The diversity of oomycetes associated with cotton seedlings in Alabama and the assembly of the spermosphere microbiome of cotton and soybean

Abstract

Cotton and soybean are major crops in the US grown for fiber and protein. Fungal and oomycete pathogens can affect cotton and soybean seedlings resulting in seed and seedling damping-off. Many different oomycete species may be associated with seedling disease, but the species diversity is not as well characterized in cotton compared to soybean. Several surveys of the oomycetes associated with cotton seedlings in Alabama and the US precede the development of molecular tools for species identification and recent changes in oomycete taxonomy. Chapter 1 reviews the relevant literature for seedling diseases, management, and oomycete taxonomy. Then in chapter 2, we seek to identify the diversity of oomycete species associated with cotton seedlings in Alabama using molecular tools, determine the pathogenic species using a seed virulence assay and correlate oomycete diversity with edaphic factors. We hypothesize the precise identification of the oomycete pathogens and their correlation with field properties will help inform better management strategies to maximize yield. We identified a total of 339 oomycete isolates associated with cotton seedlings in North, Central and South Alabama in 2021 and 2022. The identified oomycetes included 28 different species of which 25, including an unnamed species, and species with diverse ecological roles like mycoparasites that have not been previously reported to be associated with cotton seedlings in Alabama and the US as confirmed by the USDA ARS fungal database. Surprisingly, we did not collect any G. ultimum isolate contrary to previous studies. Although Globisporangium irregulare and G. sylvaticum have been previously reported in past studies. Six species were pathogenic to cotton seeds and those isolated in frequencies from cotton seedlings in both years were G. irregulare and P. nicotianae. Species composition and richness varied by soil type. Northern fields with higher cation exchange capacity had higher oomycete richness, which reduced as we sampled in southern soils with lower cation exchange capacity. Some species like P. nicotianae prefer soils with low sand content while other species like G. irregulare were isolated across all soil types irrespective of the sand content. Additionally, many damping-off pathogens residing in soil are stimulated by exudates released by seeds shortly after planting in an environment called the spermosphere. Pathogens move by chemotaxis towards exuding seeds, colonize the seeds and cause disease in a few hours. Therefore, understanding microbial interactions in a spermosphere may give insight into seedling disease control and identification of native microbes that may be potential biocontrol. Interestingly, microbial interactions in the spermosphere are not well understood compared to later phases and plant-associated environments after which seedling disease must have occurred. A major challenge linked with this could be the absence of an easy and reliable method to collect spermosphere samples for high throughput sequencing like we have for the rhizosphere or phyllosphere. In chapter 3, we developed a simple reliable sampling method to collect the spermosphere and used this method to determine the changes in microbial diversity in the spermosphere of cotton and soybean. We developed a sampling method that sufficiently collects spermosphere samples as defined in space and time for high throughput sequencing. Spermosphere microbial communities differed between soybean and cotton and from bulk soil. These communities develop as early as twelve hours after seeds are sown. Major changes observed in microbial communities was a reduction in the evenness of taxa from time point 0 to 18 after seeds were planted. Particularly, soybean spermosphere had the greatest reduction in taxa evenness, followed by cotton and bulk soil, which only had a slight reduction in taxa evenness over time. Significant indicator taxa enriched in soybean spermosphere included Bacilli and Gammaproteobacteria while cotton spermosphere was enriched in Planctomycetes compared to bulk soil. Additionally, we identified some genera with long history of plant growth promotion such as Paenibacillus and Brevibacillus to be enriched in soybean spermosphere. Overall, this study demonstrates the need for prescriptive control strategies tailored to fields based on apparent soil properties and disease pressure history. It serves as a baseline study for future application in developing or determining better seed treatments and resistant varieties for oomycete pathogens in cotton seedlings. The method developed for spermosphere microbiome sequencing could be utilized for various future studies and some of the microbes enriched in soybean spermosphere could be further explored for improved plant growth. In chapter 4 we discuss the impacts and significance of these experiments. Thus, this thesis facilitates precision agriculture for a more sustainable control of seedling diseases caused by oomycetes and improves knowledge of the plant microbiome.