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Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons

Description

We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungal community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation.

ContributorsPenton, Christopher (Author) / Gupta, Vadakattu V. S. R. (Author) / Yu, Julian (Author) / Tiedje, James M. (Author) / College of Integrative Sciences and Arts (Contributor)

Created2016-06-02

Characterization of Lipid Transport Mutants that Overcome the Iron-Transport Defect in Escherichia coli

Description

When limited for iron, Escherichia coli secretes a siderophore, enterobactin, to solubilize and intake extracellular Fe3+ by a TonB-dependent high-affinity pathway. Consequently, E. coli tonB mutants grow poorly on a medium limited for iron. Upon longer incubation, however, faster growing colonies emerge and overcome this growth defect. The work presented in this paper reports and characterizes these faster growing colonies (revertants) in an attempt to dissect the mechanism by which they overcome the TonB deficiency. Genomic analysis revealed mutations in yejM, a putative inner-to-outer membrane cardiolipin transporter, which are responsible for the faster growth phenotype in a tonB mutant background. Further characterization of the revertants revealed that they display hypersensitivity to vancomycin, a large antibiotic that is normally precluded from entering E. coli cells, and leaked periplasmic proteins into the culture supernatant, indicating a compromised outer membrane permeability barrier. All phenotypes were reversed by supplying the wild type copy of yejM on a plasmid, suggesting that yejM mutations are solely responsible for the observed phenotypes. In the absence of wild type tonB, however, the deletion of all known of cardiolipin synthase genes (clsABC) did not produce the phenotype similar to mutations in the yejM gene, suggesting the absence of cardiolipin from the outer membrane per se is not responsible for the increased outer membrane permeability. These data show that a defect in lipid biogenesis and transport can compromise outer membrane permeability barrier to allow siderophore intake and that YejM may have additional roles other than transporting cardiolipin.

ContributorsQiu, Nan (Author) / Misra, Rajeev (Thesis director) / Bean, Heather (Committee member) / Yu, Julian (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)

Created2019-05

Characterization of Tropical Agricultural Soil Microbiomes After Biochar Amendment

Description

Modern agriculture faces multiple challenges: it must produce more food for a growing global population, adopt more efficient and sustainable management strategies, and adapt to climate change. One potential component of a sustainable management strategy is the application of biochar to agricultural soils. Biochar is the carbon-rich product of biomass pyrolysis, which contains large proportions of aromatic compounds that influence its stability in soil. Concomitant with carbon sequestration, biochar has the potential to increase soil fertility through increasing soil pH, moisture and nutrient retention. Changes in the soil physical and chemical properties can result in shifts in the soil microbiome, which are the proximate drivers of soil processes. This dissertation aims to determine the compositional and functional changes in the soil microbial community in response to the addition of a low-volatile matter biochar. First, the impact of biochar on the bacterial community was investigated in two important agricultural soils (Oxisol and Mollisol) with contrasting fertility under two different cropping systems (conventional sweet corn and zero-tillage napiergrass) one month and one year after the initial addition. This study revealed that the effects of biochar on the bacterial community were most pronounced in the Oxisol under napiergrass cultivation, however soil type was the strongest determinant of the bacterial community. A follow-up study was conducted using shotgun metagenomics to probe the functional community of soil microcosms, which contained Oxisol soil under napiergrass two years after the initial addition of biochar. Biochar significantly increased total carbon in the soils but had little impact on other soil properties. Theses analyses showed that biochar-amended soil microcosms exhibited significant shifts in the functional community and key metabolic pathways related to carbon turnover and denitrification. Given the distinct alterations to the biochar-amended community, deoxyribose nucleic acid (DNA) stable isotope probing was used to target the active populations. These analyses revealed that biochar did not significantly shift the active community in soil microcosms. Overall, these results indicate that the impact of biochar on the active soil community is transient in nature. Yet, biochar may still be a promising strategy for long-term carbon sequestration in agricultural soils.

ContributorsYu, Julian (Author) / Penton, C. Ryan (Thesis advisor) / Cadillo-Quiroz, Hinsby (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Hall, Sharon (Committee member) / Arizona State University (Publisher)

Created2020