Performing Department
(N/A)
Non Technical Summary
Fungi and bacteria can be found within plant roots, shoots, and leaves, some of which can cause disease or be plant growth promoting. The interactions of fungi and bacteria can change whether and how much a single microbe can be beneficial or detrimental to a plant, impacting the yield of crops and health of forests. Recently, many plant-associated fungi were found to have bacteria living inside of them; these "endohyphal" bacteria (EHB) can impact whether the fungus can make toxins or reproduce. However, there have been no broad screens for the presence or consequences of EHB in pathogenic Fusarium spp., despite the major diseases caused by these fungi across many crops in the United States, from cereals to vegetables. We proposeto surveypathogenic Fusarium spp. for the presence of EHB and determine if EHB contribute to pathogenicity. Additionally, we will identify bacterial genes necessary for fungal association using RB-TnSeq, a high throughput transposon mutagenesis technique. Making agriculture more sustainable and resilient relies on improved tool development to overcome mounting disease pressure, changing climate, increasing public opposition to chemical usage, and pathogen resistance to synthetic chemicals. A more thorough understanding of the prevalence, impact, and genetic underpinnings of interactions between destructive fungi and EHB could provide new avenues for control, which is of increasing importance given decreasing efficacy of reliable antifungal treatments.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The major research goalof this progress is to understand the prevalence and relationship of bacterial symbionts in fungal plant pathogenicFusariumspp. that infect important crops in the USA. To do that, three research objectives were outlined:Objective I. Survey and identify endohyphal bacteria (EHB) from two agriculturally relevant Fusarium spp. that are from key pathogen groups.Objective II. Determine if pathogenicity and virulence of selected F. oxysporum isolates depends on EHB presence.Objective III. Investigate bacterial genes required for association of EHB with their hosts.In addition to the research objectives, in line with the original training and mentorship goals of the postdoctoral fellowship that this grant was previously awarded as, the supported graduate student will have a short set of professional development goals: 1. Mentorship of at least one undergraduate student 2. Membership in the American Phytopathological Society and attendance at the Southern Division meeting or other appropriate society and local meeting 3. An outreach or teaching training activity of their choice
Project Methods
Research Objective I.Fusarium isolates will be identified morphologically and genomic DNA will be extracted for verification and EHB screening by PCR using the 16S rDNA 'universal' primers 10F/1507R and 27F/1492R following previous studies. Amplicons from positive samples will be sent for Sanger sequencing.Fungal mycelia will be stained with a Live/Dead fluorescent stain or subjected to fluorescent in situ hybridizationand imaged them to visually inspect for bacteria by microscopy. Bacteria will be isolated by grinding fungi suspended in buffer and plating on media amended with the antifungal cycloheximide. The fungal partner will be cured by successive plating on media mended with ciprofloxacin or a mixture of antibiotics. Basic fungal morphology on plates will be assessed to determine any phenotype between fungi with and without EHB. Partnerships will be reformed by coculturing cured fungi and bacteria in liquid synthetic minimal media.Based on the diversity of bacteria identified, the genomes of select isolates will be sequenced (PacBio or Oxford Nanopore) to compare to previously sequenced facultative EHB from endophytic fungi using whole genome phylogenetic analysis and by identification of secretion systems and secondary metabolites.Research Objective 2We will continue investigating pathogenicity of Fusarium spp. when associated with bacteria using typical inoculation methods. Because of the change in research location, any further pathogenicity assays will be designed so that the plants can be maintained in a growth chamber, unlike the lettuce greenhouse assays that we originally proposed. Based on progress in Objective 1, we may switch to other pathosystems of Fusarium should a useful symbiosis be identified.Research Objective 3Using the sequenced RB-TnSeq Luteibacter library, a reassociation assay with both its original host Pestalotiopsis and an isolate of Fusarium graminearum will be conducted,selecting for mutants that are still able to form the relationship. Recoveredbacteriawill be the template for DNA purification, PCR amplification, DNA library preparation, and sequencing of the bar codes. The amplification of relevant DNA sequences from the insertion sites reduces efficiency effects from excess bacterial or fungal genomic DNA. Sequence analysis will be done following the publicly available computation pipeline for RB-TnSeq. Fitness values will be calculated by dividing the relative abundance of a barcode after associating the library with a host by the relative abundance of a barcode before association.Milestones for Evaluation: (1)The graduate studentwill make at least one presentation at a scientific conference (2)Wewill aim to publish two papers, one on the firsttwo objectives proposed here and another on the third objective, and to have at least one submitted by the end of the grant period. These studies will be published in relevant journals of interest to a diversity of plant and microbial-related fields, to promote my work and to distribute our findings as broadly as possible.