Progress 07/15/24 to 07/14/25

Outputs
Target Audience:The target audience served by this project during the reporting period include extension plant pathologist in the southeast US, domestic and international academic researchers, federal government researchers, undergraduate and graduate students studying plant pathology, and cotton producers in the southeast US.This work matters to my target audience for multiple reasons. For the extension plant pathologist and cotton producers in the southeast US, this work provides information about pathogen presence and what environmental factors can drive infection across the cotton belt, a key piece of information that was missing prior to this work. Understanding what pathogens are where and what factors drive infection will allow for more targeted disease management approaches that are location specific and more effective, thus increasing cotton yield and profits. Efforts to reach extension plant pathologist and cotton producers included giving educational talks for extension agents who contributed tothis projectat extension agent focused conferencesand publicationof a report on the Crop Protection Network, a wesbite where research articles are summarized in a more approachable manner specifically for growers and the general public.For academic and federal researchers, this work provides foundational information about pathogen and functional gene content abundance and biogeography of an understudied field crop, cotton. This target audience will be able to expand upon any work done as well as identify any additional avenues of research or knowledge gaps associated with cotton disease management and microbiome community assembly and function. Efforts to reach academic and federal researchersgiving educational talks at conferences focused on advancing the science of plant pathology in addition to a conference focused on microbial ecology. Lastly, this work matters to students studying plant pathology because they will be exposed to the types of research being conducted and possible jobs within plant pathology as well as understanding what we know and still do not know pertaining to cotton seedling disease, providing ideas for future funding opportunities and research studies. Efforts to reachstudents studying plant pathology include giving educational talks at department sponsored symposiums. Changes/Problems:Based on feedback given by the review panel,objective 3 has been improved to use synthetic communities in two greenhouse experiments to investigate how oomycete community composition and dominance structureimpact disease phenotypes in cotton seedlings. By incorporating these changes to objective 3, we will be better able to understand what oomycete diversity and community composition means in relation todisease outcomes in cotton seedlings. This adjustment will better link objective 3 to the two additional objectives of this project. To address how oomycete community composition impacts disease phenotypes, we will construct severalrandom assemblegesofoomycete communities (approximately 12). Each assemblege will contain the same number of oomycete species and each oomycete species will be present at randomabundances. Each assemblege will have five replecates, set up in a random block designand the experiment will last 30 days.To address how dominance structure in oomycete communities impacts disease phenotypes, we will manipulatemultiple variations of oomycete community composition. For example, variation of communitieswould includeone highly pathogenic species at high abundance and multiple non-pathogenic/weakly pathogenic species at lower abundance, a highly pathogenic species at low abundance and multiple non-pathogenic/weakly pathogenic at higher abundance, andmultiple weakly pathogenic species present at equal abundances. Similarly, each community variation will have five replecates, set up in a random block design and the experiment will last 30 days.Previous work by the Noel lab has identified which oomycete species are highly, weakly or non-pathogenic to cotton seeds (Olofintila et al. 2023;https://doi.org/10.1094/PDIS-06-23-1159-RE) and there arelong term culture stocks of each species tested. We will use this previous knowledge and culture collection to construct the oomycete synthetic communities. During bothexperiments, we will take soil samples prior to inoculation of synthetic communities, one day after inoculation, seven days after inoculation, 14 days after inoculation and at the end of the experiment to provide evidence that our manipulated synthetic community is what we designed and that it colonized in the soil. In addition, at each time point, we will collect plant height, growth stage/number of nodes, and provide a disease severity index score. Once the experiment has finished, plant weight, root weight, androot discoloration will be collected in addition to photos being taken of the plants ineach pot and washed roots. We plan to analyze plant healthdata using generalize linear models in R. What opportunities for training and professional development has the project provided?Training opportunities provided by this project include one-on-one work with my mentor to provide insights on handling of and analysis troubleshooting withlarge, complex datasets, laboratory and grant management and mentoring of students. Professional development provided by this project include participation at the 2024 International Symposium of Microbial Ecology,American Phytopathological Society Plant Health 2024 conference, and 2025 Beltwide Cotton Conference. Additionally, I have participatedThe SoutheastEmerging Pathogen Working Group sponsored by Cotton Inc. How have the results been disseminated to communities of interest?We have publishedan article to the Crop Protection Network which summarizes our work on the prevalence of the oomycete species on cotton in Alabama for growers and the general public. In addition, results have been disseminated via poster or oral presentation at threescientific conferences and one professional working group. What do you plan to do during the next reporting period to accomplish the goals? We plan to conduct analyses with our bacterial amplicon sequence data to fully elucidate any biogeographic patterns present in cotton soils, rhizosphere, and roots. We plan to re-conduct dataanalyses using 2023 and 2024 shotgun sequence data to support or rejectany preliminary biogeographic patterns in functional gene content in cotton rhizosphere and soils. We plan to conduct two greenhouse experiments to identify how oomycete community composition and dominance structure in oomycete communities impacts disease phenotypes. We plan to write manuscripts on identified biogeographic patterns associated with pathogen abundance and functional gene contentin addition to results from our greenhouse experiments to disseminate our results. Right now, the objective leastmet is objective 3. This is the first reporting periodso work is ongoing to determine the most appropriate protocols and design thevariation of oomycete communitiesto be deployedin objective 3.

Impacts
What was accomplished under these goals? Obj 1: We want to establish biogeographic patterns associated with fungi, bacteria, andoomycetes in cotton soils, rhizosphere, and roots to understand where pathogens occur and what environmental factors can contribute to infection. Obj 1 Response: Fungi, oomycetes and bacterial amplicon sequencing has been completed for 2023 and 2024 soil, rhizosphere and root samples. Total number of samples was 205 and 225 from 2023 and 2024, respectively. Soil variables for each site were gathered using the USDA Web Soil Survey and weather information was collected using NASA POWER Project funded through the NASA Earth Science/Applied Science Program. Sequences were quality filtered prior to analyses.Differences in alphaand beta diversity were seen between soil, rhizosphere and root tissue therefore, we analyzed each environment separately. For soil, we documented a negativerelationship between the number of fungal species present and average rainthree days post planting (3DPP). A negativerelationship was documented betweenhow evenly distributed fungal species are andjulien day,longitude, and average rain3DPP. We found no relationships between fungal community composition and any variable of interest.A negative relationship was seen between number of oomycete species present and percent organic matter and average percent stand but a positive relationship with longitude. We reported anegativerelationshipbetweenhow evenly distributed oomycetespecies are and percent organic matter.Oomycete community composition was influenced by average root zone soil wetness 3DPP. For rhizosphere, a negative relationship between number of fungal species present and julien day was reported. We described a positive relationship between how evenly distributed fungal species are and average root zone soil wetness 3DPP. Fungal community composition was influenced by latitude and average root zone soil wetness 3DPP. We documented a positive relationship between number of oomycete species present and latitude, average earth surface temperature 3DPP and average root zone soil wetness 3DPP. There was no relationship between how evenly distributed oomycetespecies are and any variables of interest.Oomycete community composition was influenced by latitude, longitude, percent sand, average root zone soil wetness 3DPP, presence of oomycete disease pressure and average seedling percent stand. For root tissue, we observed a negative relationship between the number of fungal species present and julien day, percent sand, presence of oomycete disease pressure and average seedling percent stand but a positive relationship with average root zone soil wetness 3DPP and presence of fungal disease pressure. Apositive relationship between how evenly distrubuted fungal species are and percent organic matter and presence of fungal disease pressure was reported. Fungal community composition was influenced by percent organic matter and presence of fungal disease pressure. A postitive relationship was noted between number of oomycete species present and latitude however,a negative relationship between how evenlydistrubuted oomycetespecies are,average earth surface temperature 3DPP and average seedling percent stand was documented. Oomycete species composition was influenced by average earth surface temperature 3DPPand average seedling percent stand. One interesting pattern noted in both years was that there was a split in the average relative abundance ofGlobisporangium irregulareandG. ultimumaround 90°W, which is where the Mississippi River runs.Globisporangium irregularewas less abundant west of the Mississippi River in states like OK and TX, whereG. ultimumwas more prevalent. Both of these oomycete species are known pathogens to cotton. Obj 2: We want to determine patterns in functional gene content by region or environmental factors. Obj 2 Response: We have shotgun sequenced 77 samples (53 rhizosphere + 24 soil) and 76 rhizosphere samples from 2023 and 2024, respectively. Samples from 2023 and 2024 have been processed downstream analysis with environmental and soil variables and we are currently working on analyzing both years of shotgun sequence data in relation to environmental and soil variables. Based on 2023 shotgun sequence data alone, we reported that even distribution of functional gene pathway abundance increased as the number of seedlings that came out of the ground at 30 days post planting increased. We noted that presence of disease pressure in general (fungal or oomycete) was associated with less even gene pathway abundance compared to sites without disease pressure. Presence of general disease pressure also influenced the presence-absence of functional gene families found within cotton rhizosphere and soils. The composition of functional gene families contained large amounts of variation around 90 degrees west, which is approximately where the Mississippi River runs.Sites that had general disease pressure possessed 317 unique functional gene families compared to sites without general disease pressure. Obj 3: We want to use null and ecological models to identify microbes that improve cotton seedling health and productivity and create synthetic communities to use in inoculation experiments on cotton seedlings. Obj 3 Response: Based on feedback given by the panel, we have improvedobjective 3 to use synthetic communities in two greenhouse experiments to investigate how oomycete community composition and dominance structureimpact disease phenotypes in cotton seedlings. To address how oomycete community composition impacts disease phenotypes we will construct severalrandom assembleges ofoomycete communities and each species will be present at randomabundances. To address how dominance structure in oomycete communities impacts disease phenotypes, we will manipulatemultiple variations of oomycete community composition. For example, one variation of acommunity would includeone highly pathogenic species at high abundance and multiple non-pathogenic/weakly pathogenic species (and vice versa) while another variation of a community would include multiple weakly pathogenic species present at equal abundances. Previous work by the Noel lab has identified which oomycete species are highly, weakly or non-pathogenic to cotton seeds (Olofintila et al. 2023;https://doi.org/10.1094/PDIS-06-23-1159-RE) and there arelong term culture stocks of each species tested. We will use this previous knowledge and culture collection to construct the oomycete synthetic communities. We are currently conducting vigor testing of the cotton seedas well as testing inoculation protocolswe plan to use in these experiments. Obj 1, 2, 3 Outcomes: This study has collected cotton soils and seedlings from across the southeast US from 2023 and 2024, conducted laboratory protocols to sequence the associated microbiome and its functional gene content and link them to environmental factorsto provide foundational knowledge on biogeographic patterns associated with cotton pathogen abundance and functional gene content as well as increasing the awareness of the importance of cotton seedling diseases. Additionally, this work has enhanced research, methods, writing, and presenting skills of an early career scientists. Obj 1, 2, 3 Impact: Extension agents and cotton growers will be most immediately helped by this work because it provides a molecular survey of pathogen abundance across the cotton belt and with this knowledge, extension agents can aid growers with choosing pathogen and location specific management choices that will, in turn, increase cotton yield and profits. Additionally, the scientific community will be helped by this work because it provides foundational knowledge about the microbial communities and functional gene content present in cotton soil, rhizosphere and root tissue and their association with cotton seedling disease and environmental factors which can then be expanded upon by others.

Publications