Project : USDA ARS

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

Location: Genetic Improvement for Fruits & Vegetables Laboratory

2022 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
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 pepper in the U.S. 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. In collaboration with North Carolina State University, RNAi constructs targeting tospoviruses are being investigated in pepper as a novel means to confer TCSV resistance. Virus-induced gene silencing (VIGS) studies are in progress to identify efficacious constructs to be used as candidates in Agrobacterium-mediated plant transformation. 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 resumed in 2021. Evaluations of pepper populations developed for fresh-cut applications and specialty snack markets are underway. A new scientist to lead the Potato Variety Development Program for the Eastern U.S. 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, as well as increasing the field trial sizes which were greatly reduced in the old scientist’s absence. In winter of 2021/22, the new scientist successfully generated true potato seed at the greenhouse in Orono, Maine. Approximately 300 different crosses were successfully made, totaling more than 70,000 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 breeding program year 1 plots more than doubled to 28,000 entries. Year 2 entries increased by 20-fold (from 23 entries in 2021 to 447 entries in 2022). Additional achievements in the field include establishing a dripline irrigation system at the Chapman Research Farm in Chapman, Maine. This work is critical towards restoring the full capacity of the potato breeding program. Host plant responses to pathogen challenge result from activation and suppression of numerous genes. Change in gene expression is mediated mainly through transcriptional regulating proteins. We have developed transgenic potato lines overproducing and underproducing the potato transcriptional regulator StZFP2, which is responsive to late blight infections. While there were limited changes in susceptibility to late blight, the architecture of the plants was altered. Overexpression of StZFP2 resulted in dwarf, compact plants, while suppression resulted in enhanced plant height and extensive aerial stolon production. These changes support a previously unknown role for StZFP2 in regulating potato plant growth and provide new plant material to further investigate downstream genes involved in potato plant growth and stolon production. Plant infecting fungi secrete various proteins that can weaken the physical barrier of the plant cell wall. Additionally, they may release proteins that can suppress host responses. We are investigating two individual systems where a pair of proteins interact to recognize and activate host defense responses. The EIX1/EIX2 proteins recognize specific regions of cell-wall degrading enzymes released by pathogens such as Phytophthora. The Ve1/Ve2 proteins recognize a small protein from the pathogen Verticillium. Initial investigations on the sequences of each gene copy in various tetraploid potatoes reveal that some have internal stop codons or frameshifts that would result in a non-functional protein. We are currently identifying expression levels of the functional and non-functional copies to determine their relationship to pathogen recognition and resistance. This will provide molecular screens useful in future cultivar selection. Detection of common scab-causing Streptomyces in potato fields is a tool needed by potato growers to predict disease impact prior to making cultivar planting decisions. In collaboration with North Dakota State University, Pennsylvania State University, and Michigan State University, a molecular diagnostic assay for phytopathogenic Streptomyces was optimized, validated to work against five different target phytopathogenic species in controlled greenhouse settings, and validated to distinguish infested from non-infested soils in field sites providing an ex ante estimate of common scab disease impact prior to planting. In collaboration with Brigham Young University, the efficacy of five non-pathogenic Streptomyces strains to act as biocontrol agents against Helminthosporium solani (silver scurf) and Pythium ultimum (Pythium leak) were tested in a field trial. The strains had previously been shown by the research team to have antagonistic activity against these pathogens. None of the strains significantly limited impact of the disease in the field suggesting that the strains are either not viable in the field or the antagonistic activity failed to materialize in the field. The strains were shown in controlled greenhouse settings to be able to readily colonize the tuber surface, but field environmental conditions may limit growth or colonization. Improved genome assemblies of Streptomyces were developed in collaboration with Oregon State University. Eighteen Streptomyces strains, including the Type strains of all of the known phytopathogens, were selected for in-depth genome sequencing using both the Illumina and Minion platform. A hybrid assembly pipeline has been developed and generation of nearly finished or fully finished genome assemblies is in progress. These genomes will serve as valuable reference genomes for improved genomic analyses. Additionally, 40 more Streptomyces genomes were sequenced on the Illumina platform to add more genomic resources for predicting disease-associated genes. Transcriptome sequencing is a valuable tool for decoding the plant response to environmental stimuli. Scientists in GIFVL employed RNA-seq to profile the transcriptome of potato plants following low-dose treatments of the herbicide 2,4-D and tomato treatments of nitrogen. Low-dose 2,4-D treatments are a potential tool for management of the disease potato common scab, but inconsistency of the treatment limits the real-world efficacy of this potential tool. Through analysis of potato transcriptome response to 2,4-D, we identified several activated metabolic pathways that may help to explain the mechanism through which 2,4-D exposure limits common scab severity. Nitrogen treatments of tomato can limit the impact of the disease early blight. A transcriptome profiling approach was employed to dissect tomato pathways potentially associated with response to nitrogen and the pathogen, but so few differentially expressed genes were identified that the approach is not promising for continuation.

Accomplishments
1. Identification of fungal antimetabolites and their metabolic genes associated with tomato fruit resistance. Glycoalkaloid Metabolic Enzyme (GAME) gene expression is associated with tomato fungal antimetabolite accumulation and anthracnose resistance. Anthracnose caused by Colletotrichum species is a serious disease of Solanaceous crops worldwide where precipitation during the growing season is favorable to infection. Genetic resistance to anthracnose is desirable because cultural techniques including fungicide application provide incomplete protection. High levels of genetic resistance to anthracnose exist in wild or non-adapted tomato, but this resistance has been difficult to transfer using traditional breeding. ARS scientists in Beltsville, Maryland, identified (GAME) genes in tomato and determined that their expression is associated with fungal antimetabolite accumulation and anthracnose fruit rot resistance. The antimetabolites accumulated at levels sufficient to inhibit fungal fruit rot without detrimental effects on fruit safety. This fundamental discovery validates association of fungal antimetabolites and their metabolic genes with fruit rot resistance and provides new metabolic genes that breeders and biotechnologists can use to speed development of elite cultivars resistant to anthracnose fruit rot.

2. Discovery of regulatory gene QTL for anthracnose resistance. Discovery of regulatory genes associated with tomato fungal resistance. Identification and characterization of regulatory genes that influence expression of metabolic pathways is critical in developing targeted strategies to regulate pathways that influence crop attributes. In cooperation with scientists at West Virginia State University, ARS scientists in Beltsville, Maryland, identified genetic markers termed QTL associated with regulatory genes that may modulate the metabolic pathway responsible for synthesis of tomatine and its derivative compounds that exhibit antifungal activity in tomato. Two genetic techniques called Genotyping-By-Sequencing and Genome Wide Association Study were utilized to identify a QTL on chromosome 6 with large influence on fungal resistance and additional minor QTL on chromosomes 3, 5 and 10. Genes within these genetic markers encode regulatory regions for genes including a Cytochrome P450 protein, a bHLH transcription factor, an AP2/ERF transcription factor, and multiple disease resistance proteins. These QTL provide breeders with selectable markers in tomato for marker-assisted disease resistance breeding.

3. New yeast endophyte with antifungal biocontrol activity. Potato plants, like most other plants, have various fungi that live inside the plant. These fungi, part of what is referred to as the endobiome, can have beneficial effects on tolerance of potato plants to environmental and disease-induced stresses. Research conducted by ARS scientists in Beltsville, Maryland, provided the first understanding of the spectrum of fungi living inside potato plants. Endophyte fungal populations were limited to a few population structures in individual plants of a cultivar, with greater variations found between cultivars, indicating that the host genetic background is important in defining the host endobiome. Via transfer of fungal endophytes from disease resistant cultivars to susceptible cultivars, scientists identified a yeast with biocontrol properties that inhibits fungal disease-causing organisms in planta through natural processes. Knowledge of fungal endobiome diversity aids discovery of biocontrol organisms, including the novel yeast bioconotrol organism that we identified. The results are important to the scientific community and for commercial application in development of novel disease control products for organic production systems as well as conventional potato production.

4. A new diagnostic detection assay for the causative organism of potato common scab. Common scab is an endemic disease in many potato fields. The causative agent of the disease are Streptomyces bacteria that produce the phytotoxin Thaxtomin A. Detection of the Thaxtomin A biosynthetic operon, TxtAB, has been proposed as a molecular diagnostic tool for these phytopathogens. In collaboration with scientists at North Dakota State University, Pennsylvania State University, and Michigan State University, ARS scientists in Beltsville, Maryland, developed and validated a TxtAB qPCR detection assay for the pathogens in inoculated greenhouse pots and naturally infested field soil. In the greenhouse, the qPCR detection assay was able to detect the inoculum concentration of five distinct phytopathogenic Streptomyces species with high accuracy. In the field, the qPCR detection assay successfully distinguished between infested fields, in which common scab disease occurred on tubers, and non-infested soil, in which disease was rarely observed. These results provide validation for use of the TxtAB qPCR detection assay by research scientists and university extension personnel to detect disease-causing Streptomyces in field soil and support management of common scab in grower production fields.

U.S. DEPARTMENT OF AGRICULTURE

Genetic Improvement for Fruits & Vegetables Laboratory: Beltsville, MD