Location: Genetic Improvement for Fruits & Vegetables Laboratory
2023 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
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 trials of advanced breeding lines for specialty markets. In particular, snack pepper lines bearing fruit with low seed count, superior shape and flavor, in multiple fruit colors are being readied for release at the culmination of 2023 trials.
Building upon research that identified key steroidal glycoalkaloid (SGA) metabolites in tomato responsible for fungal fruit rot resistance, analytical and sensory evaluations of recombinant inbred tomato lines with varied SGA profiles provided new evidence that these compounds did not negatively effect flavor and acceptability of fruit with SGA levels sufficient to inhibit fungal fruit rot. The results strengthen transfer of this technology for practical use by scientists in crop improvement.
Preliminary studies are underway to evaluate novel genes for enhancing photosynthetic efficiency and crop productivity in tomato. These genes reduce the detrimental effects of photorespiration. Early results provide evidence for increased plant biomass in plants expressing these genes. Tests are underway to evaluate the novel technology in an array of tomato lines with varied plant habit, fruit size and plant architecture.
Molecular detection of common scab was further improved through in-depth identification of minimum infectious doses and detection limits for five species of the pathogen. At several infested field sites in collaboration with researchers at North Dakota State University and Pennsylvania State University, common scab severity was shown to be significantly correlated with abundance of the pathogen based on the developed molecular diagnostic assay, providing a valuable tool to growers for making informed planting decisions about the need for using cultivars with higher degrees of resistance to common scab. Additionally, at several field sites tubers were collected for Streptomyces isolation and more than 200 putatively pathogenic Streptomyces isolates have been added to the ARS culture collection.
2,4-D when applied to potato at very low doses can induce anthocyanin production and increase resistance to common scab. Transcriptional changes in the metabolic pathways for fatty acid and sucrose/glucose were found to be associated with plant response to 2,4-D dependent on the presence of the pathogen. Sucrose and glucose abundances were found to be significantly shifted in the 2,4-D treated plants. Since glucose abundance has previously been shown to be correlated with common scab resistance, this is a potential mechanism to explain the efficacy of low-dose 2,-4D treatments for common scab management.
Accurate translation of RNA sequences into functional proteins is dependent on the correct coupling of amino acids with tRNAs. In collaboration with researchers at Yale University, a family of tRNAs in Streptomyces with non-canonical amino acid coupling were characterized. The alternative tRNAs are associated with the mistranslation of Pro in place of either Thr, Ala, or Asn. The alternative tRNAs are present throughout the genus including in multiple lineages of phytopathogenic Streptomyces. Expression of the alternative tRNAs is associated with improved response to some specific acute stresses.
The xylem-infecting fungal pathogen Verticillium produces a small protein of unknown function within infected plants. To determine the function of the plant encoded gene, transgenic overexpression was developed in potato plants. A striking phenotype was observed, where stem internodal tuber-like structures formed, and plant height was greatly reduced. An apparent shift in metabolite partitioning occurred with reduced below-ground tuber formation and development of aerial tubers on shortened stems, suggesting that the pathogen can shift metabolite accumulation to regions it is colonizing. This addresses potential gene function and provides new insight into altering potato plant development.
Melatonin is produced in plants and has been reported to play multiple roles in plant development and resistance to biotic and abiotic stresses, mainly indirectly through alteration of other hormone pathways. Initial studies with foliar applications of melatonin to potato revealed that certain varieties responded with altered tuber morphology, exhibiting multiple secondary tuber initiation sites. Manipulation of one of the melatonin biosynthetic genes in transgenic potato also resulted in extensive secondary tuber growth. Considering that some abiotic stresses can result in secondary tuber initiation, it may be possible to alter the melatonin levels to overcome the secondary tuber formation.
In winter of 2022/23, the new potato breeder successfully generated true potato seed at the greenhouse in Orono, Maine. Approximately 250 different crosses were successfully made, totaling more than 65,000 seeds. ~30,000 of these seeds are currently being germinated and seedlings will be grown out to supply the 2023 field season with minitubers. These crosses include tetraploid clones in the table and chipping market classes, as well as diploid clones.
Potato field work has continued to increase in size. Compared with the previous year, breeding program year 1 plots increased 12.5%. The number of breeding program year 2 entries increased by 28%. The number of breeding program year 3 entries increased by 593% increase. In addition to increases in the breeding program, conservation of historical varieties increased with the addition of 96 new clones.
Project summary of past 60 months:
Novel approaches for disease management of Anthracnose (Colletotrichum) of pepper and tomato, Early Blight (Alternaria) of potato, and Common Scab (Streptomyces) of potato were discovered and developed. For tomato Anthracnose, multiple QTLs associated with disease resistance were discovered which enabled the development of molecular markers for more rapid breeding of Colletotrichum resistance. Additionally, a steroidal glycoalkaloid biosynthetic pathway was identified in an Anthracnose-resistant tomato genotype and shown to be essential for disease resistance, suggesting that manipulation of that pathway could confer resistance in susceptible tomato cultivars. For potato Early Blight, multiple plant stress-related genes were identified that confer resistance to Alternaria infection when overexpressed in plants providing knowledge of genetic loci than can increase Early Blight resistance. Related, novel Alternaria toxins responsible for plant cell death were discovered providing additional targets for research on Early Blight disease management. Finally, several fungal endophytes of potato were discovered that increase plant resistance to Alternaria and have the potential to be used as biofungicides to improve disease resistance. For potato Common Scab, the diversity of the pathogen across the United States was further characterized including the identification of several novel species. Research also confirmed that distinct Streptomyces species are differentially virulent and that there are significant strain-by-cultivar interactions, emphasizing the need for knowledge about the local population of phytopathogenic Streptomyces. A molecular diagnostic assay was developed for field use for prediction of Common Scab disease severity to provide a tool to help growers make informed planting decisions. Finally, the efficacy of low-dose 2,4-D treatment for managing common scab was confirmed to be effective across multiple white potato cultivars. Other agronomic traits for potato, pepper, and tomato were also improved in the past 60 months of this project. For potato, important traits such as heat tolerance and resistance to biotic stress were identified in breeding lines and wild relatives of potato and specific genetic loci identified. The wild relatives of potato were shown to have high heterozygosity, suggesting that they can be useful tools for exploiting heterosis. In pepper, snack pepper lines with reduced seeds were developed to improve consumer demand for snack peppers. Additionally, ozone treatment of pepper seeds was developed as an efficacious and cost-effective tool for control of viral diseases. For tomato, novel cherry tomato lines were developed with multiple improved agronomic traits and will be a valuable germplasm resource for breeders developing the next generation of tomato cultivars.
Accomplishments
1. Novel cherry tomato lines developed. Tomato flavor and nutritive value are important determinants of consumer acceptance. ARS scientists in Beltsville, Maryland, completed development of novel cherry tomato lines with compact habit that are productive, do not require staking and are adapted to ground or raised bed culture. Two of these lines carry the recessive y locus for pink colored fruit and accumulate high concentrations of anthocyanin in fruit peel, resulting in purple-pigmented ripe fruit. Fruit are flavorful and provides nutritive benefits of both lycopene and anthocyanin. The third line produces red cocktail-size cherry tomatoes with superior flavor and crack resistance. These lines will be released for use as public cultivars and as a germplasm source of novel characters for use by commercial breeders.
2. Genomic epidemiology reveals that maintenance of pathogen-free potato seed stock effectively curtails common scab transmission. Strategies to control the spread of potato common scab disease among grower fields and selection of cultivars with suitable disease resistance has been hindered by poor knowledge of pathogen diversity and transmission. ARS scientists in Beltsville, Maryland, in collaboration with Oregon State University, classified the primary virulence determinants of Streptomcyes, the causative agents of potato common scab disease, into multiple types and subtypes. Novel subtypes of known virulence factors were newly identified showcasing the flexibility of Streptomyces to express key plant phytotoxins. Contrary to long-held dogma, further analysis demonstrated that horizontal gene transfer of the key virulence gene clusters is much less common than previously presumed. Genomic epidemiology revealed that physical transmission of the pathogen between grower sites is rare but was common until about 100 years ago. This result suggests that the implementation of certified disease-free seed potatoes, which started in the United States in the 1920s, has been largely effective at curtailing spatial transmission of common scab pathogens. These fundamental research findings describing pathogen diversity and host-pathogen interactions will help growers make informed planting decisions based on resistance against the local populations of the pathogen and guide plant breeders in development of disease resistant cultivars.
3. Discovery of cell death inducing components of Alternaria infections. Although one of the most economically important diseases of potato and tomato, development of host resistance remains difficult due to a lack of knowledge on host-pathogen interactions. The fungus is thought to kill plant cells through production of toxic compounds. ARS scientists in Beltsville, Maryland, have identified two specific chemical toxins and one protein produced by Alternaria that account for the cell death. Our discovery that multiple fungal products are responsible for cell death and characterization of these products will allow scientists active in breeding to develop targeted approaches for screening plant germplasm with durable multicomponent resistance to Alternaria.
4. Discovery of a new biofungicide for Alternaria management. Current management practices for early blight of potato and tomato, caused by the fungus Alternaria solani, relies on fungicide applications. Existing synthetic fungicides cannot be used in organic production, and A. solani is developing resistance to these fungicides. During a study of endophytic fungi from potato and other plants a fungus was identified that could produce a compound highly inhibitory to Alternaria. The highly water soluble, non-enzymatic compound is readily produced in simple liquid medium and can be diluted and applied directly to plant leaves. Control of Alternaria was as good as existing organic treatments, with the advantage of being able to be introduced along with other organic methods and with fungicides used in conventional grower applications. This provides an immediate application benefit that can be used by potato and tomato growers for early blight management.
Review Publications
Jones, R.W., Perez, F.G. 2022. Differential plant response to toxins and elicitor proteins released by Alternaria solani. Journal of Plant Pathology. https://doi.org/10.1007/s42161-022-01286-w.
Fabian, M.L., Zhang, C., Sun, J., Price, N.P., Chen, P., Clarke, C.R., Jones, R.W., Stommel, J.R. 2023. Steroidal glycoalkaloids contribute to anthracnose resistance in solanum lycopersicum. Journal of Experimental Botany. 74(12):3700-3713. https://doi.org/10.1093/jxb/erad108.
Stommel, J.R., Dougherty, L.E., Collins, P.J., Wien, C., Uva, R. 2023. Plant type and antidesiccants influence longevity of cut pepper stems for floriculture applications. HortTechnology. 33:215-224. https://doi.org/10.21273/HORTTECH05144-22.
Schuntermann, D., Fischer, J., Bile, J., Gaier, S., Shelley, B., Jahn, M., Hoffman, K., Westhof, E., Soll, D., Clarke, C.R., Vargas-Rodriguez, O. 2023. Mistranslation of the genetic code by a new family of bacterial transfer RNAs. Journal of Biological Chemistry. https://doi.org/10.1016/j.jbc.2023.104852.
Nguyen, H., Mowery, J.D., Clarke, C.R. 2022. Description of Streptomyces griseiscabiei sp. nov. and reassignment of Streptomyces sp. strain NRRL B-16521 to Streptomyces acidiscabies. International Journal of Systematic and Evolutionary Microbiology. https://doi.org/10.1099/ijsem.0.005574.
Collins, P.J., Tan, R., Wen, Z., Boyse, J., Chilvers, M.I., Wang, D. 2022. Genetic mapping of host resistance to soybean sudden death syndrome. Crop Science. https://doi.org/10.1002/csc2.20689.
