Research Project: Potato and Other Solanaceous Crop Improvement and Disease Management

Location: Genetic Improvement for Fruits & Vegetables Laboratory

2020 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
Colletotrichum species are responsible for the fungal disease anthracnose that reduces marketable yield of tomato and pepper fruit. In support of Objective 3, a high-density SNP-based tomato linkage map was developed using recombinant inbred lines susceptible and resistant to the Colletotrichum fungus. We have validated five genes using qRT PCR after integrating 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. Whole genome sequencing of differential isolates that infect immature and/or mature pepper fruit is completed and transcriptome profiling is underway 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 relatively new to the U.S. and now established in Florida, a major production area for both tomato and pepper. In support of Objective 4, we completed a second round of screening of Capsicum chinese pepper accessions that exhibited varying levels of resistance to TCSV. 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. While we were able to support trials off-site, evaluations at Beltville Agricultural Research Center (BARC) were limited to pot evaluations in the summer of 2020 due to restrictions on establishing transplants for field trials. With cooperator support, evaluations of advanced pepper lines under simulated commercial use conditions were completed. Growth of potato in tissue culture was further optimized. Apical cuttings were determined to give much more consistent growth and were, therefore, determined to be better for screening the effects of thaxtomin against in vitro potato plantlets. The apical cutting plantlets were used to redo the thaxtomin sensitivity screen for 31 cultivars of potato in the tissue culture collection of ARS Beltsville, Maryland. Additionally, spores of Streptomyces directly applied to in vitro potato plantlets were determined to lead to stunted potato growth. Screening all 31 cultivars of potato using the spore assays is currently underway and expected to correlate better with actual common scab resistance than the thaxtomin screening assay. Accurate and exhaustive characterization of the species of pathogenic Streptomyces present in any region is critical for disease management. 150 Streptomyces strains in the ARS culture collection were recently sequenced. In collaboration with Oregon State University, scientists at ARS Beltsville, Maryland, have determined the phylogenetic relationship of these strains. Multiple novel lineages of Streptomyces were identified. A novel species of phytopathogenic Streptomyces was characterized (see Accomplishments section). Within this novel species group, strains that are aggressive pathogens were very closely related to strains that were only weakly virulent or even avirulent on potato. Within the genomes, there were no polymorphisms in known virulence genes between the highly virulent and the avirulent strains suggesting that the differential phenotypes are due to unknown virulence genes. A formal description of the species is being developed to validly name and publish this novel phytopathogenic Streptomyces. Low-dose treatment of the herbicide 2,4-D was recently shown by scientists at ARS Beltsville, Maryland, to provide some protection against common scab in multiple cultivars of potato in production in the Eastern United States. The mode-of-action and optimal treatment methodology are currently unknown. A first-year field trial designed to optimize the timing of 2,4-D treatment was completed in collaboration with Penn State University. The results indicated that the timing of 2,4-D treatment isn’t a critical factor, but the efficacy of control remains variable site-to-site and year-to-year. A transcriptomics project identifying differentially regulated genes in potato following 2,4-D treatment is currently underway to improve understanding of the mode-of-action. The cultivar RH89, which has a publicly available genome sequence, was confirmed sensitive to common scab and therefore valuable for this project. While the economic impact of common scab is largest on potato, several specialty tuber and root crops are affected by this disease. The primary virulence determinant for Streptomyces to cause disease on potato is the phytotoxin thaxtomin. The virulence determinants that lead to common scab on other root crops are unknown. Scientists at ARS Beltsville, Maryland, determined that radish, turnip, beet, and carrot are all sensitive to the phytotoxin thaxtomin. Streptomyces mutants defective in the production of thaxtomin were unable to elicit common scab symptoms on any tested plant. Therefore, the same phytotoxin primarily responsible for common scab of potato is also responsible for common scab on these other crops. Management strategies that limit common scab on these other crops may also be applicable to potato. Non-pathogenic Streptomyces strains in the ARS Streptomyces culture collection are promising potential biocontrol agents against pests of potato due to their ability to thrive in 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, scientist at ARS Beltsville, Maryland, have identified multiple Streptomyces strains with antagonistic activity against several pests of potato. The level of chitinase activity has been quantified for 85 Streptomyces strains. Chitinase activity is expected to correlate with biocontrol activity against multiple pests of potato due to the presence of chitin in the cell walls of fungi and nematodes. Twenty Streptomyces strains have been screened for activity against potato root-knot nematodes with two strains having strong inhibitory activity in a soil-based plant galling assay. Eighty-two Streptomyces have been screened for activity against Pythium leak in vitro with three strains having strong inhibitory activity. Two of these three strains also have inhibitory activity against Pythium leak on harvested potatoes. Late blight, caused by Phytophthora infestans, and early blight, caused principally by Alternaria solani, generate the majority of economic losses in potato production. Unique strategies are being developed to manage these diseases in an economical and environmentally benign manner. Phytophtora infestans is being targeted through RNAi mediated suppression of a critical cell wall protein. The protein has a highly conserved region that is found amongst all Phytophthora species. Bacterial expression of the RNAi has been developed using a double T7 promoter. Induction of the T7 promoter allows forward and reverse copies of the RNA to be made. The dsRNA shows activity in crude sonicates of the bacterial cells, demonstrating a low-cost source for application to foliage. Testing of foliar treatments is underway. Alternatively, we have prepared constructs containing an antisense copy of the target region for developmental testing in transgenic potato plants. Introduction of the construct into Kennebec and Bintje potato is underway. Cause of potato early blight cell death identified. Early blight of potato, caused by the fungus Alternaria, is often the most important problem in potato production. There are no known resistance genes that can be used in breeding efforts to control early blight, therefore, fungicides are the only current method of control. Potato foliage is killed after infection with the early blight fungus, which then feeds on the dying leaf tissue. In a study of proteins secreted by Alternaria, ARS scientists in Beltsville, Maryland, found that certain proteins result in death of the leaf tissue. The death result from the potato recognizing the protein and then attempting to kill the invading pathogen. This reaction aids the infection by Alternaria, which colonizes the dying tissue and produces more of the cell death eliciting protein. Future identification of the potato genes that recognize the cell death eliciting proteins may allow for development of potatoes lacking the recognition and subsequent cell death, resulting in potatoes resistant to Alternaria. Alternaria solani is being targeted through gene disruption studies where individual necrosis eliciting proteins are being removed. Selection is based in part on fungal gene expression patterns, and on presence/absence genotyping of pathogenic and saprophytic isolates of Alternaria. The corresponding receptor genes in the potato are being tested through Agrobacterium-mediated overexpression to determine if they become increasingly sensitive to the elicitor proteins. Recent reports suggest that Alternaria species can inhabit plants in an endophytic manner. Studies have been initiated to determine the endophyte population of potato varieties and wild germplasm. Changes in endophyte populations are also being examined relative to clonal generation, fungicide treatments and plant age. This may eventually lead to manipulation of potato endophyte populations to manage various diseases.

Accomplishments
1. Resistance genes identified in tomato for anthracnose fruit rot. Anthracnose fruit rot causes major crop losses for tomato and pepper growers. ARS scientists in Beltsville, Maryland, working in collaboration with scientists at West Virginia State University, used a high-density tomato genetic linkage map to develop breeding lines of tomato resistant to the fungus that causes fruit rot. The scientists identified five genes that offer fruit rot resistance to tomatoes. These new genetic lines will help breeders transfer fruit rot resistance to advanced tomato breeds. The same methods can be used to provide resistance genes in pepper and other related crops, thus enhancing marketable yield and reducing cost and environmental concerns related to conventional chemical-based crop disease management.

2. Novel Streptomyces bacteria identified that causes potato common scab. Through genome sequencing of bacterial strains in the ARS Streptomyces culture collection, ARS scientists in Beltsville, Maryland, identified a novel Streptomyces species that included pathogenic strains that cause common scab disease of potato and non-pathogenic strains. As expected, non-pathogenic strains of this novel species did not produce the plant toxin thaxtomin, which is considered necessary for common scab development. Unexpectedly, non-pathogenic strains of this species were also identified that produced toxin. This novel species confounds existing dogma for mechansims of common scab disease development and provides evidence that new disease-causing determinants elicit common scab. This novel species is valuable to researchers developing effective disease management strategies for potato.

Review Publications
Haynes, K.G., Zotarelli, L., Christensen, C., Walker, S. 2019. Early generation selection within a diploid hybrid solanum tuberosum groups phureja and stenotomum population for the intense yellow-flesh creamer potato market. HortScience. 54(12):2118–2124. https://doi.org/10.21273/HORTSCI13576-18.
Natarajan, P., Akinmoju, T.A., Nimmakayala, P., Lopez-Ortiz, C., Garcia-Lozano, M., Thompson, B.J., Stommel, J.R., Reddy, U.K. 2020. Integrated metabolomic and transcriptomic analysis to characterize cutin biosynthesis between low- and high-cutin genotypes of Capsicum chinense Jacq. International Journal of Molecular Sciences. Int. J. Mol. Sci. 2020, 21(4). https://doi.org/10.3390/ijms21041397.
Garcia-Lozano, M., Haynes, J., Lopez, C., Natarajan, P., Pena-Garcia, Y., Nimmakayala, P., Stommel, J.R., Alaparthi, S.B., Sirbu, C., Balagurusamy, N., Reddy, U.K. 2020. Effect of pepper-containing diets on the diversity and composition of gut microbiome of Drosophila melanogaster. PLoS One. Int. J. Mol. Sci. 2020, 21(3), 945. https://doi.org/10.3390/ijms21030945.
Ortiz, C.L., Garcia, Y.P., Natarajan, P., Bhandari, M., Abburi, V., Dutta, S., Yadav, L., Stommel, J.R., Nimmakayala, P., Reddy, U. 2020. The ankyrin repeat gene family in Capsicum spp: Genome-wide survey, characterization and gene expression profile. Scientific Reports. Sci Rep 10, 4044 (2020). https://doi.org/10.1038/s41598-020-61057-4.
De Frias, A., Luo, Y., Zhou, B., Zhang, B., Ingram, D., Vorst, K., Brecht, J., Stommel, J.R. 2019. Effects of door opening pattern of an enclosed refrigerated display case on product temperature and energy consumption. Food Control. https://doi.org/10.1016/j.foodcont.2019.107044.
Clarke, C.R., Tegg, R.S., Thompson, H.K., Frederick, C., Haynes, K.G., Kramer, M.H., Wilson, C. 2020. Low-dose foliar treatments of the auxin analog 2,4-D reduce potato common scab and powdery scab for multiple potato cultivars and enhance root development. Crop Protection Journal. https://doi.org/10.1016/j.cropro.2020.105208.
Clarke, C.R., Park, S., Tuosto, R., Jia, X., Mcgough, A., Vanmullekom, J., Westwood, J.H. 2020. Multiple immunity-related genes control susceptibility of Arabidopsis thaliana to the parasitic weed Phelipanche aegyptiaca. PeerJ. https://doi.org/10.7717/peerj.9268.
Haynes, K.G., Qu, X. 2018. Three cycles of recurrent maternal half-sib selection continue to reduce foliar late blight in a diploid hybrid Solanum phureja-S. stenotomum population. American Journal of Potato Research. https://doi.org/10.1007/s12230-018-9681-x.
Fleisher, D.H., Haynes, K.G., Timlin, D.J. 2020. Cultivar coefficient stability and effects on yield projections in the SPUDSIM model. Agronomy Journal. 2020:1-16.
Christensen, C.T., Zotarelli, L., Haynes, K.G., Kelly, C.E. 2019. Solanum chacoense to Comparative evaluation of the effects of gibberellic acid concentrations on dormancy break in tubers of solanum chacoense. HortTechnology. https://doi.org/10.21273/HORTTECH04448-19.