Location: Genetic Improvement for Fruits & Vegetables Laboratory
2021 Annual Report
Objectives
Objective 1: Develop potato germplasm with improved levels of resistance to
biotic stressors, particularly late blight, common scab, and soft rot. [NP301, C1, PS1A, 1B; C2, PS2A].
Objective 2: Develop potato germplasm with improved levels of resistance to abiotic stressors, particularly for heat tolerance and reduced nitrogen input. [NP301, C1, PS 1A, 1B].
Objective 3: Use existing knowledge of the gene conservation between tomato and pepper to identify and develop markers for tomato anthracnose resistance in pepper, develop and implement effective marker assisted selection within Capsicum, and release new anthracnose resistant pepper germplasm. [NP301, C1, PS1A and PS1B]
Objective 4: Characterize the inheritance of resistance to tomato chlorotic spot virus in Capsicum, and develop and release adapted breeding lines suitable for breeding resistant hybrids. [NP301, C1, PS1A and PS1B]
Objective 5: Develop and release pepper breeding lines and cultivars with improved quality attributes for the culinary, culinary/ornamental, and minimally processed fresh-cut market. [NP301, C1, PS1B]
Objective 6: Discover pathogen gene function through use of functional genomics techniques. [NP303, C2, PS2A]
Objective 7: Characterize underlying mechanisms of resistance in solanaceous hosts in response to pathogen infection. [NP303, C2, PS2B]
Objective 8: Develop novel strategies for genetic improvement to manage disease in solanaceous crops. [NP303, C3, PS3A]
Approach
Late blight resistance genes in diploid and tetraploid potato will be identified via single nucleotide polymorphisms and incorporated into tetraploid germplasm. Tetraploid germplasm resistant to common scab will be identified via field testing and introgressed into commercial quality germplasm. A tissue culture assay using thaxtomin will be developed to identify scab resistance early in the breeding program at the seedling stage. Diploid germplasm with resistance to soft rot and blackleg will be identified via inoculations with the main bacterial species causing the disease. Resistance will be introgressed into advanced lines for varietal release. Diploids from cultivated and wild potato species will be evaluated for heat tolerance via tissue culture and validated in field tests. Wild species segregating for nitrogen uptake efficiency have been crossed into cultivated diploids. Progeny will be evaluated for nitrogen uptake efficiency and tuberization. Genotype by sequencing will be used to map anthracnose resistance loci in tomato using a recombinant inbred line population that we developed. Genetic stocks with resistance loci will be released. Tomato markers will be used to identify resistance homologues in pepper. Additional loci may be identified via linkage disequilibrium mapping of Capsicum baccatum accessions that we previously characterized. Loci will be transferred to C. annuum using bridge lines. Tomato chlorotic spot virus resistant lines identified in initial screening of C. chinense will be field tested, inheritance characterized, and resistance introgressed into C. annuum. Selection for high-value specialty peppers has combined desirable fruit and plant attributes for culinary/ornamental and strict culinary use. Breeding is required to refine/stabilize selections and conduct multi-location trials. Diverse bell and jalapeno Capsicum germplasm we selected for fresh-cut attributes will be used to develop a selection index. Combining ability will identify superior backcross lines for release. Functional genomics will be used to discern pathogen gene function for glycosyl hydrolase enzymes having multiple roles in initiation of plant disease. Genes encoding glycosyl hydrolases from Alternaria and Streptomyces will be identified in infected hosts via RNA-seq based gene expression profiling. Candidate genes will be cloned and tested via transient expression. Functionality will be further evaluated via RNAi suppression. Nitrogen treatments will be tested to generate RNA-seq host/pathogen expression profiles and identify means to reduce Alternaria infection. To reduce common scab severity, auxin analogues will be applied to potato foliage followed by host/Streptomyces gene expression profiling to identify gene targets for reduced susceptibility. We will evaluate methods for weakening Phytophthora and Alternaria cell walls to reduce pathogen ability to colonize hosts. Enzymes for protoplast generation, plant defense, and enzymes the pathogen uses to alter its own cell wall will be evaluated using Agrobacterium-mediated expression and tissue inoculations.
Progress Report
A new scientist to lead the potato breeding and genetics research program was hired in January 2021 and has resumed research at a new duty station in Orono, Maine. Much of the early work of the new scientist is related to setting up a functional lab and greenhouse space. Tissue culture chambers, biosafety hood, and DNA extraction equipment have been established. The USDA has two greenhouses in Orono, Maine. Significant work was required to restore these greenhouses to be functional. The new scientist coordinated work to repair exhaust fans, fix electrical issues and assembled and installed a fertilization system. The greenhouses are now functional for greenhouse activities, including growing seedlings and setting up crossing blocks for the Potato Varietal Development for the Eastern U.S.
Since the retirement of the prior scientist in FY19, the support staff has continued field activities. As a vegetatively prorogated crop, it is necessary to plant, harvest, and replant breeding lines not in tissue culture to maintain this critical germplasm. The support staff and the new scientist coordinated the planting of 11 field trials at Aroostook Research Farm in Presque Isle, Maine, and 20 field trials at the Chapman Farm in Chapman, Maine. 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 in support of varietal development research.
The new scientist contacted six collaborators and received true potato seed shipped to Orono, Maine. This seed was combined with seeds generated by the prior scientist before retirement to germinate 300 true potato seed families. From these 300 families, approximately 28,000 breeding lines have been planted in the greenhouse. These lines will produce minitubers in the greenhouse that will be planted in field trials in 2022 to reestablish the first-year breeding program selection, which was severely cut back since the retirement of the prior scientist in FY19. This work is critical towards restoring the full capacity of the breeding program.
Colletotrichum species are responsible for the fungal disease anthracnose that reduces the marketable yield of tomato and pepper fruit. We previously developed a high-density SNP -based tomato linkage map using recombinant inbred lines susceptible and resistant to the Colletotrichum fungus. We further characterized and validated five genes using qRT PCR in conjunction with RNAseq and QTL data. These QTL-related genes display differential expression between resistant and susceptible tomato lines. The research is collaborative with West Virginia State University. Transcriptome profiling of the plant host and differential fungal isolates is ongoing to identify mechanisms whereby aggressive isolates can proliferate in normally resistant host genotypes.
New genes for virus resistance must be identified in pepper to address the emergent tospovirus Tomato Chlorotic Spot Virus (TCSV) that is now established in Florida, a major production area for tomato and pepper. Collaboration with North Carolina State University was established to obtain RNAi constructs targeting tospoviruses (including TCSV) and reduce symptoms in tomato. Vectors are currently being transferred to Agrobacterium for plant transformation. Characterization of C. chinense accessions exhibiting TCSV phenotypes ranging from resistant to very susceptible is ongoing with ARS cooperators in Fort Pierce, Florida. Inheritance studies are planned to characterize resistance and develop breeding strategies for introgressing resistance to C. annuum.
Value-added crops can be very profitable in comparison to conventional forms of the commodity. In support of Objective 5, breeding for specialty peppers with improved flavor, color, and related consumer valued attributes continued this summer with multilocation trials of advanced breeding lines for specialty markets. After missing field trials in 2020 due to pandemic restrictions, trials have resumed in 2021. Evaluations of pepper populations developed for fresh-cut applications and specialty snack markets are underway.
In collaboration with Oregon State University, scientists at ARS Beltsville, Maryland, continued to analyze a large collection of Streptomyces genomes to characterize the diversity of phytopathogens in the genus. Three novel species groups have been identified and are being formally named and described. For the most prevalent species, Streptomyces scabiei, in-depth genomic epidemiology revealed that potato growing sites across the United States have endemic populations of the pathogen and that relatively recent transmission events have occurred between grower sites. Genomic analyses of the primary pathogenicity determinant, the Thaxtomin pathogenicity island, revealed horizontal gene transfer of the pathogenicity island leading to novel pathogen emergence.
The phytotoxin Thaxtomin is necessary for Streptomyces to cause common scabs of potato. Scientists at ARS Beltsville, Maryland, cloned the Thaxtomin biosynthetic genes into a Thaxtomin-deficient mutant and confirmed rescue of virulence in the complemented strain on potato. Using this validated toolset, scientists were able to confirm that Thaxtomin is also necessary for Streptomyces to cause common scab of radish, carrot, turnip, and beet. Therefore, disease management strategies that are effective against common scab of one of these crops should be tested for the other crops given the similar mode of pathogenicity.
Low-dose treatments of the herbicide 2,4-D have been shown to reduce the severity of common scab of potato in field settings. Following up on recent field trials, scientists at ARS Beltsville, Maryland, have confirmed the ability of 2,4-D to protect against common scab in a greenhouse setting following optimized treatment conditions. Whole transcriptome sequencing is being employed to identify genes associated with response to 2,4-D that may be responsible for the observed effect of reducing disease severity.
Endolysins are lytic enzymes encoded in phage genomes responsible for the lysis of bacterial cell walls and the release of phages from bacteria. Scientists at ARS Beltsville, Maryland, have identified more than 500 phage endolysins present in phytopathogenic Streptomyces strains. Crude protein extracts from E. coli expressing these phage endolysins were shown to be highly lytic to multiple phytopathogenic species of Streptomyces. With extramural funding support, ARS scientists are collaborating with Florida A&M University to test the ability of 12 candidate endolysins to lyse Streptomyces.
Non-pathogenic Streptomyces strains are promising potential biocontrol agents against pests of potato due to their ability to thrive in the soil in close association with potato and the known antimicrobial production capacity within the genus. In collaboration with scientists at Washington State University and Brigham Young University, scientists at ARS Beltsville, Maryland, identified more than 10 Streptomyces with inhibitory solid activity against Pythium leak and identified more than 20 Streptomyces with inhibitory solid activity against silver scurf disease of potato. The four most promising strains are currently being tested in field trials in Idaho. Five Streptomyces that strongly inhibit potato root-knot nematode egg hatch and galling of plant roots have also been identified. Four of these strains have been confirmed to colonize potato tubers and survive in the soil for at least 14 weeks.
Late blight, caused by Phytophthora infestans, and early blight, caused by Alternaria solani, are responsible for most disease losses in potato production. Studies underway are directly addressing the functional role of specific genes used by the pathogens for infection and specific genes used by the plant for defense or lack of susceptibility. The principal challenge is that both diseases need to be controlled to provide full grower application, yet the two pathogens are very different in the way they cause disease and their microbial cell wall structure.
A series of potential necrosis eliciting proteins from Alternaria solani have been cloned from A. solani and expressed through Agro-infiltration of tobacco and potato leaves. Since A. solani causes host cell death in advance of infection, limiting necrotic cell death may limit the pathogen. Proteins were selected based on secretion from the fungus or presence on the spore or hyphal surface. Various members of GH 12 hydrolases, xylanases, and Alt-A antigenic protein elicited no plant response, while NEP produced consistent necrosis. Identification of the host receptor responsible for NEP recognition and subsequent necrosis could be useful for altering the necrotic host response and limiting A. solani infections.
An overlooked source for potential control of Phytophthora infestans is the endobiome of potato and tomato. Trichoderma species have been isolated as endophytes from potato and tomato, along with other fungi, that may prove useful as biological control microbes. Testing is underway to determine if and how fungal isolates can be transferred between cultivars of potato to limit disease. One endophyte had been found to have strong antifungal activity due to antibiosis. Additional studies underway will determine how fungicides affect the endobiome, in either a positive or negative manner.
Accomplishments
1. Ozone treatment reduces seed viral contamination but is insufficient to disinfest seed. Viral diseases of crop plants transmissible by contaminated seed result in significant crop loss without efficacious treatments to rid transmissible by contaminated seed, resulting in significant crop loss without efficacious treatments to rid the seed of virus. One such disease is pepper mild mottle virus (PMMoV) which is localized to the seed coat of sweet and hot pepper. Conventional treatments reduce but do not eliminate the incidence of seed-born PMMoV. We evaluated the use of ozone as a treatment to disinfest pepper seed naturally contaminated with PMMoV. Exposure to purified virus and contaminated seed demonstrated that ozone could reduce concentrations of viable PMMoV and that virus destruction increased with the length of ozone exposure time. Levels of viral inactivation were insufficient to recommend ozone as a standard treatment to reduce PMMoV incidence in contaminated pepper seed lots substantially. These results are of practical use in updated guidelines for seed disinfestation and will benefit seed producers and plant researchers .
2. Discovery, characterization, and formal description of three novel phytopathogenic Streptomyces species. Three new novel species of Streptomyces that cause common scabs of potato were identified through high diversity sampling and genome sequencing of strains in the ARS Streptomyces culture collection. Two of the novel species were confirmed to be present in multiple potato-growing regions in the United States. Finished genome sequences were developed, and biochemical and pathogenic properties of representative strains for each of the species were characterized. The three novel species are officially named S. caniscabiei, S. griseoscabiei, and S. aureuscabiei, respectively. Strains for each of the three species were selected and deposited into international culture collections to be available to other researchers and served as references for the Type strains. Characterization of the species-level diversity of phytopathogenic Streptomyces is critical for understanding the distribution of the disease in the United States and the specific risks of disease faced by potato growers in different locations.
3. Thaxtomin phytotoxin is responsible for common scab of radish, carrot, and beet. Thaxtomin is produced by multiple species of Streptomyces and is the primary virulence determinant for common scabs of potato. Select species of Streptomyces are also known to cause common scabs on other root crops such as radish, carrot, beet, and turnip, but the molecular details of virulence in these pathosystems are unknown. Using purified Thaxtomin toxin and various plant immunity assays, radish, carrot, beet, and turnip were all shown to be sensitive to Thaxtomin. Additionally, a complemented strain of a Thaxtomin-deficient mutant strain of Streptomyces was generated to confirm the importance of Thaxtomin production for disease development on these vegetables. The wildtype Streptomyces strain was able to cause disease on radish, carrot, and beet in greenhouse pots. The Thaxtomin-deficient mutant strain was unable to cause disease, but the Thaxtomin-complemented strain caused wildtype-levels of disease. These results unambiguously confirm that Thaxtomin is necessary and the key determinant of common scab symptom development of radish, carrot, and beet. Therefore, new disease management tools developed for potato that mitigate common scabs can now be employed with good certainty to manage this devastating disease on these other vegetable crops .
Review Publications
Stommel, J.R., Dumm, J.M., Hammond, J. 2021. Effect of ozone on inactivation of purified pepper mild mottle virus and contaminated pepper seed. Phytofrontiers. 1: 85-93. https://doi.org/10.1094/PHYTOFR-09-20-0020-R.
Lopez-Ortiz, C., Pena-Garcia, Y., Bhandari, M., Abburi, V., Stommel, J.R., Nimmakayala, P., Reddy, U.K. 2021. Identification of miRNAs and their targets involved in flower and fruit development across domesticated and wild capsicum species. International Journal of Molecular Sciences. U.K. 2021. https://doi.org/10.3390/ijms22094866.
Weisberg, A., Kotha, R., Kramer, C., Chang, J., Luthria, D.L., Clarke, C.R. 2020. Whole-genome sequencing of five Streptomyces isolates identifies a new pathogenic species group that causes common scab disease of potato. Molecular Plant-Microbe Interactions. 34(1):39-48. https://doi.org/10.1094/MPMI-06-20-0164-R.
Mechan-Llontop, M.E., Tian, L., Sharma, P., Heflin, L., Bernal-Galeano, V., Haak, D.C., Clarke, C.R., Vinatzer, B.A. 2021. Experimental evidence pointing to rain as a reservoir of tomato phyllosphere microbiota. Phytobiomes Journal. e-ISSN:2471-2906. https://doi.org/10.1094/PBIOMES-04-21-0025-R.
Die, J.V., Jones, R.W., Ogden, E.L., Ehlenfeldt, M.K., Rowland, L.J. 2020. Characterization and analysis of anthocyanin-related genes in wild-type blueberry and the pink-fruited mutant cultivar 'Pink Lemonade': New insights into anthocyanin biosynthesis. Agronomy. https://doi.org/10.3390/agronomy10091296.
