Location: Genetic Improvement for Fruits & Vegetables Laboratory
2019 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
Diverse Colletotrichum species are responsible for the fungal disease anthracnose that reduces marketable yield of tomato and pepper fruit. In support of Objective 3, Genotype-by-sequencing (GBS) analysis of tomato recombinant inbred lines susceptible and resistant to the Colletotrichum fungus has been completed. We have identified two chromosomal regions associated with host plant response to infection by Colletotrichum. Fine mapping of these chromosomal regions has been initiated. The research is collaborative with West Virginia State University. Tomato transformants have been generated to evaluate putative candidate genes that we have identified which may condition resistance in tomato. Additional research has been initiated to characterize fungal isolates that differentially cause immature and/or mature pepper fruit rot. Whole genome sequencing of differential isolates has been 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, prior years research identified pepper accessions that exhibited varying levels of resistance to TCSV. A field screen for replicated evaluation of promising accessions was planned for the fall/winter production season in Florida, but was postponed due to the extended government shutdown. Plans are in place to resume tests this winter.
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 multi-location trials of advanced breeding lines for the conventional culinary and specialty markets. The inheritance of low seed count in pepper was characterized and is being leveraged to develop and release snack pepper breeding lines. Renewed interest by commercial growers has enabled similar trials of high-value ornamental peppers for use as cut stems. Advanced breeding lines were transferred to four commercial growers who expressed interest in product development. Development of pepper populations segregating for fresh-cut quality attributes is ongoing. Summer trials are designed to assess combining ability of promising lines. Breeding lines being developed from these populations will be valuable for development of improved cultivars for minimally processed fresh-cut market applications.
Our previous work, assessing possible potato resistance mechanisms for use in limiting early blight, identified cell death genes as potential candidates. Further studies have now identified a fungal toxin produced during infection that results in cell death. Only one toxin caused cell death, out of a panel of potential pathogen-produced toxins. We are now testing our bio-engineered cell death resistant potato plants, along with other potato germplasm, to determine how resistant they are to the toxin. This may allow direct screening through use of the toxin.
Cell wall specific genes of Phytophthora infestans are being targeted through use of dsRNA constructs expressed in a bacterial system, and through bioengineered plants. The dsRNA constructs produced from crude bacterial preps caused a reduced growth of Phytophthora in culture. Production in plants is being assessed to determine the optimal production methods. Bacterial production may be useful as a surface protectant, while bioengineered plants may offer more long term disease control.
Several experimental techniques to study the molecular details of infection of potato by Streptomyces scabiei were optimized in FY19. These include: greenhouse and growth chamber quantitative infection assays, field trials, molecular cloning, qPCR, toxin purification and quantification, potato tissue culture, and molecular plant defense response assays. These optimized assays were used to address several critical questions related to common scab disease of potato: A two-year of a field trial testing the ability of extremely low-dose 2,4-d treatment to control common scab disease was completed. This work confirmed that low-dose 2,4d is an efficacious control for common scab of white potatoes in field growth conditions (see Accomplishments section). Field trials to optimize the timing of the treatment are on-going.
Cultivar resistance to common scab disease of potato is dependent on the pathogen population. Common scab disease symptoms are predominantly a result of a single toxin produced by the pathogenic agent. Therefore, the current paradigm in common scab pathology is that a potato cultivar resistant to one species of the common scab pathogen should be resistant to all species. However, cultivar resistance to common scab disease identified in one breeding program is often not durable when tested in other potato breeding programs across the United States. Over 50 potato cultivars were infected with three distinct species of the common scab pathogen and cultivars that are resistant or susceptible to all three species and cultivars that have varying resistance dependent on the pathogen species were identified. This result showcases several cultivars of potato that are expected to be resistant to the majority of common scab populations but also highlights that many potato cultivars are resistant to only specific species of the pathogen. Furthermore, these results emphasize that it is critical for extension specialists and growers to consider their local population of the common scab pathogen when determining which cultivars to plant for common scab resistance.
Several experiments were optimized to rapidly quantify plant cell death elicited by the primary phytotoxin associated with common scab. These experiments led to the identification of multiple genetic pathways critical for the elicitation of cell death and the identification of chemical stimulants that attenuate the severity of the toxin response in plants which may lead to novel common scab disease control.
An emerging species of the common scab pathogen not previously confirmed in the United States was identified in the ARS Streptomyces strain collection. Several isolates of this species were confirmed to be pathogenic on multiple potato cultivars. In general, strains of the emerging pathogenic species manifested less severe disease symptoms than the predominant species of pathogenic Streptomyces prevalent in the United States. Full genome sequences were acquired for five isolates of the emerging species and comparative genomics experiments are on-going to understand why these strains are less pathogenic than the Streptomyces pathogens already established in the United States.
Tissue culture plantlets of 31 diverse cultivars of potato (including tetraploids and diploids) have either been obtained from the potato Genbank or placed into tissue culture from tuber shoot tissue and established contamination free in the tissue culture collection. An improved growth medium has been developed to support the growth of all the cultivars in tissue culture including the challenging-to-grow diploids. Assays to determine sensitivity of all cultivars in the collection to the phytotoxin thaxtomin were developed. Additionally, plant infection assays of tissue culture plantlets with living pathogen spores were developed. Both of these assays are being used to screen all 31 potato cultivars in the collection, with the screen approximately 40% complete.
Several non-pathogenic Streptomyces isolates that were collected from potato tuber surfaces have been characterized for potential biocontrol activity. The chitinase activity of more than 70 non-pathogenic Streptomyces strains has been quantified. Several strains with extremely high chitinase activity, which is expected to enable degradation of the cell walls of nematodes and fungal pathogens of potato, have been identified. 20 of these strains have been tested for the ability to inhibit potato root knot nematode. 30 of these strains have been tested for the ability to inhibit the potato rot pathogen Pythium ultimum. 12 of these strains have been tested for the ability to inhibit the potato tuber surface pathogen Helminthosporium solani. Non-pathogenic Streptomyces strains that have great potential as biocontrol agents based on their ability to kill the target pathogen and thrive in soil in close association with tubers have been identified for all three of these problematic pests of potato.
Accomplishments
1. Reduced seed count in pepper improves versatility for specialty markets. Small/miniature sweet and hot peppers such as snack peppers are a rapidly growing specialty market. Low seed count is an important attribute for consumer acceptance of small-fruited specialty peppers. ARS scientists in Beltsville, Maryland, identified novel small fruited breeding lines with quality attributes suitable for the snack pepper market and determined the inheritance of low seed count in this material. Our data support the presence of a single gene together with minor genes that condition the low seed count attribute. Simple inheritance of this trait will facilitate breeding of improved specialty pepper varieties with low seed content valued by consumers. This research will benefit plant breeders developing new pepper varieties and ultimately, consumers with availability of new high quality specialty peppers.
2. Identification of stress-related genes providing protection from a potato disease. Plants exposed to stress from infection Alternaria, one of the most important disease-causing fungi on potato plants, can undergo localized cell death, which aids Alternaria infections. Assessment of a series of potato genes that counter stress-induced cell death identified two candidate genes that may be involved in limiting disease. Testing of these genes through elevated expression in cis-genic potato plants identified a cell-death inhibitor gene that substantially reduced infection by Alternaria. Selection of breeding lines with enhanced stress tolerance, based on reduced cell death, may provide a new direction for Alternaria resistance development in potato.
3. Regulator of elevated gene expression increases resistance to a potato disease. Plants have numerous genes that show elevated expression after infection by disease-causing fungi. This response may occur too late after infection, allowing the pathogen to overwhelm plant defenses. ARS scientists in Beltsville, Maryland, previously identified a regulator gene that is activated upon infection by the major potato disease-causing organism Phytophthora. Overexpression in potato plants resulted in improved resistance to Phytophthora. This discovery indicates that plants may be selected for high levels of expression of this regulator to limit disease.
4. Low-dose potato herbicide substantially increases the marketable yield of field-grown potatoes. This project tested the efficacy of a low dose herbicide for seven cultivars of white potatoes grown primarily on the east coast. Two years of field trials were performed at ARS-operated Aroostock Farm in Presque Isle, Maine. One-year field trial was performed in Scaramento, Pennsylvania, in collaboration with Sterman Masser Inc. Across all of the tested potato cultivars, treatment with 2,4d led to a 25% increase in the marketable yield of potatoes due to substantial suppression of disease lesion formation. Additionally, this project was the first to confirm that the new lower volatility amine formulation of 2,4d is an effective control of common scab when sprayed at low dose. Results are relevant to growers for reduction of common scab in commercial production acreage.
Review Publications
Jones, R.W. 2019. Plant vascular system-feeding Psyllidae (Hemiptera) and Nematoda genomes encode family 12 glycosyl hydrolases. The Canadian Entomologist. 151(3):291-297. https://doi.org/10.4039/tce.2019.11.
Jones, R.W., Perez, F.G. 2019. Assessing possible mechanisms of resistance to early blight caused by Alternaria solani. Potato Research. https://doi.org/10.1007/s11540-019-9420-9.
Novak, N.G., Perez, F.G., Jones, R.W., Lawrence, S.D. 2019. Detached leaf assays: A simplified approach to study gene expression in potato during infestation by the chewing insect Manduca sexta. Journal of Visualized Experiments. 147:e59153.
Clarke, C.R., Kramer, C., Kramer, M.H. 2019. Cultivar resistance to common scab disease of potato is dependent on the pathogen species. Phytopathology. https://doi.org/10.1094/PHYTO-09-18-0368-R.
Stommel, J.R. 2019. Reduced seed count improves versatility and propagation of small-fruited peppers (Capsicum annuum L.) for specialty markets. HortScience. 54(4):652-655. https://doi.org/10.21273/HORTSCI13602-18.
Xiao, Z., Rausch, S.R., Luo, Y., Sun, J., Yu, L., Wang, Q., Chen, P., Yu, L., Stommel, J.R. 2018. Microgreens of Brassicaceae: Genetic diversity of phytochemical concentrations and antioxidant capacities. LWT - Food Science and Technology. 101:731-737.
Schmitz Carley, C.A., Coombs, J.J., Clough, M.E., DeJong, W.S., Douches, D.S., Haynes, K.G., Higgins, C.R., Holm, D.G., Miller, J.C., Navarro, F.M., Novy, R.G., Palta, J.P., Parish, D.L., Porter, G.A., Sathuvalli, V.R., Thompson, A.L., Zotarelli, L., Yencho, G.C., Endelman, J.B. 2018. Genetic covariance of environments in the potato national chip processing trial. Crop Science. 58:1-8.
Lawrence, S.D., Novak, N.G., Perez, F.G., Jones, R.W. 2019. Over expression of the Q-type ZFP StZFP2 in potato increases resistance to potato late blight (Phytophthora infestans) infection. Journal of Plant Interactions. 14(1):129-136. https://doi.org/10.1080/17429145.2018.1562109.
Guedes, M.L., Haynes, K.G., Vinyard, B.T., Pinto, C.A. 2019. Heat tolerance in diploid wild potato species. American Journal of Potato Research. 96:294-302. https://doi.org/10.1007/s12230-019-09716-9.
