Progress 05/01/24 to 04/30/25
Outputs
Target Audience:The target audience included undergraduate andgraduate students, postdoctoral fellows, and the scientific community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Ph.D. students had the opportunity to conduct 16S rRNA sequence analysis and gain hands-on experience in microbial community profiling and analyzing soil physico-chemical properties. How have the results been disseminated to communities of interest?Results collected so far were presented in the American Society of Plant Biologists-Southern Section meeting held in Raleigh, NC, from March 28-30. The published papers were incorporated into undergraduate and graduate courses taught by the PI. What do you plan to do during the next reporting period to accomplish the goals?1.The 16S rRNA and fungal ITS sequences from Andropogon virginicus samples collected across multiple sites will be analyzed to identify the key factors driving the separation of bacterial and fungal communities. 2.Functional potential of bacterial and fungal communities will be inferred based on 16S rRNA and ITS sequence data. 3.A similar analysis will be conducted for the rhizobiome transfer experiment in maize under drought conditions to assess microbial shifts and their predicted functional roles. 4.Leaf and root primary metabolites, secondary metabolites, and hormone analysis will be done to get more insights into the plant response to different inocula under drought stress. 5.Shotgun metagenomics of rhizosphere soils from the maize greenhouse experiment will also be done to identify the key microbial strains and their functions. 6.The second greenhouse experiment will be conducted to screen the cover crops that have a similar root exudate profile to that of Andropogon virginicus. The resulting findings will be compiled and submitted for publication.
Impacts
What was accomplished under these goals?
1. Andropogon virginicus soil sampling and soil characteristics analysis Soil samples were collected from various sites across different states where Andropogon virginicus L., commonly known as broomsedge bluestem grass, naturally grows. The growing sites were identified through the SERNEC (SouthEast Regional Network of Expertise and Collections) data portal, and metadata was gathered to obtain the necessary permissions. Rhizospheric and bulk soil samples were collected from all sites and classified as either mesic or xeric based on soil particle proportions and water-retaining capacity. Total soil DNA was extracted using the Quick-DNA Fecal/Soil Kit (Zymo Research), following the manufacturers' instructions. The V3-V4 region of the 16S rRNA gene was PCR-amplified, purified, and sent for 16S rRNA gene amplicon sequencing. 16S rRNA sequences were analyzed to determine the bacterial composition of soil samples. 2. 16S rRNA sequence analysis of Andropogon virginicus rhizosphere and bulk soil samples This study explored microbial community dynamics in soils dominated by Andropogon under different environmental conditions, including moisture stress (mesic vs. xeric), soil compartment (bulk vs. rhizosphere), texture, and geographic location. The dominant microbial classes were Acidobacteriae, Alphaproteobacteria, Verrucomicrobiae, and Gammaproteobacteria, with notable variation across treatments. Venn diagrams demonstrated significant OTU overlap among groups, with unique contributions from SC Xeric and NC Mesic samples. Beta diversity analyses using Bray-Curtis, Jaccard, and UniFrac distances revealed significant shifts in microbial composition across all grouping factors, with the strongest variation explained by stress and texture combined (Weighted UniFrac R² = 0.426). Alpha diversity was highest in mesic, rhizosphere, and loamy textured soils, while significantly lower under xeric stress. Differential abundance analyses (KW-Dunn, LEfSe, ANCOM-BC2) identified key taxa distinguishing stress and soil groups. Xeric environments favored stress-tolerant genera such as Candidatus Udaeobacter, Acidibacter, Mycobacterium, and Paenibacillus. LEfSe results further showed that several genera, including Bacillus, Pseudarthrobacter, and Methylobacterium-Methylorubrum, were enriched in bulk soils, whereas rhizosphere-enriched taxa included Aquicella, Bryocella, and Flavisolibacter. ANCOM-BC2 identified 59 xeric-enriched genera, many of which had significant correlations with environmental factors. Correlation heatmaps demonstrated strong relationships between enriched taxa and variables like organic matter, pH, phosphorus, potassium, calcium, and micronutrients such as Zn, Cu, and Mn. For example, Acidibacter correlated with organic matter and Cu, while Mycobacterium correlated with pH and Mg. Ordination analyses validated environmental filtering as a key factor shaping microbial communities. RDA explained 69.2% of the constrained variation at the phylum level, with organic matter, potassium, and calcium identified as significant contributors. CCA, confirmed by a DCA axis length over 6, explained 71.6% of the constrained variation. ANOVA and envfit results indicated organic matter (r² = 0.51), calcium (r² = 0.38), and copper (r² = 0.25) as the strongest environmental factors influencing microbial assemblages. Overall, these results emphasize how stress and physicochemical properties drive microbial community composition and function across different Andropogon sites. 3. Multiplication of the Andropogon inoculum under greenhouse conditions The Andropogon inoculum collected from various locations was multiplied by growing Andropogon in the same collected soil within greenhouse settings under drought conditions (40% of field capacity). 4. Morphological and physiological response of maize to different Andropogon inocula under drought stress After multiplying the inoculum, a greenhouse experiment was conducted where maize was inoculated with both sterilized and non-sterilized Andropogon inoculum from different locations under drought (40% of field capacity) and ambient conditions (80% of field capacity). The results showed that maize plants inoculated with non-autoclaved Andropogon inoculum outperformed those with sterilized inoculum under drought stress, in both morphology and physiology. These trends were consistent across all locations. Plant height was significantly greater in maize inoculated with non-sterilized inoculum compared to sterilized under drought conditions (p<0.001). Specific root length was higher (p<0.001), and root tissue density was lower (p<0.001) in non-sterilized drought-stressed plants, indicating longer, thinner roots that enhanced water and nutrient uptake. The net photosynthetic rate was higher in non-sterilized drought-stressed plants relative to sterilized ones (p<0.001). Similar patterns were observed for other physiological parameters, including stomatal conductance, leaf transpiration rate, and leaf water potential (p<0.001). 5.16S rRNA sequence analysis of the Andropogon rhizobiome-inoculated maize drought experiment This study examined the impacts of drought stress and soil sterilization on microbial community composition and diversity across six experimental sites. Soils were subjected to four treatments: auto-claved ambient, non-autoclaved ambient, auto-claved drought, and non-autoclaved drought. Community composition was dominated by Acidobacteriae, Alphaproteobacteria, Verrucomicrobiae, and Gammaproteobacteria, with drought treatments showing higher relative abundances of stress-adapted taxa such as Thermoleophilia and Gemmatimonadetes. Venn diagrams identified a core microbiome common across treatments and sites, with unique sequences notably enriched under drought conditions. Beta diversity analyses using Bray-Curtis, Jaccard, and UniFrac distances indicated significant separation between drought and ambient treatments (PERMANOVA R² = 0.12-0.24, p < 0.001), with treatments explaining more variation than site differences. Weighted UniFrac distance demonstrated the strongest separation. Alpha diversity metrics (Observed, Chao1, ACE, Fisher) showed no significant changes in richness (p > 0.2), but Shannon and Simpson indices were significantly reduced under drought (p = 6.2e−06 and 7.4e−06). Site-level differences were also significant for Shannon (p=0.033) and Simpson (p=0.0095). Differential abundance analysis (KW-Dunn) identified Burkholderia-Caballeronia-Paraburkholderia and Rhodanobacter as key genera enriched under ambient and drought conditions, respectively. Heatmaps for plant growth-promoting bacteria (PGPB) revealed enrichment of Mycobacterium and Pseudomonas in drought autoclaved soils, whereas Bradyrhizobium and Rhizobacter were more abundant in ambient non-autoclaved samples. Location-specific patterns showed increased presence of Paenibacillus, Rhodopseudomonas, and Mycobacterium in more drought-prone areas. LEfSe analysis further highlighted ambient-enriched taxa (e.g., Burkholderia-Caballeronia-Paraburkholderia, Mucilaginibacter) and drought-associated genera (e.g., Pseudarthrobacter, Sphingomonas, Candidatus Udaeobacter). The effects of sterilization were also clear, with autoclaved soils favoring Pseudomonas and Ralstonia, while non-autoclaved soils supported Pandoraea and WPS-2. Collectively, these findings demonstrate how drought and sterilization significantly reshape soil microbial communities.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Zhang Z, Jatana B, Nawaz MS, Campbell B, Suseela V, Tharayil N. 2025. Cross-inoculation of Andropogon virginicus rhizobiome enhances fungal diversity and network complexity in maize (Zea mays) rhizosphere under drought. Climate Smart Agriculture doi.org/10.1016/j.csag.2025.100056.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Goyal, M!., Campbell, B., Tharayil, N., & Suseela, V. 2025. Deciphering the role of rhizobiome associated with a ruderal plant in enhancing drought stress tolerance in maize (Zea mays). 86th Annual Southern Section-American Society of Plant Biologists Meeting, Raleigh, NC
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Progress 05/01/23 to 04/30/24
Outputs
Target Audience:The target audience included graduate and undergraduate students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Ph.D. students had the opportunity to conduct 16S rRNA sequence analysis and gain hands-on experience in microbial community profiling and analyzing soil physico-chemical properties. Undergraduate students got experiential learning opportunities in microbial and soil analysis. How have the results been disseminated to communities of interest?The results are being generated and are not yet ready for dissemination. The study results related to this project,published in the Plant Journal in the last reporting period, were incorporated into the undergraduate and graduate soil coursetaught by the PI. What do you plan to do during the next reporting period to accomplish the goals?The ITS sequencing did not yield satisfactory results this time, so the plan is to repeat the sequencing for fungal ITS genes. In the next reporting period, we will plant Andropogon in soil from selected sites and subject it to drought and ambient moisture conditions. Fromthe collected rhizosphere soil, the site that will provide the maximum resilience to drought-stressed Andropogon plants will be selected for thesubsequent maize experiment. In the maize experiment, wewill measure all morphological and physiological plant parameters, plant secondary metabolites, and the diversity and function of rhizosphere microbial communities in maize subjected to drought and ambient conditions.
Impacts
What was accomplished under these goals?
Soil samples were collected from various sites across different states, such as South Carolina, Georgia, Texas, Alabama,Louisiana, Florida, and Oklahoma,where Andropogon virginicus L., commonly known as broomsedge bluestem grass, naturally grows. The growing sites were identified through the SERNEC (Southeast Regional Network of Expertise and Collections) data portal, and metadata was collected to obtain the necessary permissions. Rhizosphereand bulk soil samples were collected from all sites and classified as mesic or xeric based on soil particle proportions and water-retaining capacity. Total soil DNA was extracted using the OMEGA Soil DNA Kit (Omega Bio-tek, USA) and the Quick-DNA Fecal/Soil Kit (Zymo Research), following the manufacturers' instructions. The V3-V4 region of the 16S rRNA gene was PCR-amplified, purified, and sent for 16S rRNA gene amplicon sequencing. The 16S rRNA sequences were analyzed to determine the bacterial composition of soil samples. The relative abundance of various phyla was observed, and alpha diversity metrics such as Chao1, Fisher, Observed, and Shannon indicated significant differences between rhizosphere and bulk soil samples, though no significant differences were observed between xeric and mesic soils. Beta diversity analysis comparing Andropogon virginicus with other grasses revealed that Andropogon rhizosphere samples formed a distinct cluster separate from the other grasses. Additionally, a linkage clustering tree of Andropogon virginicus beta diversity showed that most rhizosphere samples formed a separate cluster from bulk soil samples, with a few exceptions. Taxonomic comparisons at the phylum level indicated differences in abundance, particularly with higher Bacteroidetes and lower Planctomycetes in earlier samples compared to current ones. Soil nutrient and texture analysis of samples from all sites wascompleted. The soils from all sites were processed by sieving through a 2mm sieve to prepare for growing Andropogon and subsequentlymaize in the next season.
Publications
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Progress 05/01/22 to 04/30/23
Outputs
Target Audience:The target audience includes the scientific community, undergraduate and graduate students, and postdoctoral fellows. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training opportunities for threegraduate studentson various aspects of soil microbiology and analytical techniques, as well as several undergraduates. How have the results been disseminated to communities of interest?Study 1 was published in the Plant Journal (Zhang Z, Jatana B, Campbell B, Gill J, Suseela V, Tharayil N. 2022. Cross inoculation of rhizobiome from a congeneric ruderal plant imparts drought tolerance in maize (Zea mays) through changes in root morphology and proteome. The Plant Journal, doi:10.1111/tpj.15775.) The PI incorporated the results of Study 1 into courses taught at Clemson. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will collect soil of Andropogon virginicus from different regions of the southern states of the USA and focus on exploring the microbiome composition and its potential to impart drought tolerance to maize. This will help us understand the effect of different climatic and edaphic conditions on the rhizobiome composition of ruderal plants and how it, in turn, affects the rhizobiome efficiency to impart drought tolerance in crops.
Impacts
What was accomplished under these goals?
Study 1: To identify the rhizobiome that imparts drought tolerance to ruderal plants, we selected the ruderal plant Andropogon virginicus and collected its rhizobiome from the rhizosphere. We cross-inoculated this rhizobiome into maize and subjected the maize to drought to analyze the potential of the Andropogon rhizobiome to confer drought tolerance to maize. Study 2: To explore general patterns of plant responses to drought stress and assess the microbial diversity among different plants, we conducted a meta-analysis using data from the NCBI database derived from published articles. We compared this data with a study conducted in our lab by Zhang et al., 2022. All the published studies involved different host plants. We found that the microbiomes inhabiting crop plants and grass species are influenced more by host plants than by environmental factors like drought. As host plants release root exudates, specific microbial taxa colonize the rhizosphere and endosphere. The results further reinforce our understanding that microbial communities are actively and dynamically shaped by genetic variation in host plants.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2022
Citation:
Zhang Z, Jatana B, Campbell B, Gill J!, Suseela V, Tharayil N. 2022. Cross inoculation of rhizobiome from a congeneric ruderal plant imparts drought tolerance in maize (Zea mays) through changes in root morphology and proteome. The Plant Journal, doi:10.1111/tpj.15775.
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