeding superior cultivars, fruit size, wall thickness, and skin toughness which can be easily selected for in a breeding program were identified as predictive measures of Jalapeno fresh-cut quality. Genotypes that are useful as parents in transferring superior fresh-cut traits to progeny were also identified. This research provides valuable tools to plant breeders and the fresh-cut industry to help sustain fresh-cut market growth.
Review Publications
Lopez-Ortiz, C., Reddy, U., Zhang, C., Natarajan, P., Nimmakayala, P., Benedito, V., Fabian, M., Stommel, J.R. 2023. QTL and PACE analysis identify candidate genes for anthracnose resistance in tomato. Theoretical and Applied Genetics. https://doi.org/10.3389/fpls.2023.1200999.
Park, E., Luo, Y., Zhou, B., Fonseca, J.M., Stommel, J.R. 2024. Varied attributes of jalapeño pepper cultivars influence fresh-cut product quality. Journal of the American Society for Horticultural Science. 149(3):152-161. https://doi.org/10.21273/JASHS05346-23.
Upadhyay, R.K., Motyka, V., Pokorna, E., Roberta, F., Dobrev, P.I., Handa, A.K., Mattoo, A.K. 2024. Transition from development to ripening of tomato fruit impacts abundance of endogenous phytohormones and their pathway metabolites. Plant Growth Regulation. https://doi.org/10.1007/s10725-024-01165-7.
Upadhyay, R.K., Shao, J.Y., Grace, R.E., Mattoo, A.K. 2024. Comparative genomics and evidence for an unusual functional polyamine oxidation pathway in aquatic duckweed (Spirodela polyrhiza L.). Current Plant Biology. 39: Article e100359. https://doi.org/10.1016/j.cpb.2024.100359.
Behling, W., Coombs, J., Collins, P.J., Douches, D. 2023. An analysis of inter-endosperm balance number crosses with the wild potato solanum verrucosum. American Journal of Potato Research. 101:34-44. https://doi.org/10.1007/s12230-023-09937-z.
Weisberg, A., Pearce, E., Kramer, C.G., Chang, J., Clarke, C.R. 2023. Diverse mobile genetic elements shaped the evolution of streptomyces virulence. Microbial Genomics. https://doi.org/10.1099/mgen.0.001127.
Shelley, B., Pandey, B., Sarwar, A., Douches, D., Collins, P.J., Qu, X., Pasche, J., Clarke, C.R. 2024. The role of soil abundance of TxtAB in potato common scab disease severity. American Phytopathological Society. https://doi.org/10.1094/PHYTO-09-23-0347-R.
Alvarez-Quinto, R., Grinstead, S.C., Jones, R.W., Mollov, D.S. 2023. Genomic characterization of a new mitovirus associated with walking iris (Trimezia northiana). Archives of Virology. 168:273. https://doi.org/10.1007/s00705-023-05901-8.
