Chapter 3: EFFECTS OF LAND USE AND MANAGEMENT ON SOIL BACTERIAL
3.4 DISCUSSION
3.4.1 Bacterial community structure under different land uses at the Baynesfield
PCR-DGGE of 16S rDNA sequences from Baynesfield soils clearly differentiated bacterial community composition under all the agricultural land uses, on the basis of band number and the varying distances the PCR amplicons migrated in the gel.
However, results of analyses based on average relative intensity of bands were less definitive. The banding patterns observed in the soil bacterial DNA profiles, with numerous, low intensity, closely-spaced bands interspersed with either a single or a few bright, dominant bands, were possibly due to the high heterogeneity of directly extracted soil DNA, indicating the complexity of the bacterial community composition. The similarity in the band profiles of the replicate samples from the same land use indicated greater differences in bacterial population structure between the different land uses than within the same one. In a study of soil microbial community responses to differing management practices, Crecchio et al. (2004) reported a similar type of banding pattern when using directly isolated soil DNA.
They suggested that the bright bands possibly indicated the presence of a limited number of dominant, ecologically well-adapted bacterial types in the soil. On the other hand, the numerous light bands possibly indicated that many, equally abundant populations characterised each soil.
The bacterial community structure was compared between widely different land uses with very different vegetation, fertilizer, and tillage histories. Consequently, large differences in nutrient status, pH, acidity and organic matter content between sites were found. NMS analysis of DGGE profiles clearly separated the bacterial populations in the six land use soils, indicating substantial differences in community composition between sites. This could be due to many interacting factors for example, the rhizosphere of different plant species is known to support bacterial communities of different species composition (Marschner et al., 2001; Miethling et al., 2003). It was shown by CCA that a range of physicochemical soil properties (P, acidity, Mg, ECEC, K and organic C) had influenced bacterial community composition, with P and acidity showing the strongest correlations. Nonetheless, CCA1 and CCA2 accounted for only 22.8% and 15.7% respectively, of the total variability, suggesting that other unmeasured factors were also influential.
Surface soils are physically, chemically and structurally heterogeneous and may provide numerous micro-environments for the survival and growth of microbes (Yao et al., 2006). Analysis of selected soil variables by MRPP separated all the soils under the different land uses from each other, except those under sugarcane and maize and those under kikuyu pasture and native grassland. Despite this, visual assessment and NMS analysis of DGGE profiles of bacterial communities, clearly distinguished between both maize and sugarcane and also between kikuyu and native grassland. As was shown by CCA, soils under the sugarcane and maize monocultures had a high P and K content (from fertilizer additions) and the lowest organic C content of all the land use types. In an earlier study at this site, soils under sugarcane and maize were found to have lost organic matter due to repeated tillage, and had a smaller microbial biomass in comparison with soils under kikuyu, native grassland and exotic forests (Haynes et al., 2003). In the DGGE gels, sugarcane and maize bacterial communities displayed fewer bands per lane than the other land uses. Therefore, as each band theoretically represents a different bacterial population (group of species), the soil
communities under sugarcane and maize had a lower richness and genetic diversity than those under the other land uses. This could largely be due to the effects of tillage, which cause intensive mixing of soil with organic matter and organic residues from the field (e.g. roots, straw and stubble). Tillage mechanically loosens the soil, resulting in increased mineralization of organic matter, causing a lowering of organic matter content, an increase in mineral-available nitrogen (nitrate), and increased erosion (Honermeier, 2007). More easily decomposable C fractions are lost preferentially when land use or management leads to the promotion of organic matter decomposition. These labile C fractions are thought to support a substantial proportion of the heterotrophic microbial community and their loss could, therefore, lead to a disproportionate decline in microbial diversity (Degens et al., 2000: Graham, 2003).
Tillage may also cause a reduction in microbial diversity by, for example, mixing the soil, which results in a more homogeneous soil volume, thus reducing the number of microhabitats or “unit communities” (species assemblages of soil organisms which occupy each distinct organic resource) and thereby reducing the structural diversity of the community (Giller et al., 1997). In contrast, in the relatively undisturbed surface soils of land uses such as permanent kikuyu pasture or exotic forests, the soil volume is likely to be extremely heterogeneous in both the size and continuity of pores, and in the distribution of “hot spots” of microbial activity. These occur around randomly distributed decomposing litter such as dead or dying roots and earthworm casts (Beare et al., 1995). Despite the fact that at Baynesfield, the maize fields are tilled annually, whereas the sugarcane fields are tilled only at replanting (at approximately six yearly intervals) the bacterial richness and evenness were similar under the two crops, suggesting that at this site, the above-mentioned effect was of little importance.
In uncultivated soils in the study area a low P status (i.e. 1–10 mg kg-1) is common.
Conversely in the sugarcane and maize soils, exceptionally high levels of extractable P (i.e. > 150 mg kg-1, almost ten times the recommended level of 15–20 mg kg-1)were found. As suggested by CCA (Figure 3.2), P was a major factor in distinguishing bacterial communities under sugarcane and maize from those under other land uses at this site, indicating that P status had influenced bacterial community composition considerably. Thus exceptionally high P levels seemed to be associated with a
reduction in genetic diversity, which could possibly be as a result of the loss of bacterial species adapted to growth at low P levels. These results are indicative of the detrimental effects of excessive fertilizer applications on soil microbial diversity.
Soil organic C content was similar under native grassland, wattle and pine, but was greatest under permanent kikuyu pasture. Previously, Haynes et al. (2003) had reported similar findings at the same site. Under grassland, large amounts of soil organic matter accumulated characteristically, due to the turnover of the extensive root system and to above-ground inputs such as stem and leaf tissue and animal dung.
Under improved kikuyu pasture, organic matter accumulation was greater than under native grassland due to greater dry matter production (and greater organic matter inputs) induced by improved, high-yielding cultivars, irrigation and regular fertilizer applications (Haynes et al., 2003). In the present study, bacterial communities under kikuyu produced slightly fewer bands per lane in DGGE gels than did those from native grassland soils. This indicated a lower richness and genetic diversity in the soil bacterial community under kikuyu than under native grassland. Trends for richness, derived from ANOVA, confirmed this. On the basis of the number of bands present, the kikuyu soil bacterial community could be differentiated from all those studied at this site except the pine community.
The greater richness and genetic diversity of bacterial communities under unimproved native grassland, compared to that under pine, kikuyu, maize and sugarcane may reflect the physicochemical heterogeneity of undisturbed soil as well as a greater heterogeneity in the above-ground plant community. A more diverse plant community in native grassland would result in a greater variety of organic matter inputs to the soil than those of the agricultural or forestry monocultures. In grassland biomes, major plant constituents added to soils include cellulose and hemicellulose. Microbial degradation of these materials requires a diversity of microbes to break down complex substances into simple substrates for use by a variety of other organisms (Alexander, 1977). The larger number of potential substrates could account for the greater richness and genetic diversity of the bacterial community observed under native grassland than under the long-term pine, kikuyu, maize, and sugarcane monocultures. Pairwise comparisons of richness data separated native grassland from all land uses except wattle.
It is well known that the quality of soil organic matter differs between forests and grasslands, with carbohydrates tending to be higher in pasture than in forest soils, while phenolic compounds are usually significantly higher under forests (Yao et al., 2006). Carbon in forest soil enters as litter and roots, and by rhizodeposition (Hernesmaa et al., 2005). The organic C content of pine soils at site 1 was similar to that of native grassland and wattle, although the nature of the communities differed substantially between these soils as indicated by their clear separation by CCA (Figure 3.2). The separation of the communities under pine from those under the other land uses was due primarily to the effects of acidity, as pine soil had the lowest pH and the highest exchangeable acidity of all the soils at the study site. The replicate soil bacterial communities under pine produced numerous bands per lane in the DGGE gels, indicating a high richness. ANOVA analysis of this data separated pine soil bacteria from those of all the other land uses except kikuyu.
In the pine forest, a thick layer of needles covered the soil surface. Because pine needles are rich in lignocellulosic and phenolic compounds, which are recalcitrant and difficult to metabolise, a microbial succession, initiated by the fungi, is required to effect their breakdown into simple aromatic substances and, finally, into low molecular weight organic acids (Alexander, 1977; Ratering et al., 2007). The production of the latter, together with humic acids, could help account for the high acidity observed in pine soil. The pathways involved in this degradation are complex, resulting in the formation of a large number of metabolic intermediates, which could provide substrates for a wide variety of microorganisms (Alexander, 1977; Ratering et al., 2007). This may account for the observed richness in the pine prokaryotic community. The low pH, exchangeable Ca and Mg, and higher exchange acidity under pine than other land uses were probably important in differentiating the pine bacterial community from those of the other land uses. In an earlier study by Haynes et al. (2003), the microbial biomass was found to be higher under pine than under native grassland although that study did not take community richness or genetic diversity into account.
In wattle soil, organic C content was higher than under maize and sugarcane; similar to native grassland soil; slightly lower than under pine; and much lower than in kikuyu soil. Wattle soil had the highest pH and the highest Ca and Mg content of all
the land use types at this site. Replicate samples of DNA amplicons from wattle soil bacteria consistently produced most bands per lane in DGGE gels when compared to those of all the other land uses. MRPP tests of community profiles clearly differentiated between the wattle and pine communities at this site. A difference in richness between wattle and pine and all the other land uses except native grassland, was also shown by ANOVA. This was unexpected because of the greater variety of plant organic matter inputs to native grassland soil than to those of the wattle monoculture. However, the wattle plantation at Baynesfield had been established for
< 10 years and was situated on the site of an old Eucalyptus (gum tree) plantation.
Litter on the forest floor included wattle leaves and twigs, with rotting gum tree stumps still visible. These could all provide substrates for microbial metabolism. In addition, as wattle is a legume, nitrogen fixing bacteria associated with their root nodules would be released into soil in rotting root material. Wattle is an acacia species and, while it is an exotic, many other acacias are indigenous to the area. Therefore soil microbes associated with these acacias could possibly adapt to growth in the rhizosphere of wattles. All these factors could account for the high richness found under this land use. The observed lower organic C content under wattle than under pine is probably associated with loss of organic matter following the harvest of the Eucalypt plantation previously on this site.
Based on the Shannon Weaver Diversity index, calculations from the DGGE data showed an overall difference in diversity but not in evenness in the various soil bacterial communities. The Shannon Weaver index is a general diversity index, which increases with the number of species and is higher if the mass is distributed evenly over the species. Evenness is independent of the number of species and is lower if a small number of bands are dominant, and highest if the relative abundance of all bands is essentially similar. The equitability correspondingly indicates the presence of dominant bands (Dilly et al., 2004). As the banding profiles of all the different soil communities contained a large number of light, low intensity bands, together with one or a few, bright dominant bands, a low evenness resulted. Nonetheless, an overall difference in diversity was shown. Pairwise comparisons separated maize bacterial communities from those under pine and wattle, although no differences were found in pairwise comparisons of the communities under the other land uses. Tebbe and Schloter (2007) reported a case study, where soil microbial diversity of
conventionally-managed fields was compared to fields under short-term and long- term organic farming, respectively. The microbial biomass of the investigated field plots from the different treatments did not differ from each other, whereas significant changes in the microbial community were detected at all taxonomic levels that were analysed. However, the overall diversity as calculated by the Shannon index was not significantly different between treatments. In addition, strain (ecotype) diversity showed that although the overall diversity (Shannon index) was unaffected, a clear shift in the type of strains could be detected. As the Shannon index is dependent on both ‘richness’ (for which clear, reproducible assignments of individual organisms to species level are required) and ‘evenness’ (for which a reliable quantification method is needed) and as neither of these parameters can be unequivocally determined for soil prokaryotes (Tebbe and Schloter, 2007), this method of determining bacterial diversity has some shortcomings. In the present study, analyses based on the presence or absence of bands or on band numbers clearly separated the various communities under the different land uses, whereas the Shannon Weaver diversity index based on the average relative intensity of the bands (mass), showed only slight differences, or none at all, because of the low evenness.