Molecular Biological Studies of Soil Microbial Communities Under Different Management Practices in Forest Ecosystems of Queensland

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Hughes, Jane

Xu, Zhihong

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2005
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Abstract

Soil microorganisms play important roles in maintaining soil quality and ecosystem health. Development of effective methods for studying the composition, diversity, and behavior of microorganisms in soil habitats is essential for a broader understanding of soil quality. Forest management strategies and practices are of vital significance for sustainable forest production. How the different forest management measures will influence soil microbial communities is a widespread concern of forest industry and scientific communities. Only a small proportion (~0.1%) of the bacteria from natural habitats can be cultured on laboratory growth media. Direct extraction of whole-community DNA from soil, followed by polymerase chain reaction (PCR) and other analysis circumvents the problems of the culture-dependent methods and may shed light on a broader range of microbial communities in the soil. DNA-based molecular methods rely on high quality soil microbial DNA as template, and thus extraction of good quality DNA from soil samples has been a challenge because of the complex and heterogeneous nature of the soil matrix. The objectives of this research were to establish a set of DNA-based molecular methods and to apply them to investigate forest soil microbial composition and diversity. Soil samples were collected from different forest ecosystems, i.e., the natural forest (YNF) and the first rotation (~ 50 years) (Y1R) and the second rotation (~ 1 year) (Y2R) of hoop pine plantations at Yarraman, and from different forest residue management practices (the experiments had established 6.4 years before the samples were collected) at Gympie, two long-term experimental sites of the Queensland Department of Primary Industry-Forestry in subtropical Queensland, Australia. Some DNA-based molecular techniques, including DNA extraction and purification, PCR amplification, DNA screening, cloning, sequencing and phylogenetic analyses, were explored using Yarraman soil samples, which were high in organic matter, clay and iron oxide contents. A set of methods was assembled based on the recommendations of the method development experiments and applied to the investigations of the microbial composition and diversity of the Yarraman and Gympie soil samples. Four soil DNA extraction methods, including the Zhou method (Zhou et al., 1996), the Holben method (Holben, 1994), the UltraClean (Mo Bio) and FastDNA (Bio 101) soil DNA extraction kits, were explored. It was necessary to modify these methods for Yarraman soil. I designed and introduced a pre-lysis buffer washing step, to partially remove soil humic substances and promote soil dispersion. This modification greatly improved the quality of the extracted DNA, decreasing co-extracted humic substances by 31% and increasing DNA yield by 24%. The improved Holben method was recommended for fungal community studies, and the improved Zhou method for bacterial community studies. The extracted DNA was good in quality, with a consistent size of ~20 kb and a yield of 48-87 g g-1 soil, and could be successfully used for 16S (Zhou method) and 18S (Holben method) rDNA amplifications. For less difficult environmental samples, UltraClean kits could be a good option, because they are simple and fast and the extracted DNA are also of good quality. Screening of the DNA PCR products using TGGE, Heteroduplex-TGGE and SSCP was also explored. These methods were not so effective for the screening of the soil DNA PCR products, owing to the difficulty in interpretation of the results. Cloning was a necessary step to obtain a single sequence at species level in soil microbial community studies. The screening of the clone library by TGGE, Heteroduplex-TGGE and SSCP could only separate the clones into several major bands, although SSCP gave better separation. Sequencing of selected clones directly from the clone library obtained ultimate results of microbial taxonomic composition and diversity through well-established sequence analysis software packages and the databases. It was recommended that, in this project with the target of microbial community composition and diversity, soil DNA PCR products were directly cloned to construct clone libraries and a sample of clones were sequenced to achieve an estimate of the taxonomic composition of the soil. Fungal communities of the Yarraman soil samples under the natural forest (YNF) and the hoop pine plantations (YHP) were investigated using 18S rDNA based cloning and sequencing approaches. Twenty-eight clone sequences were obtained and analysed. Three fungal orders, i.e., Zygomycota, Ascomycota and Basidiomycota were detected from the YNF and YHP samples. By contrast, culture-based analyses of fungi in the literature were mostly Ascomycetes. YNF appeared to have more Ascomycota but less Zygomycota than YHP, and within the Zygomycota order, YHP had more unidentified species than YNF. Bacterial communities of Yarraman soil samples of YNF, Y1R and Y2R were investigated using 16S rDNA-based cloning and sequencing approaches. 305 16S rDNA clone sequences were analysed and showed an overall bacterial community composition of Unclassified bacteria (34.4%), Proteobacteria (22.0%), Verrucomicrobia (15.7%), Acidobacteria (10.2%), Chloroflexi (6.9%), Gemmatimonadetes (5.6%), and Actinobacteria (5.2%). There was a significant difference among YNF, Y1R and Y2R in the taxonomic group composition. YNF had a greater proportion of Acidobacteria (18.0%), Verrucomicrobia (23.0%) and Chloroflexi (9.0%) than Y1R and Y2R (corresponding to 6.3%, 12.1% and 5.9%, respectively), while Y1R and Y2R had a higher percentage of the Unclassified group (38.5% for Y1R and 46.5% for Y2R) than YNF (18.0%). For the Proteobacteria group, YNF had more Alpha-subdivision but Y1R and Y2R had more Delta-subdivision. From YNF to Y1R to Y2R, the clone sequence variable site ratios, 5% and 10% OTU numbers and Shannon's diversity index H' values tended to decrease, indicating the soil bacterial diversity decreased from the natural forest to the first and the second rotation hoop pine plantations. The large amount of unclassified clone sequences could imply a novel group of bacteria in the soil, particularly in the hoop pine soil samples. Alternatively they may result from artefacts during the PCR process. Bacterial communities of the Gympie soil under different residue management practices, i.e., residue (litter plus logging residue) removed (G0R), residue retained (G1R), and residue doubled (G2R), were also investigated using the 16S rDNA-based cloning and sequencing approaches. Acidobacteria (37.6%) and Proteobacteria (35.6%, including Alpha-subdivision of 29.9% and Gamma-subdivision of 5.7%) were dominant components of the communities, followed by Actinobacteria (14.7%), Verrucomicrobia (7.3%) and Unclassified bacteria. There was no significant difference among G0R, G1R and G2R in the bacterial community compositions and diversity. These findings provided an in-depth vision of the soil microbial communities under different forest management practices. Their combination with other soil analysis results, such as physical and chemical properties, and forest production data, could provide an improved understanding of sustainable forest management strategies.

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Thesis (PhD Doctorate)

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Doctor of Philosophy (PhD)

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Australian School of Environmental Studies

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Soil microorganisms

forest management strategies

soil samples

soil DNA

forest ecosystems (Queensland)

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