Progress 09/01/23 to 08/31/24
Outputs
Target Audience:This year, targeted audiences included scientific audiences at international and national science meetings. Respectively, these included audiences at: International Phytobiomes Conference, St. Louis, USA; "Primer Encuentro Nacional Sobre Microorganismos benéficos para la agricultura" [First National meeting on beneficial microorganisms for agriculture], Morelia, Mexico; Genetics of Maize-Microbiome Interaction Workshop, Raleigh, USA; and 66th Annual Maize Genetics Meeting 2024, Raleigh, USA. Details concerning these presentations are below (see Other products/outputs). Changes/Problems:We had originally proposed to use both enrichments for inoculum development. Unfortunately, the FAW control properties were limited and inconsistent. So, instead as a contingency, we started screening isolates from another study and introduced them to B73 and Tx77 maize inbreds. We found four promising isolates that showed both plant growth and FAW resistance benefits. The most beneficial isolate so far is a methylotrophic bacterium that enhances plant growth and development, and resistance against FAW and pathogenic fungi in greenhouse experiments. We are currently concentrating on this isolate to refine methods to enhance it growth so that we can produce sufficient volume to evaluate it at field scale. The field evaluation will be carried out in the first half of 2025. What opportunities for training and professional development has the project provided?The project provided training opportunities in advanced microbiome analysis techniques, including DNA extraction, next-generation sequencing (MinION), and bioinformatic analysis using platforms like EZBIOME to two Visiting Scholars (E. de la Vega Camarillo, S. Rodrigues Ferreira), in addition to the PhD student supported in part by the project and other laboratory personnel. The Visiting Scholars and project graduate and undergraduate students gained experience in complex experimental design and data analysis, including canonical centroid analysis and multivariate statistics for analyzing plant-microbe interactions. Additionally, participants developed skills in standardized protocols for insect handling, plant phenotyping, and rhizosphere sampling techniques. Finally, one REEU (USDA Research and Extension Experiences for Undergraduates) student received hands-on training for insect (FAW) resistance screening and microbiome analysis within the project during summer 2024. How have the results been disseminated to communities of interest?Thus far, results have been disseminated through presentations at scientific meetings and one preprint associated with a manuscript submitted for publication. These include the following: Topcu, Ilksen, Julio S Bernal, Sanjay Antony-Babu (2024) Dysbiosis in Maize Leaf Endosphere Microbiome is Associated with Domestication, 12 September 2024, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-4850295/v1] Bernal, JS; Ilksen Topcu; Amanda Quattrone; Sanjay Antony-Babu (2024) [Bioprospecting for microbial inoculants for climate-smart maize production: Maize evolution and maize landrace ecology offer clues]. Conference at Primer Encuentro Nacional sobre Microorganismos Benéficos para la Agricultura, Morelia, Mexico, September 2024. Topcu, Ilksen, Amanda Quattrone, Ariel Black, Tristan Gregory, Julio S Bernal, Sanjay Antony-Babu (2024) Crop Domestication in Zea mays has Contrasting Effects on Microbiome Community Structure in Above and Below-Ground Microbiota. Genetics of Maize-Microbiome Interaction Workshop - February 29th, 2024, Raleigh, North Carolina, USA. (Oral presentation). Topcu, Ilksen, Amanda Quattrone, Ariel Black, Tristan Gregory, Julio S Bernal, Sanjay Antony-Babu (2024) Crop Domestication in Zea mays has Contrasting Effects on Microbiome Community Structure in Above and Below-Ground Microbiota. The 66th Annual Maize Genetics Meeting 2024, Raleigh, North Carolina, USA. (Poster presentation). Topcu, Ilksen, Julio S Bernal, Sanjay Antony-Babu (2024) Domestication and Breeding of Zea mays Shaped Fungal Microbiome Recruitment. International Phytobiomes Conference, November 20th, 2024, St. Louis, Missouri, USA. (Poster presentation). Topcu, Ilksen, Peiguo Yuan, Tristan Gregory, Julio S Bernal, Sanjay Antony-Babu (2024) Domestication of Zea mays Results in Divergent Impacts on Leaf Endosphere Microbiota. International Phytobiomes Conference, November 20th, 2024, St. Louis, Missouri, USA. (Oral presentation) Topcu, Ilksen; Amanda Quattrone; Sanjay Antony-Babu; Julio S. Bernal (2024) [Bioprospecting for microbial inoculants for climate-smart maize production: Maize evolution and maize landrace ecology offer clues]. Proceedings Primer Encuentro Nacional sobre Microorganismos Benéficos para la Agricultura, Morelia, Mexico, September 2024, p. 24. What do you plan to do during the next reporting period to accomplish the goals?During the remaining months our focus will be on Objective 3, "Isolate, scale-up, reconstitute and evaluate an effective, reconstituted enrichment." In particular, under Objective 3.1 ("identify the bacterial species in the enrichment that best enhance FAW resistance") we plan to continue screening as many as possible of the ca. 400 isolates that we have obtained, guided by published reports of insecticidal activity. Additionally, we will concentrate efforts on refining methods to enhance growth of four promising bacterial isolates (determined in lab and greenhouse experiments) in anticipation of field evaluation (under Objective 3.3). Under Objective 3.2 ("isolate individual species, and reconstitute the enrichment with original ratios of relevant species") we plan further greenhouse evaluation of four promising isolates that have thus far shown both plant growth and FAW resistance benefits. Finally, under Objective 3.3 ("evaluate the reconstituted enrichment in field trials") we plan on conducting field trials in spring (March) or early summer (June) 2025 once we have determined which single isolate or isolate consortia of the four promising isolates is most promising for field trials, and have successfully scaled-up their production.
Impacts
What was accomplished under these goals?
Objective 1: Last year as a contingency response we decided to isolate endosphere bacteria from teosinte accessions and recovered strains of Methylobacterium, Microbacterium and Streptomyces that showed high promise in both growth promotion and fall armyworm (FAW) resistance. This year we isolated bacterial strains from teosinte mucilage using both conventional methods and GALT Prospector™ technology, with the latter yielding 379 isolates that were characterized for nitrogen fixation, phosphate solubilization, and IAA production capabilities using colorimetric microplate assays. Taxonomic analysis revealed a diverse bacterial community dominated by Pantoea (58 isolates), Pseudomonas (58 isolates), Stenotrophomonas (55 isolates), Rhizobium (41 isolates), Bacillus (36 isolates), and Microbacterium (36 isolates), with their phenotypic characteristics analyzed using K-means clustering to group isolates based on their functional traits. The Prospector™ technology revealed unique genera not detected through conventional methods, including Erwinia and Roseateales, while also showing Microbacterium as the dominant genus (28.5%) and demonstrating a higher prevalence of previously underrepresented genera like Curtobacterium and Stenotrophomonas. Objective 2: Last year, screening focused on uncovering resistance to fall armyworm (FAW) in teosinte and maize accessions, and we found three accessions (one of Zea diploperennis perennial teosinte, two of Zea mays parviglumis Balsas teosinte) to be most effective sources of endophytic isolates for inoculating FAW resistance to B73 maize. This year, we screened 16 Balsas teosinte and four Mexican landrace maize accessions and B73 inbred maize (= reference control) for western corn rootworm (WCR) resistance. These 21 accessions were ranked from most to least resistant to WCR based on insect and plant phenotypic traits. For example, the most resistant teosinte accession (Los Naranjos) showed only 23% WCR larva survival compared to 79% in least resistant accession, and suffered only a 20% tissue loss in roots. Overall, WCR-resistant accessions maintained significantly higher microbial diversity under WCR stress and demonstrated selective enrichment of beneficial bacteria, particularly among Proteobacteria. Resistant accession phenotypes were characterized by enhanced expression of multiple defense-related pathways and preserved microbial network integrity under WCR stress. After screening, we employed advanced techniques including next-generation sequencing (MinION) and bioinformatic analysis (EZBIOME) to characterize the rhizosphere and WCR microbiomes, and LC-MS/MS phytohormone profiling of root tissue of one pair each of most resistant-least resistant accessions for Balsas teosinte, landrace maize, and inbred maize (B73 vs B73-mutant known to be resistant to WCR). We are currently analyzing these data for microbial correlates of resistance in plants and insects, and microbial-phytohormone correlates of resistance. We anticipate that these results will be available in early 2025. Our goal is to uncover microbial taxa associated with maize resistance to WCR, whether directly or through phytohormones, which would be targets for isolation. Below (under Objective 3) we provide some preliminary findings concerning promising microbial taxa for maize resistance to WCR. With complementary support from non-project resources we were able to compare bacterial community composition and assembly patterns in known fall armyworm (FAW)-resistant Balsas teosinte and -susceptible landrace maize in a common garden in Mexico. We used MinION full-length 16S rRNA sequencing and EZBIOME platform analysis to compare five compartments (bulk soil, rhizosphere, mucilage, leaf endosphere, seed endosphere) between Balsas teosinte and landrace maize samples collected within a common field (common garden) in Mexico. While the seed endophyte results are pending (early 2025), the findings so far revealed that despite sharing soil conditions, host (maize vs Balsas teosinte) genetic background significantly influences microbiome assembly, with Balsas teosinte showing enrichment of plant growth-promoting and other bacteria, suggesting targets for isolation for future evaluation for FAW resistance as well as other traits. Objective 3: Under Objective 3.1, we found in our WCR screening that key bacterial species for isolation should include Pseudomonas putida (3.0-3.2 fold increased enrichment in resistant accessions), Stenotrophomonas maltophilia (2.7-2.9 fold increase), and Enterobacter species (2.5-2.7 fold increase), which showed consistent enrichment in resistant accessions. Beneficial soil bacteria, including Bacillus subtilis and Rhizobium species, comprised 18-22% of enriched communities in most resistant accessions compared to 8-12% in least-resistant accessions. These bacterial communities demonstrated significant enhancement of defense-related pathways, including chitinase activity (3.8-fold increase), peroxidase production (3.5-fold), and systemic resistance signaling (3.2-fold). Separately, under Objective 3.2 we began screening microbial consortia targeting fall armyworm (FAW) and consisting of combinations made from four bacterial strains obtained from the teosinte leaf endosphere. The results of this effort will be analyzed in early 2025. Also under Objective 3.1, we studied the rhizosphere microbiota of Balsas teosinte, and Mexican and US landrace and elite inbred maize genotypes. Maize landraces and elite inbreds demonstrated greater microbial diversity and richness compared to teosintes, and US landraces and inbred lines harbored more biomarker taxa compared to teosintes. However, teosintes hosted higher numbers of unique taxa compared to the maize genotypes. Maize genotypes showed greater abundance of nitrogen cycle (N-cycle) gene nirK compared to teosintes. In contrast, teosintes and Mexican landrace maize hosted higher abundance of the nosZ gene compared to Mexican inbred and US maize genotypes. No significant differences were observed among genotypes for the nirS gene. These results will be important for identifying microbial taxa that may enhance the FAW tolerance (compensation for growth loss to insect feeding) of maize. Separate analyses revealed significant genotype × compartment differences in fungal communities of teosinte and maize genotypes. Beta diversity analysis revealed that teosintes in the leaf endosphere clearly separated from maize in the same compartment, though no clear separation was observed among genotypes in the rhizosphere or bulk soil. Other analyses showed that Perennial teosinte and Balsas teosinte were enriched with unique fungal families such as Didymellaceae and Ceratobasidiaceae in the rhizosphere, and Davidiellaceae and Aspergillaceae in the leaf endosphere. Overall, the teosintes exhibited higher diversity and richness in the leaf endosphere compared to maize, as shown by both diversity and richness metrics.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Under Review
Year Published:
2025
Citation:
Topcu, I, Bernal, JS, Antony-Babu, S (2024) Dysbiosis in Maize Leaf Endosphere Microbiome is Associated with Domestication. Environmental Microbiology (ms currently being revised following first-round of review)
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:Thus far, targeted audiences have included scientific audiences at international, national, and local science meetings. Invited symposium papers include: (i) Antony-Babu, S., Topcu, I., Black, A., Gregory, T., Bernal, J. Dysbiosis by domestication: Evidence of microbiome perturbations in modern maize caused by crop domestication. International Society for Chemical Ecology Annual Meeting, July 2023, Bangalore, India; and. (ii) Bernal, J., Fontes-Puebla, A., Topcu, I., Antony-Babu, A. The evolution of maize farming explains maize-herbivore and maize-microbiome interactions: Implications for pest management. Entomological Society of America Annual Meeting, November 2023, National Harbor, MD. Volunteered papers include: (iii) Bernal, J. Crop ancestors as sources of pest management insights and solutions. Zeavolution online seminar, May 2023, hosted by North Carolina State University; (iv) Topcu, I., Black A., Gregory, T., Bernal J.S., Antony-Babu S., Effect of Maize Domestication and Breeding on Phyllosphere Microbial Communities. Department of Plant Pathology and Microbiology Annual Symposium 2023, Texas A&M University, College Station; and, (v) Topcu, I., Black, A., Gregory, T., Bernal, J., Antony-Babu, S. Dysbiosis by Domestication: Evidence of Microbiome Perturbations Caused by Crop Domestication in Maize. The American Phytopathological Society, Plant Health 2023, Denver. Changes/Problems:Last year we proposed to implement two changes to address problems encountered during the project's first year, and these problems were addressed: We began isolating individual bacterial strains from teosinte endosphere and screening some of the isolates which have shown promise in both plant growth and tolerance to fall armyworm herbivory damage. Importantly, this process has been greatly facilitated with acquisition of the Prospector Platform (see above). We investigated whether the inconsistent results (preliminary vs first-year results) obtained in the first year were due to divergent responses to bacterial enrichments among maize cultivars. As noted above, we are presently finalizing our data analysis for this experiment but can see that the responses indeed differ among maize cultivars for some but not all plant and insect traits that we measured. The only significant change relative to what we originally planned is the incorporation of the more precise and efficient Prospector Isolation and Discovery Platform to replace isolation using cell-sorter. What opportunities for training and professional development has the project provided?One graduate student (Ilksen Topcu) continued her training within the project and with project support (project support ended this year). Additionally undergraduate students Katy Heaney, Haden Ball, Amrita Gabyu, David Martinez, Cassandra McCallum, Nahuel Levy, and Arturo España were trained in basic microbiology methods. The latter student (España) was (still is) supported by aUSDA REEU program based in University of Texas Rio Grande Valley (EFAS - Empowering Future Agricultural Scientists, Dr. Teresa Feria). Finally, one PhD visiting scholarship application (CAPES Foundation Scholarship for sandwich doctoral program) is pending for one student (Sarah Rodrigues Ferreira, Universidade Estadual do Norte Fluminense). How have the results been disseminated to communities of interest?Thus far, results have been disseminated through presentations at scientific meetings. One presentation at an international meeting (International Society for Chemical Ecology Annual Meeting, Bangalore, India), two at national meetings (Entomological Society of America Annual Meeting, National Harbor; American Phytopathological Society, Plant Health 2023, Denver), and two at local meetings (Zeavolution online seminar, North Carolina State University; Department of Plant Pathology and Microbiology Annual Symposium 2023, Texas A&M University). The graduate student, and undergraduate students as warranted, coauthored the presentations. What do you plan to do during the next reporting period to accomplish the goals?Activities during the third year will focus mostly on Objectives 2 and 3. Objective 2: We will continue screening bacterial isolates from teosinte accessions individually and in combinations to identify those that are most promising for evaluating under Objective 3 (field experiment), and for further development within future research projects. Additionally, we will continue developing optimal culture methods for enriching promising, highly fastidious bacteria (e.g., methylotrophs). Objective 3: We will optimize use of the Prospector Platform to replace bacterial isolation using a cell-sorter to fully exploit the platform's advantages and optimize our workflow. We anticipate conducting the field experiment in the spring 2024 growing season.
Impacts
What was accomplished under these goals?
Objective 1: Last year we found that use of direct enrichments as microbial community yielded inconsistent results in terms of plant growth promotion and tolerance to fall armyworm (detailed in our response to question 5). This year we tested whether plant responses to direct enrichments varied with maize cultivar. For this we compared the responses among five maize cultivars (two landraces, three inbred lines). While we are presently analyzing the experiment's results, preliminary analyses indicate that direct microbial enrichments affected the maize cultivars differently for some (but not all) plant-based and insect-based traits. Also, we made progress on our contingency plan to isolate and screen individual and combinations of endosphere bacteria from teosinte accessions. Within the isolates, we found the strains that belonged to the genera Methylobacterium, Microbacterium and Streptomyces to show high promise in both growth promotion and fall armyworm tolerance. Objective 2: We are currently screening four bacterial isolates (Methylobacterium, Microbacterium, two Streptomyces) from teosinte accessions and the four-isolate combination. The results of this screening will be available in the early weeks of 2024 and will inform our field experiment under Objective 3. Culture methods to enrich isolates are being developed. Methylotrophs are highly fastidious and require specific culture optimization. Currently, the isolates are being tested for efficacy to increase herbivory tolerance in B73 and two Texas inbred lines. Objective 3: This objective was modified to include Prospector Isolation and Discovery Platform (https://isolationbio.com/prospector/) instead of isolation using cell-sorter. This platform provides significant advantages (e.g., allows us to cultivate rare and slow-growing bacteria, generate large, diverse libraries, grow thousands of microcolonies in parallel, and automatically pick and transfer single isolates into a standard multi-well plates) that will optimize our workflow. We anticipate conducting the field experiment in the spring 2024 growing season.
Publications
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:Thus far, targeted audiences have included scientific audiences at international, national, and local science meetings. Respectively, these include audiences at 53rd Annual Meeting of the Society for Invertebrate Pathology, American Phytopathological Society Plant Health 2022, and Texas A&M Genome Editing Symposium 2022. Efforts entailed invited symposium (Reclaiming an ancestor's legacy: Fortifying the maize microbiome against fall armyworm herbivory using teosinte microbiota) and volunteered national/local meeting (Effect of Maize Domestication and Breeding on Rhizosphere and Phyllosphere Microbial Communities and Network Assembly) presentations. Changes/Problems:We had proposed to use microbial enrichments as a community inoculum from foliar endosphere of teosinte towards improving growth and fall armyworm tolerance in maize. We had also presented our preliminary findings that this microbiome transplantation was effective when it was transferred to Mexican landrace maize accession. However, when this was attempted on a Corn Belt inbred maize accession (B73), we encountered inconsistency in germination rates, growth parameters and most importantly in fall armyworm tolerance. We are resolving this issue by executing two changes. We have started isolating individual bacterial strains from teosinte endosphere. We have also screened some of the isolates which have shown promise in both growth and tolerance to fall armyworm herbivory damage. We are also investigating whether the inconsistency of results with B73 is a result of a maize genotype x enrichment interaction effect. That is, whether the enrichment community can still be used for a variety of maize inbreds. However, as mentioned in #1, we have already started executing a contingency plan under which we are evaluating a variety of Corn Belt and Texas maize inbred accessions and hence do not anticipate any delay in meeting the milestones. What opportunities for training and professional development has the project provided?As part of the project, one graduate student (Ilksen Topcu) and four undergraduate students (Shelby Hamilton, Megan Chaney, Kevin Qian and Haden Ball) were trained. The students learned plant growth methods, infestation protocols for fall armyworm and western corn rootworm, extraction of DNA from tissues and soils, culture endophytic bacteria, seed inoculation methods, and damage assessment on plants. The graduate student was also trained in microbiome sequencing methods, biostatistics and microbial ecology. They also presented their research findings in national and local scientific conferences (elaborated in response to question 4c). How have the results been disseminated to communities of interest?Thus far, results have been disseminated through presentations at scientific meetings. One presentation at an international meeting (53rd Annual Meeting of the Society for Invertebrate Pathology), and two others at national and local meetings (American Phytopathological Society Plant Health 2022, Texas A&M Genome Editing Symposium 2022). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: Use of direct enrichments as microbial community was found to yield inconsistent results in terms of plant growth promotion and tolerance to fall armyworm (detailed in our response to question 5). Hence, we moved on to contingency plan to isolate endosphere bacteria from teosinte accessions. Within the isolates, we found the strains that belonged to the genera Methylobacterium, Microbacterium and Streptomyces to show high promise in both growth promotion and fall armyworm tolerance. Objective 2: We are currently in the process of screening isolates from teosinte accessions. As for now, we find the Zea diploperennis (perinial teosinte) and two accessions of Balsas teosintes (Zea mays parviglumis) to be most effective source of endophytic isolates that can impart FAW tolerance in B73 maize. Other accessions are currently being tested.
Publications
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