Location: Crop Improvement and Protection Research
2022 Annual Report
Objectives
This project will create new ways to manage plant diseases of strawberries and vegetables. New approaches to disease management are needed because many preferred crop varieties are susceptible to common diseases. To develop effective controls, we need to increase knowledge of pathogen disease cycles as well as pathogen and plant genomes. This information will enable new ways to detect pathogens and breed disease resistant crops. In addition, we need to know more about microbes in soil and on plants that suppress disease. Our goal is to fill these research needs. Overall, this project will lead to disease control methods that do not use fungicides or fumigants. We will focus on these following major objectives and subobjectives during the next five years.
Objective 1: Integrate understanding of pathogen genomes into new tools for pathogen diagnostics.
Sub-objective 1.A: Develop molecular markers for diagnostic assay development for formae speciales of Fusarium oxysporum by analysis of genomic sequence data.
Sub-objective 1.B: Develop diagnostic markers and tools for rapid identification of oomycetes using comparative analysis of mitochondrial genomes.
Sub-objective 1.C: Determine if strawberry-pathogenic isolates of Macrophomina phaseolina are polyphyletic within the context of the broad diversity of this species.
Objective 2: Gain a better understanding of strawberry and vegetable pathogen life cycles and how microbial communities can be used for new management strategies.
Sub-objective 2.A: Identify genes required for microsclerotia formation in Verticillium dahliae.
Sub-objective 2.B: Functionally characterize the role of a predicted pathogenicity chromosome in F. oxysporum f. sp. fragariae on the strawberry host-specific pathogenicity phenotype.
Sub-objective 2.C: Investigate the role of oospores in the life cycle of Peronospora effusa.
Sub-objective 2.D: Determine if genetically diverse Macrophomina phaseolina isolates from strawberry exhibit differential virulence on tolerant strawberry varieties.
Sub-objective 2.E: Identify the role of the strawberry root microbiome in Verticillium wilt of strawberry.
Objective 3: Integrate management strategies for improved control of strawberry and vegetable pathogens.
Sub-objective 3.A: Determine if an avirulence gene identified in race 1 strains of F.o. fragariae is the avirulence gene specific for FW1-mediated resistance (called AvrFW1).
Sub-objective 3.B: Characterize gene expression in spinach-downy mildew interactions in response to two races of Peronospora effusa.
Sub-objective 3.C.1: Identify the effect of biofumigant green manure soil amendment type and application rate on soil microbiome function and soilborne pathogen inoculum survival.
Sub-objective 3.C.2: Identify the effect of pre-plant application of antimicrobial producing Streptomyces bacteria on Fusarium wilt diseases of vegetables.
Sub-objective 3.D: Evaluate nitrogen dioxide (NO2) as a seed treatment for two pathogens carried on spinach seeds.
Approach
Sub-objective 1.A: Unique sequences suitable as targets for developing taxon-specific markers will be identified in genomic sequence data.
Sub-objective 1.B: Mitochondrial genomes will be used for selection of loci for development of diagnostic markers.
Sub-objective 1.C: Phylogenies will reveal if strawberry-pathogenic strains of M. phaseolina are polyphyletic within the broader diversity of this species.
Sub-objective 2.A: Comparative analyses of genomes of Verticillium species will reveal sequences required for microsclerotia formation.
Sub-objective 2.B: The loss of the chormosome will be induced and/or transferred to a non-pathogenic strain, and disease examined.
Sub-objective 2.C: Oospores of P. effusa will be examined for their ability to infect roots or leaves in growth chambers and humidity tents.
Sub-objective 2.D: A broad diversity of isolates will be tested for pathogenicity on 4 tolerant and 2 susceptible varieties.
Sub-objective 2.E: We will assess if microbial taxa in the root microbiome that correlates with pathogen biomass in the roots or disease severity significantly affects Verticillium wilt of strawberry.
Sub-objective 3.A: Quantitative trait loci (QTL) and genome-wide association studies (GWAS) to map genomic regions associated with resistance.
Sub-objective 3.B: RNASeq analysis of gene expression will be performed.
Sub-objective 3.C.1: Five weed or crop species will be tested against Verticillium dahliae as biofumigants.
Sub-objective 3.C.2: Biological control will be tested by applying Streptomyces spores to field soil as pre-plant treatments for control of Fusarium wilt.
Sub-objective 3.D: Varying percent of NO2 will be applied in chambers with seeds harboring two pathogens.
Progress Report
This report documents progress for project 2038-22000-019-000D, Disease Management and Improved Detection Systems for Control of Pathogens of Vegetables and Strawberries, which started in March 2022 and continues research from project 2038-22000-016-000D, Management of Pathogens for Strawberry and Vegetable Production Systems.
Progress was made in support of Objective 1. Relating to Sub-objective 1.A, additional genomes of Fusarium oxysporum were prepared for genomic sequencing. For Sub-objective 1.B, mitochondrial diagnostic markers for detection of Peronosclerospora spp. were developed. In support of Sub-objective 1.C, more than 400 isolates of Macrophomina spp. were collected from 24 countries and 90 host plant species. High quality DNA was extracted from all of these and submitted for high throughput sequencing. Additional isolates are being collected.
Progress was also made in support of Objective 2. Relating to Sub-objective 2.A, comparative genomics analyses were initiated with Verticillium dahliae and Verticillium alfalfae, Verticillium species that produce or do not produce microsclerotia, respectively. In support of Sub-objective 2.B, protocols were developed for chromosome deletions and transfers. Leaves with Peronospora effusa oospores were collected in support of Sub-objective 2.C. Under Sub-objective 2.D, pathogenicity assays were conducted. Strawberry bare-root transplants were obtained for analyzing the effect of Verticillium dahliae disease pressure on the strawberry root microbiome related to Sub-objective 2.E.
Finally, progress was also made on Objective 3. A pathogenicity assay was conducted with seedlings segregating for the Fusarium resistance gene in support of Sub-objective 3.A. Under Sub-objective 3.B, RNA-Sequencing was performed on two near isogenic lines of spinach and spinach cultivar Viroflay. Related to Sub-objective 3.C.1, generation of Verticillium dahliae microsclerotia for soil infestation was initiated. In support of Sub-objective 3.C.2, soil was sampled for isolation and characterization of Streptomyces bacteria for management of fusarium wilt of lettuce. Evaluations of 23 commercial spinach seed lots for oospores of P. effusa and microsclerotia of V. dahliae were initiated in Sub-objective 3.D.
Accomplishments
1. High quality DNA extracted from a wide diversity of Macrophomina spp. isolates. Macrophomina is a fungal genus that was long thought to contain a single species, M. phaseolina, which causes devastating disease on a wide variety of crops. Despite the economic importance of this pathogen, the genomic diversity remains relatively unexplored. As a prerequisite to an investigation of genomic diversity, an ARS scientist in Salinas, California, established international collaborations and collected Macrophomina spp. isolates from 24 countries and 90 host plant species. High quality DNA was extracted from all of these and the DNAs submitted for high-throughput sequencing. Building this collection is a major step that will enable impactful descriptions of the global diversity of this genus. Knowledge of the global diversity is useful to growers and plant breeders that aim to limit the pathogens from this genus on a wide variety of crops.
