Bacterial Diversity of Australian Exotic Pine Forest Soil and Leaf Litter

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Patel, Bharat

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Greene, Tony

Xu, Zhihong

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Forest plantations, widely grown for wood production, involve the selective promotion of single tree species, or replacement of natural species by exotic tree species. Slash pine (Pinus elliottii) has been chosen for reforestation of infertile sandy soils in southeast Queensland, Australia. These exotic pine plantations minimise soil and water losses, and are important scientific study sites. The soil environment of these plantations, though devoid of sufficient nutrients, oxygen and other factors, harbours innumerable bacteria that may play a crucial role in maintaining soil quality and ecosystem functions. These soil microorganisms also have the potential for use as sensitive biological indicators to reflect environmental changes. It is therefore essential to understand the interrelationships amongst bacterial communities and their environment by assessing their structural and functional diversity, and their responses to disturbances. The microbial community of an exotic pine plantation of subtropical Australia was analysed by both culture-dependent and culture-independent methods. In this study, a leaf litter-soil core sample (25 cm x 40 cm) was collected from a 22-year-old slash pine plantation in southeast Queensland, Australia in October, 2003. The core sample was divided into three fractions, namely, L layer leaf litter, F layer leaf litter, and forest soil 0-10 cm. In the culture-independent study, a modified DNA extraction and purification method was used to obtain highly purified high-molecular-weight DNA. This DNA was successfully used to amplify bacterial 16S rRNA genes with universal primers Fd1 and R6, to produce products of approximately 1500 bp. PCRamplified 16S rRNA genes were subsequently cloned and a total of 194 clones from leaf litter and soil were partially sequenced (about 510 bp). The 16S rRNA gene sequences were analysed and grouped into several phylogroups (the sequences with a similarity value ¡Ã 98 % were regarded as phylogenetically similar and grouped into one phylogroup). Sequencing representatives (¡Ã– 1400 nucleotides) from each phylogenetic group confirmed that five bacterial phyla were represented in the forest soil clone library. Phylum Acidobacterium was the most abundant phylogenetic group in terms of the number of clones and accounted for 42 % of all examined soil clones. The Verrucomicrobiales and Proteobacteria were the second and third most abundant phylogenetic groups found in the soil clone libraries, accounting for 12 % and 11 % of the soil clones, respectively. About 8 % of all examined soil clones were Planctomycetes and 27 % of soil clones were phylogenetically unidentified. The large amount of unclassified clone sequences could imply that novel groups of bacteria were present in the forest soil. When the two fractions of leaf litter clone libraries were compared, Firmicutes was the only phylum represented in the L layer leaf litter clone library. Similarly, Firmicutes dominated the F layer leaf litter (79 % of the library), was followed by Proteobacteria (21 %). For the culture-dependent study, a total of 21 isolates which were considered to represent 334 colonies from the leaf litter and forest soil were identified by 16S rRNA gene sequence analysis, indicating that L layer leaf litter and F layer leaf litter were dominated by Firmicutes (48 %) and Proteobacteria (69 %) respectively, and 91 % of the isolates from the forest soil were Firmicutes. Using culture-independent methods, Actinobacteria appeared to be absent from the L and F layer leaf litter and forest soil samples. The results implied that either the nucleic acids of Actinobacteria were difficult to extract or Actinobacteria were over represented in the culture-dependent examinations. Phylum Acidobacteria appeared to be numerically dominant and active members in most soils. However, only one named species had been isolated from an acid mine drainage site and reported by Kishimoto and Tano (1987). Analysis by culture-dependent methods revealed a different bacterial diversity, compared to the bacterial diversity from the 16S rRNA gene clone sequences. The most significant result was the observation that, as revealed by both culture-dependent and culture-independent methods, the bacterial diversity presented in the leaf litter was greatly different from the community of the soil. During the culture-dependent bacterial diversity study, four novel strains were isolated from the forest soil and leaf litter samples and complete characterisations of these novel strains were carried out. Reports on the descriptions of Bacillus decisifrondis strain E5HC-32T from forest soil and Frondicola australicus strain E1HC-02T from L layer leaf litter have been published (appendix). The information provided by assessing the microbial communities in different fractions of leaf litter and forest soil improves our understanding of the phylogenetic relationship between soil and leaf litter. It is suggested, in this study, to perform both culture-dependent and culture-independent methods to characterise the bacterial structure and diversity in forest litter and soil samples, particularly in response to different forest management practices and global change. This study also provides the basis for further functional studies of the forest soil and leaf litter of exotic pine plantation in subtropical Australia.

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Thesis (PhD Doctorate)

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Doctor of Philosophy (PhD)


School of Biomolecular and Biomedical Sciences

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