Verticillium dahliae is a fungal soilborne pathogen with a worldwide distribution. It is the main causal agent of Verticillium wilt, a vascular disease that affects about 400 different plant species. Because of the limited availability of resistant cultivars, disease management is performed through soil disinfestation with soil fumigants. In Northeast potato agroecosystems, chloropicrin and 1,3-dichloropropene are sometimes used to control Verticillium wilt and other soilborne pests. On the contrary, crop rotations with non-host species are a more sustainable and a more common management practice that is employed despite mixed results. The goal of crop rotations is to minimize inoculum build-up in soil by separating disease cycles as much as possible through time. It is crucial to select rotational crops that are both not susceptible to the disease and non-hosts of the pathogen. However, crop rotations have often rendered conflicting results in Verticillium wilt incidence, severity and soil inoculum density. Some studies have reported not only V. dahliae asymptomatically infecting weeds and rotational crops such as oat, barley, and mustard species, but the formation of microsclerotia (i.e. survival fungal structures that serve as inoculum) on the host. Previous work in our lab reported similar results from a case-study in a potato field located in Pennsylvania, where the fungus was isolated from asymptomatic oats being used as a rotational crop with potato (17).
Recent studies have highlighted the highly clonal structure of V. dahliae populations (22). In fact, each clonal lineage correlates with each of the Vegetative Compatibility Groups (VCGs) described in V. dahliae, regardless of host or geographical origin of the fungal isolates. In filamentous fungi, vegetative compatibility is the genetic ability of two isolates to undergo hyphal fusion and form a stable heterokaryon. Two fungal isolates of the same species are said to be vegetatively compatible when they share the same alleles at the het (heterokaryon incompatibility) loci. Traditionally, VCGs have been used as markers for population biology studies because they align with clonal lineages. Variations in pathogenicity and virulence have been observed in isolates from different VCGs. For instance, VCG 2A and 2B isolates are more aggressive (i.e. cause more disease) to mint, tomato and lettuce than isolates of VCG 4A or 4B (5, 27, 13). In the U.S., VCGs 2A, 2B, 4A and 4B are often associated with potato agroecosystems, with VCG 4A isolates being highly aggressive to all commercial potato cultivars. Little is known about V. dahliae as an endophyte (i.e. an organism that is able to infect a host without causing disease), what factors determine this interaction and how it affects disease management when using crop rotation. If VCG 4A isolates, highly aggressive to potato, are able to infect rotational crops as endophytes and produce microsclerotia, then crop rotations are not fulfilling the disease management goal. Consequently, this could seriously affect the dynamics of future epidemics by increasing levels of inoculum from highly aggressive VCGs, and increasing the incidence and severity of subsequent epidemics in potato crops.
Traditionally, genetic diversity studies in V. dahliae involves isolating the fungus from environmental samples; extracting the DNA and confirming species using molecular methods; and conducting tests to characterize the isolate’s VCG using international-reference tester strains. These methods which rely on culturing are tedious, labor-intensive, and limited by the slow growth of the fungus and the lack of selective nutritional media. Results may vary depending on the isolates as well as laboratory and experimental conditions. The highly clonal structure of the species and the correlation of lineages with VCGs make it ideal for the development of lineage- or VCG- specific PCR-based markers. The development of VCG-specific PCR-based markers will facilitate the study of the asymptomatic infections in rotational crops and weeds in potato fields for the purpose of helping growers make more informed management decisions.
The purpose of this project is to understand the VCG-specificity of the endophytic interactions of V. dahliae with asymptomatic rotational crops and weeds in potato fields. In this project, we will identify not only VCGs of the fungus that infect asymptomatic rotational crops, but also asymptomatic host species commonly grown in rotation with potatoes. This knowledge will provide growers and agriculture professionals a solid groundwork for designing more efficient and effective crop rotations for a sustainable management of Verticillium wilt of potato. In addition, the VCG-specific molecular tools generated in this project will also serve as a diagnostic tool for detection of V. dahliae VCGs infecting plant material, seed tubers and soil samples and a tool for the quantification of inoculum in infected/infested samples.
In this project, we will focus on Verticillium wilt of potato although concepts, rationale and results can be adapted to Verticillium wilts affecting other crops. In fact, the conceptual framework here described can potentially be applied to the management of other soilborne pathogens.
In this project, we will address the following hypothesis that is based on preliminary research conducted by our group and existing peer-reviewed literature: Lineages of V. dahliae as characterized by their VCG infect asymptomatic rotational crops and weeds in potato fields. These interactions allow the fungus to complete its life cycle, produce inoculum and shift the genetic diversity of V. dahliae populations present in the soil. The VCG diversity maintained by the asymptomatic hosts harbors potential to dictate the severity and incidence of the epidemic in the subsequent potato crops.
The main objectives of this project are to:
1. Develop VCG-specific PCR-based markers for V. dahliae VCGs 2A, 2B, 4A and 4B to be used for detection in environmental samples such as plant material and soil.
2. Identify which crop species used in rotation with potato are asymptomatically infected with V. dahliae and more specifically, which pathogen VCGs infect them.
Development of molecular detection protocols for main V. dahliae lineages. The first objective of this project is to develop VCG- or Lineage-specific markers for molecular detection of V. dahliae 2A, 2B, 4A and 4B . PCR-based markers for lineages 2A and 2B previously developed by other researchers are available and will be used in this project. PCR-based markers for lineages 4A and 4B will be developed and optimized in this project. Four isolates of V. dahliae that has been previously characterized for pathogenicity, VCG and genotype, were selected for genome sequencing and de-novo assembly: isolates T003 and S-55 from VCG 4A and, U073 and S-39 from VCG 4B. Spore and mycelia were harvested from single-spore cultures grown on PDA for 7 days. Two milliliters of sterile water were added to the surface of the plate and then, scrapped with sterile tools to release mycelia and spores. The supernatant was pipetted into 1.7 ml Eppendorf tubes and centrifuge at 12,000 rpm for 1.5 min. Liquid was poured out and tissue pellets in the tubes were lyophilized. DNA will be extracted from lyophilized fungal tissue using the DNeasy PowerPlant Pro Kit (QIAGEN, Hilden, Germany) following manufacturer’s instructions. High-quality DNA samples will be sent to Genomic Core facility in the Huck Institute of Life Sciences (The Pennsylvania State University, University Park, PA) for whole genome sequencing using Pac Bio and Illumina MiSeq. This is a method for sequencing and creation of de-novo reference genomes. A total of four V. dahliae genotypes will be sent for sequencing, two genotypes for VCG 4A and two for VCG 4B.
Collection of plant samples. The second objective of this project is to detect V. dahliae lineages infecting plant species cultivated under rotation in potato fields, and moreover, to identify which plant species get infected by which lineage (VCG). This is quite important since different lineages (VCGs) show different virulence (aggressiveness) to different plant hosts and therefore, they pose different threats to the sustainability of the disease management strategy. V. dahliae-infected plant samples have been collected through Bob Leiby, crop consultant at PA Cooperative Potato Growers, and Sara May, director of the Plant Disease Clinic in the Depart. of Plant Pathology and Environmental Microbiology, Penn State. Samples include different potato, pepper and watermelon cultivars grown in PA crop fields. Plant samples were washed with tap water. Stem tissues were finely sliced and lyophilized for further DNA extraction.
Education & Outreach Activities and Participation Summary
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