Culture-dependent and culture-independent microbial investigation of pine litters and soil in subtropical Australia |
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Authors: | Li Zhang Zhihong Xu Bharat Patel |
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Institution: | (1) Microbial Discovery Research Unit, School of Biomolecular and Physical Sciences, Griffith University, 170 Kessels Road, Brisbane, Queensland, 4111, Australia;(2) Centre for Forestry and Horticultural Research, School of Biomolecular and Physical Sciences, Griffith University, 170 Kessels Road, Brisbane, Queensland, 4111, Australia |
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Abstract: | Background, aim, and scope 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
minimize soil and water losses and are important scientific study sites. The soil environment of these plantations, though
devoid of sufficient nutrients, organic carbon and other factors, harbors 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 among
bacterial communities and their environment by assessing their structural and functional diversity and their responses to
disturbances. The main aim of our investigation was to determine the diversity of bacterial communities in forest litters
and soil during the forest leaf litter decomposition using culture-dependent and culture-independent techniques.
Materials and methods A 25-cm (diameter) × 40-cm core sample was collected and fractionated into three subsamples designated E1 (L leaf litter layer),
E2 (F leaf litter layer), and E5 (0–10 cm soil layer). Both culture-dependent and culture-independent methods were applied
in this study. In the culture-independent study, a strategy of whole-community DNA extraction, polymerase chain reaction (PCR)
amplification followed by cloning and 16S rDNA sequence analysis was used; for culture-dependent study, the strategy included
sample plating and bacteria isolating, DNA extraction, PCR amplification, and 16S rDNA sequence analysis. The diversity similarities
between two bacterial communities and two methods are quantified using Jensen–Shannon divergence.
Results From culture-dependent study, 336 colonies in total were isolated and grouped from the three subsamples, and the 16S rRNA
sequence analysis from a representative isolate from each morphogroup (21 isolates) indicated that they belonged to the phyla
Actinobacteria, Firmicutes, and Proteobacteria. Culture-independent assessment based on 16S rRNA gene library comprising 194 clones revealed that members of the phylum Actinobacteria were absent in the culture-independent studies. Clones in libraries from E1 consisted exclusively of members of the Firmicutes. The majority of clones from E2 were related to Firmicutes (79%) and Proteobacteria (21%). Clones derived from E5 were mostly affiliated with Acidobacterium (42%), followed by unclassified bacteria (27%), Verrucomicrobiales (12%), Proteobacteria (11%), and Planctomycetes (8%).
Discussion This study showed that bacterial culturabilities in different fractions of leaf litters were similar, and both of them were
higher than the bacterial culturability in the soil. Unculturable bacterial diversity in the soil, however, was much higher
than the leaf litter bacterial diversity. The bacterial diversity on the top layer of leaf litters was slightly less than
that on the bottom layer of leaf litters. This might indicate that forest soils are a more complex environment than leaf litters
are and also that they might inhabit more unculturable microorganisms in the forest soils, which would need to be further
investigated. The leaf litter layer samples also demonstrate the significant difference between the bacterial community diversity
discovered by these two methods in this study. The information provided by assessing the different fractions of leaf litters
and forest soil has improved our understanding of the bacterial community distributions within the forest soil and the above-leaf
litters in an exotic pine plantation of subtropical Australia.
Conclusions This study represents the first attempt to examine the bacterial community in the different fractions of forest leaf litters
and soil in subtropical Australia. The data from this study show that the 16S rDNA clone libraries provided more comprehensive
phylogenetic diversity in the soil and leaf litter samples than the culture collections provided, and both the culture-dependent
and culture-independent studies revealed that the bacterial diversity present in the leaf litters was very different to that
present in the soil. The comparative analysis of bacterial communities in different fractions of leaf litters and soil samples
has also provided important baseline information about the bacterial diversity and composition in the exotic pine forest plantations.
Recommendations and perspectives The experimental data provided important information on the bacterial diversity in forest leaf litter and soil samples, though
additional surveys and comparisons at different locations would be needed to further characterize. In addition, combined methods
that can provide different parts of information on bacterial diversity are encouraged to be used in bacterial community study.
The established libraries of diverse 16S rRNA gene fragments from slash pine leaf litters and forest soil can be used to construct
specific DNA primers and probes to target bacterial groups of interest. It may then be possible to study the ecology of these
bacterial communities and the role of specific bacterial groups that contribute to the many interesting properties of these
environments. |
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Keywords: | 16S rRNA gene libraries Bacterial community Isolation Pine forest leaf litters and soil Phylogeny |
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