Influence of erosion on soil microbial biomass ... - Wiley Online Library

land degradation & development Land Degrad. Develop. 15: 183–195 (2004) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ldr.607

INFLUENCE OF EROSION ON SOIL MICROBIAL BIOMASS, ABUNDANCE AND COMMUNITY DIVERSITY J. A. MABUHAY,* N. NAKAGOSHI AND Y. ISAGI Graduate School for International Development and Cooperation, Hiroshima University, 1-5-1 Kagamiyama, Higashi-Hiroshima, 739-8529 Japan Received 28 August 2003; Revised 14 October 2003; Accepted 5 January 2004

ABSTRACT This study aimed to determine microbial biomass carbon and microbial abundance immediately after, and two years after, forest soil erosion, so as to estimate the degree of damage, including the rate of recovery of microorganisms, in each area. It also aimed to determine the community diversity, and to establish relationships between microbial biomass, microbial abundance and the physico-chemical properties of the soil. Three different study areas in Hiroshima Prefecture, Japan, were used. One undisturbed area and two eroded areas (one immediately after and one two years after erosion). The analysis of variance showed a highly significant difference in microbial biomass carbon and abundance between the study areas. The undisturbed area showed the highest value, followed by the area eroded two years ago, then lastly the area studied immediately after the erosion. The biomass carbon was highly correlated with gram positive bacteria with r2 ¼ 0983, p < 001. The biomass carbon and microbial population were shown to be significantly correlated to the soil’s physico-chemical properties, such as pH, moisture content, water-holding capacity and CN ratio. However, CN ratio proved to be closely correlated to biomass carbon with r2 ¼ 0978, p < 001, to Gram-positive bacteria with r2 ¼ 0977, p < 001, to Gram-negative bacteria with r2 ¼ 0989, p < 001 and to fungi with r2 ¼ 0977, p < 001. The undisturbed area showed a highly diverse community in both of the restriction enzymes used, followed by the area affected by erosion two years ago, then the area immediately after erosion. Copyright # 2004 John Wiley & Sons, Ltd. key words: biomass carbon; chloroform-fumigation extraction; community diversity; microbial abundance; soil erosion; TRFLP analysis

INTRODUCTION Soil erosion is the most widespread form of soil degradation. The land area globally affected by erosion is 1094 million ha by water erosion, of which 751 million ha is severely affected (Lal, 2003). Soil degradation refers to reduced soil fertility due to changes in physical, chemical and biological soil properties caused by erosion (Carpenter et al., 2001). The resulting loss in soil depth is accompanied by losses in nutrients, inorganic matter and in the diversity of living organisms comprising the soil biota carried away with the eroding sediments. Topsoil is a reservoir of bacterial and fungal spores and other propagules of organisms important for decomposition, nutrient cycling and mycorrhizal formation (Habte, 1989; Sieverding, 1991). Various land uses trigger soil erosion, which degrades soil. Land use can drastically modify resistance or increase the vulnerability to environmental change (Martinez-Fernadez et al., 1995). Consequent loss of productivity in these areas is on a collision course with increasing human population demand (Carpenter et al.,

Correspondence to: J. A. Mabuhay, Graduate School for International Development and Cooperation, Hiroshima University, 1-5-1 Kagamiyama, Higashi-Hiroshima, 739-8529 Japan. E-mail: [email protected] Contract/grant sponsor: Hiroshima University COE Programme for Social Capacity Development for Environmental Management and International Cooperation. Copyright # 2004 John Wiley & Sons, Ltd.

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2001). Geographic location and topographic factors, such as bearings and altitudes, also affect the rates of erosion. Uncovered surfaces at high altitudes, and bearings facing wet monsoon winds, are generally susceptible to erosion. Soils support critical processes such as hydrological and biochemical cycling, they contain a wide array of organisms, and they also provide a nutrient and hydrological reservoir, crucial both for below- and above-ground organism survival (Neary et al., 1999). Among the components of the soil, the living population is most dynamic, and greatly varies from one environment to another. The microbial community includes a wide range of individual species that may respond very heterogeneously to changes in the environment. Microorganisms can lose their resilience to ecosystem disturbances and become no longer able to perform their normal processes of nutrient cycling and maintaining soil structure (Brady and Weil, 1999). Past studies done on other kinds of disturbances showed that soil microorganisms are sensitive indicators of the present status of the ecosystem. Soil microbial biomass and microflora were shown to be greatly decreased by burning (Tataeshi et al., 1989a; Mabuhay et al., 2003). The studies of Ovreas and Torsvik (1998), Smit et al. (2001), Fantroussi et al. (1999) and Mu¨ller et al. (2001) showed that agricultural intensification, heavy-metal contamination, and herbicide and pesticide pollution decreased microbial diversity and activity. In addition to these, Lipson et al. (2000) and Larsen et al. (2002) stated that repeated and prolonged freezing and thawing cycles reduced the microbial biomass. Microorganisms are undeniably controlling the fertility and productivity of soil ecosystems. Greipsson and El-Mayas (1999) showed that revegetation of barren lands is usually slow because of a lack of soil microorganisms. With regards to soil erosion, most of the studies done were on its effects on soil nutrients and mycorrhizae, but not on the general effect on microorganisms. Despite these important functions of soil microorganisms, they often constitute the underestimated part of soil ecosystems (Anand et al., 2003). This study aimed to determine microbial biomass carbon and microbial abundance immediately after, and two years after soil erosion, so as to estimate the degree of damage, including the rate of recovery of microorganisms, in each area. It also aimed to determine the community diversity, and to establish relationships between microbial biomass, microbial abundance and the physico-chemical properties of the soil. MATERIALS AND METHODS Study Sites This study was conducted at three different sites in Hiroshima Prefecture. This region has a warm-temperate monsoon climate with an annual average temperature of about 176 C and average annual precipitation of about 1500 mm. The first site, in Itsukaichi (34 250 500 N, 132 190 800 E), Hiroshima City was naturally eroded, triggered by an earthquake on 24 March 2001. This area was a secondary mixed-pine forest, situated next to a dam. The land use and road construction made the rocky hill slope more susceptible to soil erosion. The total area of erosion was approximately 450 m2 (30 m 15 m). The average slope is 65 degrees. The next site, at Yasumiyama (34 140 500 N, 132 350 500 E), Kure City, was also naturally eroded in March 1999 (two years before the experiment was conducted). This area was also dominated by mixed, primarily needle-leaved, vegetation. The total area eroded was approximately 500 m2 but this was in small patches along the hill slope. The average slope is 62 degrees. The road construction to the peak of the mountain triggered the vulnerability of the slope to erosion. In both study areas, severe soil erosion occurred, removing topsoil to a depth of more than 30 cm. The third site is the undisturbed part of Kagamiyama, Higashi-Hiroshima City (34 240 200 N, 132 430 700 E). The average slope of the study area is 53 degrees. This has been a stable area for more than 30 years and is dominated by pine trees. In each of the three sites, 3 plots (10 m 10 m) were established and designated as Plot 1 (bottom of hill), Plot 2 (middle slope) and Plot 3 (ridge). Soil Sampling Soil samples were taken from 12 different points, from the top layer (0–5 cm), randomly in each of the plots of the study sites. Samples were then homogenized and big particles, such as litter, rocks and macrofauna, were removed by hand. Samples were then subjected to sieving (mesh size depending on the analysis done) and were stored at 4 C until use. Sampling was done once every month for a period of 13 months (from April 2001 to April 2002). Copyright # 2004 John Wiley & Sons, Ltd.

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Physical and Chemical Properties of Soil The moisture content was measured by oven-drying the samples at 105 C for 24 hours. The colour of the soil was determined by ocular inspection. Prior to determination, the samples used were air dried and the standard soil colour chart was used to determine the colour of the soil from each site (Oyama and Takehara, 1997; Munsell, 1976). The water-holding capacity was measured using the Hilgard method (Childers et al., 1996). Soil samples were oven-dried at 110 C for 24 hours. The soil texture was determined using the modified ‘jam jar’ experiment (Anonymous, 2000; Gardeners Supply Company, 2002). In this experiment, soil samples were sieved using a mesh size of 2 mm to remove large soil separates like rocks and cobbles. This was followed by the use of the texture triangle (Alexander, 1961). The pH at each site was determined using an electrode pH meter where a 1 : 25 slurry of soil and distilled water was used (Tateishi et al., 1989b). The total carbon and nitrogen were measured using a C–N analyser (Tateishi et al., 1989b). The soil samples were passed through a 1 mm sieve. Oven-drying was done until a constant weight was obtained. Microbial Analyses Microbial biomass carbon. The microbial biomass carbon was measured using the chloroform-fumigation and direct extraction method (Vance et al., 1987). This procedure was then followed by dichromate digestion and titration. The soil samples were passed through a 1 mm sieve to obtain mineral soil. Prior to the actual fumigation and extraction method, the moisture content and water-holding capacity were determined. The soil moisture was adjusted to 55 per cent of water-holding capacity. The organic carbon in each sample was calculated using this formula: 03 ½B S ½F=8 ð100 þ 30 W=100Þ=30 where B is blank, S is sample, 8 is 8 ml sample extract, F is the titration factor, 100 is 100 ml 05 M K2SO4, 30 is the weight of the soil sample used, and W is 55% water-holding capacity. Biomass C ¼

C extracted from fumigated soil C extracted from non fumigated soil KEC

Where: KEC is the extractable part of microbial biomass C after fumigation. The KEC for mineral soil is 0378 Microbial count. The dilution plate count technique was used in this study (Tateishi et al., 1989b). This technique is based on the principle that a complete detachment and dispersion of cells from the soil will give rise to discrete colonies when incubated on a petri plate containing nutrient media. The Gram-positive bacteria, Gram-negative bacteria, and fungi, were enumerated using different specific microbial media. For Gram-positive bacteria, an albumin medium was used. This medium was prepared using 025 g egg albumin, 1 g glucose, 05 g K2HPO4, 02 g MgSO47H2O, trace Fe2(SO4)3, and 15 g agar dissolved in 1000 ml distilled water (pH ¼ 7) (Tateishi et al., 1989b). For Gram-negative bacteria, the same albumin medium was used with 5 ml 01% crystal violet. Fungi were enumerated using Rose Bengal medium which contained 10 g glucose, 5 g peptone, 05 g MgSO47H2O, 003 g Rose Bengal and 20 g agar dissolved in 1000 ml distilled water. The homogenized soil samples were passed through a 2 mm sieve. Thirty grams of the sieved soil was dispersed in 270 ml sterile water in a 500 ml Erlenmeyer flask (Tateishi et al., 1989a). Three subsamples were used from each plot because it is much better to use many subsamples than numerous replicate plates per dilution, since the variation among duplicate soil samples is far greater than the variation between replicate plates or replicate dilutions. The flask was covered with a rubber stopper and shaken vigorously for 10 minutes. In the case of fungi, a similar procedure was followed, except that instead of being shaken vigorously, it was subjected to a rotary shaker for 10 minutes at a minimum number of revolutions per minute to avoid rupture of mycelium and sporulating Copyright # 2004 John Wiley & Sons, Ltd.


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bodies into an indeterminate number of fragments, each of which may produce a single colony (Alexander, 1961). A series of dilutions were undertaken until a 104 dilution was reached. The petri plates were then incubated, at 28 C for 48 hours for bacteria, and at 25 C for 7 days for fungi. After the required incubation period, the number of colonies was counted. The mean of the three subsamples and three replicates was obtained. The number of bacteria and fungi present per gram of soil was calculated using this formula: CFU=g ¼ average number of colonies dilution factor=volume plated ðmlÞ Where: CFU refers to colony forming units and the dilution factor is the reciprocal of the final dilution. Community diversity determined by terminal restriction fragment length polymorphism (TRFLP). Molecular genetic methods, which enable detection of microorganisms without the need to cultivate or isolate them, have offered a lot of advantages in the field of ecology. The ability to amplify genes from the total microbial population has provided the potential to study microbial community structure and population dynamics in both natural and managed environments. Direct amplification of bacterial 16S rRNA genes from extracted soil DNA provides the most comprehensive and flexible means of sampling bacterial communities (Dunbar et al., 2000). This study used the TRFLP analysis for community fingerprinting, to give information about the relative diversity in microbial communities of soil after soil erosion and to provide a quick comparison of the different communities in each area (Fuhrman et al., 2002; Tiquia et al., 2002; Dunbar et al., 2000; Lukow et al., 2000). Unfortunately, this procedure was only carried out on the final samples (April 2002), however we find it significant to include the results that we have. The DNA extraction method was based on the method described by Porteous et al. (1997). Two subsamples were used from each study area. DNA was extracted from a 500 mg soil sample. For final purification of DNA samples, a Microcon-100 microconcentrator was used. Concentration of DNA was estimated by agarose gel electrophoresis followed by ultraviolet photography. Polymerase chain reaction amplification was done following the procedure described by Furhman et al. (2002) who used 16S rRNA ‘universal primers’ of Lane et al. (1985) and Fuhrman et al. (2002) with reverse primer C (ACGGGCGGTGTGTRC) labelled with fluorochrome 50 , 6-carboxylfluorescein (FAM) on the 50 end and the forward A primer (CAGCMGCCGCGGTAATWC) unlabelled. These primers are suitable for bacterial, archaeal, and eukaryotic target sequences (Furhman et al., 2002). The enzyme amplitaq gold (Applied Biosystems) was used with standard buffers, 08 mM primers, a 9 minute 94 C hot start and 30 cycles of 94, 55 and 72 C for 1 minute each. Products were digested with restriction enzyme AfaI/RsaI and HhaI for 4 hours and then run on 3100 Genetic Analyzer (Applied Biosystems) and analysed with Genescan software. Replications were done at the level of the PCR, restriction and Genescan lane. Statistical Analysis Data for moisture content, water-holding capacity, pH, CN ratio, microbial abundance, microbial biomass carbon and community diversity were subjected to analysis of variance (ANOVA) to test the significant differences betweem the three study sites. Correlation analysis was used to test the relationships among the microbial population, microbial biomass and the soil’s physical and chemical characteristics. To compare the community diversity between the three study sites, peaks from TRF profiles were converted to binary data and a Jaccard similarity matrix was calculated. RESULTS Physical and Chemical Properties of the Soil The soil colour varied greatly at each study site. Itsukaichi is greyish-brown, Yasumiyama is bright-brown and Kagamiyama is brown (Table I). The soil texture of Itsukaichi is sandy, Yasumiyama is sandy-clay loam and Kagamiyama is loam (Table I). The amount of sand increases, while the amount of clay and silt decreases, as the topographic location increases. This trend is consistent at all the study areas. Copyright # 2004 John Wiley & Sons, Ltd.


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Table I. Physical properties of the soil in the study sites in Hiroshima Prefecture Study sites

Soil color

Texture

Itsukaichi Yasumi Kagamiyama

25YR6/2 Sandy 75YR5/6 Sandy-clay loam 10YR4/4 Loam

Sand (%) Silt (%) Clay (%) C (%) N (%) pH 885 6748 4113

101 877 3407

14 2375 248

046 131 695

004 020 146

WHC Moisture content

665 4044 511 6418 502 887

1061 1998 3392

The moisture content at each study site varied greatly and fluctuated. The moisture content was high in spring, where the highest was in Kagamiyama having a mean percentage of 3392, followed by the eroded area in Yasumiyama with a mean percentage of 1998, then the eroded area in Itsukaichi with a mean percentage of 1061 (Table I). In terms of water-holding capacity (WHC), the area in Kagamiyama had the highest value having an average of 8870 per cent, followed by the eroded area in Yasumiyama where the mean percentage was 6418, and lastly the eroded area in Itsukaichi which had a mean percentage of 4044. Little fluctuation occurred in WHC in all the study areas, because WHC is largely dependent on the soil texture and organic-matter content. The soil pH in all the study areas ranged from acidic to nearly neutral (Table I). The mean pH at Itsukaichi was 665, at Yasumiyama it was 511, and the mean pH at Kagamiyama was 502. There was not much fluctuation in the pH values at any of the sites. For the carbon and nitrogen analysis, the values greatly vary from one area to another (Table I). The Kagamiyama area showed the highest percentage of carbon and nitrogen content; 695 per cent and 146 per cent respectively. This was followed by the area at Yasumiyama, where the carbon content was 131 per cent, and the nitrogen content was 020 per cent. The lowest value was in Itsukaichi where the carbon content was 046 per cent, and the nitrogen was 004 per cent. Microbial Biomass Carbon The analysis of variance (ANOVA) shows that there is a significant difference in microbial biomass carbon between the study areas (Table II). The area in Kagamiyama showed the highest biomass carbon, followed by the area in Yasumiyama, then lastly the area in Itsukaichi. The area in Itsukaichi showed very low microbial biomass carbon immediately after the occurrence of erosion. There was a gradual increase in biomass carbon in Yasumiyama and Itsukaichi from April to July 2001 (Figure 1). A slight decrease was observed in these areas in August and a gradual increase again from September to December, and then the values decreased again until March 2002. Microbial Abundance The number of Gram-positive bacteria showed a great degree of variation from one area to another. The ANOVA shows a significant difference among the three study sites (Table II). An increasing trend was observed in all the study sites from April to July 2001, but fluctuation occurred during the peak of the summer season (Figure 2). Gram-negative bacteria showed the lowest count among the three types of microorganisms (Gram-positive bacteria, Gram-negative bacteria and fungi). The ANOVA shows that there is a significant difference in the

Table II. Summary of F-values from analysis of variance (ANOVA) for microbial biomass carbon, Gram-positive and Gram-negative bacteria, and fungi in the three study sites Source of variation

Number of observations Biomass carbon Gram-positive bacteria

Study sites Sampling time

3 13

7923** 11ns

7264** 154ns

Gram-negative bacteria 7422** 011ns

Fungi 9339** 145ns

**Significant at / ¼ 001. ns Not significant. Copyright # 2004 John Wiley & Sons, Ltd.


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Figure 1. Average microbial biomass carbon (mg C/kg dry soil) of the three plots at each study site.

Figure 2. Average Gram-positive bacterial population (CFU 104) of the three plots at each study site.

Gram-negative bacterial count between the three study areas (Table II). Little fluctuation was observed in eroded areas, but the Kagamiyama area clearly showed seasonal fluctuations in the number of Gram-negative bacteria (Figure 3). The fungal abundance also varied between the three study sites. The Kagamiyama area showed the highest fungal count, followed by the Yasumiyama area. There was a very low count of fungi immediately after erosion in Itsukaichi, but an increasing trend was observed from April 2001 to April 2002 (Figure 4). The ANOVA shows a significant difference in fungal count among the three study sites (Table II). Copyright # 2004 John Wiley & Sons, Ltd.



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Figure 3. Average Gram-negative bacterial population (CFU 104) of the three plots at each study site.

Figure 4. Average fungal population (CFU 104) of the three plots at each study site.

The microbial abundance showed a great difference between the Kagamiyama site and the eroded sites, which shows that the three groups of microorganisms (Gram-positive bacteria, Gram-negative bacteria and fungi) were significantly affected by erosion. Correlation Analysis The correlation analysis shows a significant correlation between the biomass carbon and moisture, water-holding capacity, pH and CN ratio (Table III). The biomass carbon and moisture content, and biomass carbon and waterholding capacity, showed a significant positive relationship, which shows that as the percentage of moisture Copyright # 2004 John Wiley & Sons, Ltd.


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Table III. The correlation analysis between biomass carbon, microbial population and the physico-chemical properties of the soil

Moisture content Water-holding capacity pH CN ratio Biomass carbon

Biomass carbon

Gram-positive bacteria

0920** 0941** 0646** 0978** —

0933** 0932** 0631** 0977** 0983**

Gram-negative bacteria 0897** 0907** 0630** 0989** 0975**

Fungi 0891** 0890** 0568** 0977** 0978**

**Significant at p ¼ 001.

content and water-holding capacity increase, the biomass carbon also increases. On the other hand, the biomass carbon and pH, and biomass carbon and CN ratio, showed a significant negative relationship, which indicates that as the pH value and CN ratio increase, the biomass carbon decreases. However, the CN ratio was shown to be closely related to biomass carbon with r2 ¼ 0978. The microbial abundance also showed a significant correlation with moisture content, water-holding capacity, pH and CN ratio. As with biomass carbon, microbial count (Gþ, G and fungi) showed a significant positive relationship with moisture content and water-holding capacity. Similarly, the microbial count showed a significant negative relationship with pH and CN ratio. The same was the case with microbial biomass carbon, where the CN ratio proved to be closely related to Gram-positive bacteria, Gram-negative bacteria and fungi with r2 ¼ 0977, r2 ¼ 0989 and r2 ¼ 0977 respectively. Community Diversity Based on ANOVA and on the distance matrix analysis of the community TRF profiles, the three study areas (Kagamiyama, Yasumiyama and Itsukaichi) were significantly different from each other in both of the restriction enzymes used. The replicates, which were different from other replicates in the same study area, were eliminated from the analysis and then the average was calculated. Table IV shows the average number of the terminal restriction fragments (TRFs) at each study site. Each TRF represents a different operational taxonomic group from the sample. The study sample from Kagamiyama showed the highest count; 30 for AfaI and 43 for HhaI. This was followed by Yasumiyama; 13 for AfaI and 23 for HhaI. The Itsukaichi area showed the lowest diversity, 6 and 8, for AfaI and HhaI respectively. Figures 5 and 6 show the different operational taxonomic groups in each study area. The similarity of the microbial diversity in three study sites based on distance matrix analysis of the community TRF profiles is illustrated in Figure 7. The analysis showed that the eroded sites (Yasumiyama and Itsukaichi) were most similar in composition, whereas the Kagamiyama site was the most different. DISCUSSION The correlation analysis (Table III) shows interdependence between soil microbial characteristics and the physical and chemical properties of the soil, like pH, moisture content, water-holding capacity and CN ratio. Severe erosion Table IV. The number of terminal restriction fragments (TRF) at each study site. Each TRF represents a different operational taxonomic unit from the sample Study sites

Itsukaichi Yasumiyama Kagamiyama

Copyright # 2004 John Wiley & Sons, Ltd.

Restriction enzyme Afa/RsaI

HhaI

6 13 30

8 23 43



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Figure 5. Terminal restriction fragment length polymorphism (TRFLP) analysis of the three different study sites: (a) Itsukaichi (immediately after erosion; (b) Yasumiyama (two years after erosion); and (c) Kagamiyama (undisturbed forest). For this analysis, the restriction enzyme Afa/ Rsa I was used. Each peak represents a different operational taxonomic unit from the sample. Peak height and area are related to the amount of DNA in the peak.

changes the natural habitat abruptly, which is harmful to soil biota. Ward et al. (2001) showed that decreases in organic carbon, water-holding capacity, nitrate nitrogen and overall soil quality are negative effects of soil erosion. The survival of soil microorganisms is primarily dependent on the soil system (Eldridge, 1998). On the other hand, soil resistance to erosion is itself dictated in part by microbial activity, including the binding of water-stable aggregates through polysaccharidic exudates (Sims, 1990). The surface soils are occupied by various groups of microorganisms. According to Maier and Pepper (2000), bacteria, fungi and algae, those that are aerobic and phototrophic, are abundant in most surface soils. The removal of surface soils, thus removes these microorganisms from the ecosystem. The eroded areas in this study suffered from a great loss in moisture content during the summer/dry season. The complete absence of vegetative covering made the evaporation of moisture even faster. In the eroded area in Itsukaichi (studied soon after soil erosion), there was a complete absence of vegetative covering, while very few sporadically growing herbs and mosses were found in the eroded area at Yasumiyama (two years after soil erosion). The absence of strong vegetative covering on the soil surface increases the soil’s susceptibility to further erosion. Soils are normally protected by the above and below ground parts of plants (Gow and Pidwirny, 1996). The above-ground parts of plants, like stems and leaves, act as barriers to reduce the potential of wind and rainwater to erode soils. Plants can also reduce erosion by binding and anchoring soil particles to roots. According also to Gow and Pidwirny (1996), organic matter has the ability to absorb a lot of rainwater and, without it, erosion is increased because water does not soak into the soil. Also, when soil is sandy, as in the case of the eroded area in Itsukaichi, its water holding capacity is greatly reduced. When the weather is very hot, it dries easily and its water tension is very low. Microorganisms thrive best where there is sufficient water supply (Alexander, 1961). It is supposed that soil physico-chemical properties will change gradually both in quantity and quality over time (Njorge and Morimoto, 1999), but in this study the reformation of the soil in the study areas was also very slow. Copyright # 2004 John Wiley & Sons, Ltd.


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Figure 6. Terminal restriction fragment length polymorphism (TRFLP) analysis of the three different study sites: (a) Itsukaichi (immediately after erosion; (b) Yasumiyama (two years after erosion); and (c) Kagamiyama (undisturbed forest). For this analysis, the restriction enzyme Hha I was used. Each peak represents a different operational taxonomic unit from the sample. Peak height and area are related to the amount of DNA in the peak.

Figure 7. Dendrogram based on Jaccard similarity comparisons of the three study sites’ soil microbial community diversity.

The reformation is retarded by strong winds and rain, which carry soil particles to the lower slopes and valley bottom. There was not much fluctuation observed in the pH values of all the study sites. The eroded area in Itsukaichi (immediately after erosion) showed a slightly alkaline pH during certain months but remained nearly neutral. The removal of top soil caused the organic matter to be depleted and the pH to increase. In alkaline soils, plants have difficulty in taking up tightly bound nutrients. With higher pH, bacteria are favoured over fungi. In this study, the undisturbed area in Kagamiyama showed a slightly acidic pH (Table I). In healthy forests, there is a great amount of respiration going on in the soil, not only due to the roots, but also to the decomposing organisms. This results in lots of carbonic acid being produced in the soil, thereby making it acidic. Copyright # 2004 John Wiley & Sons, Ltd.



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In this study, the eroded sites showed low percentages of soil carbon and nitrogen content, but the undisturbed site (Kagamiyama) showed a high percentage. Also, the CN ratio showed a high value especially in the Itsukaichi area. The CN ratio of organic matter is important in soil quality (Bauder, 1999). Organic matter with a high carbon to nitrogen ratio frequently has unstable soil organic matter, and there is intense competition among microorganisms for available soil nitrogen. Dry soils generally have low organic matter content, because decomposition is faster than accumulation (Bauder, 1999). When moisture content is very low, the rate of decomposition increases, which results in a lower accumulation of organic matter. If the rate of decomposition increases more rapidly than the rate of residue addition, then the soil could become a source, rather than a sink, for carbon (Haskett and Levine, 1999). In eroded sites, the addition of carbon and nitrogen from plant residues and biological fixation is almost absent due to a lack of vegetative sources. The microbial biomass carbon analysis of this study clearly revealed that erosion altered the microbial diversity of the soil ecosystem. Immediately after the occurrence of erosion, microbial biomass C was very low, indicating that microbial diversity had decreased significantly. Even two years after the occurrence of erosion, the area still showed a lower than normal microbial biomass. The soil microbial biomass is often regarded as an indicator, which may occur in the long term with regard to soil fertility and agro-ecosystem properties. Tateishi et al. (1989a) indicated that soil microbial biomass decreases with soil depth. The topsoil losses explain why eroded areas showed a lower biomass. In this study, microbial biomass C and the microbial abundance both gave the same information about the microbial diversity in eroded areas. The microbial abundance showed a higher number of bacteria than fungi. According to Sims (1990), even motile bacterial types can occur in colonies in the soil, which gives advantages, such as improved nutrient mobilization and absorption, and protection from drying toxins or ultraviolet radiation. Taylor et al. (2002) compared the microbial numbers in surface soils and subsoils where they showed that bacterial and fungal numbers generally decreased with depth. They also stated that fungi were absent from the deep-soil samples. Among the three groups of microorganisms (Gram-positive bacteria, Gram-negative bacteria and fungi), the Gram-negative bacteria showed the lowest level of tolerance against adverse environmental changes. Most bacteria cells are protected by a cell wall that contains a unique molecule called peptidoglycan (Mader, 2001). The susceptibility of Gram-negative bacteria can be attributed to the type of cell wall that-they have; it is thinner and has a thinner layer of peptidoglycan than in Gram-positive bacteria. Environmental conditions affect the density and composition of the bacterial flora, and non-biological factors can frequently alter to a large degree the nature of the population and its biochemical potential. In this study, great seasonal fluctuations in the abundance of Gramnegative bacteria were observed in Kagamiyama (undisturbed area). This shows that they are easily affected by environmental changes, seasonal availability of nutrients and overall ecosystem responses. This result is the same as in our previous study (Mabuhay et al., 2003) and the study of Tateishi et al., (1989a), on the effects of forest fires on soil microflora. The study of Fierer et al. (2003) also supported this result, where it was shown that the abundance of Gram-negative bacteria was high in the surface soil but substantially lower in the subsurface. The eroded areas showed poor microbial diversity compared with the undisturbed area. This is consistent with the expectation that microbial diversity decreases with soil depth. The similarity matrix showed that the microbial composition of Yasumiyama and Itsukaichi were most similar, while Kagamiyama was very different. This is consistent with the expectation based on the physico-chemical conditions (texture, moisture, WHC, pH, and nutrient capacity) of the three study sites. It can also be noted that many of the peaks in Figures 5 and 6, each representing taxa or groups of taxa, are not represented in all samples. Almost 50% of the peaks in the undisturbed sites are not represented in the eroded sites, which shows that many functional groups of microorganisms were removed during soil erosion. For example, the peaks near 40, 90 and 100 bps in Figure 5 are only present in the area two years after erosion and in the undisturbed area samples, but not in the area immediately after erosion. These groups of microorganisms were probably removed together with the eroded soils and had not re-established themselves by the time of our study. Also, the peak near 127 bp in Figure 6a (studied immediately after erosion) is not present in the undisturbed area and two years after erosion. This is probably a new colonizing species in the area. These results are supported by the study of Fierer et al. (2003), where they showed that the composition of soil microbial communities changes significantly with soil depth. Also, this differentiation coincides with the Copyright # 2004 John Wiley & Sons, Ltd.


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decline in microbial diversity. There have not been many studies conducted in this area, and so, comparison of results is almost impossible. In sum, this study showed that soil erosion has an adverse effect on microbial biomass, abundance and composition, by altering natural soil characteristics and removing vegetative protection. Immediately after the occurrence of erosion, a decrease in microbial characteristics was observed, and only a very slight increase was observed during favourable months in spring and autumn. Since the area was completely bare, continuous erosion took place, especially during heavy rain. The complete absence of surface covering, and the sandy soil, contributed to lower moisture content in these areas. Eroded areas cannot recover easily by themselves without proper aid. Proper knowledge and attention should be given to the areas affected by this ecological disturbance to hasten vegetative growth, and to prevent further erosion happening. acknowledgement We are grateful to Professor Takao Horikoshi of Hiroshima University and the members of his laboratory for all their help in performing the Chloroform fumigation-extraction method. We are also thankful for the hand extended to us by the members of Nakagoshi and Isagi laboratory of the same university, during the fieldwork. This study was partially funded by the Hiroshima University COE Programme for Social Capacity Development for Environmental Management and International Cooperation. references Alexander M. 1961. Introduction to Soil Microbiology. Wiley: New York, NY. Anand M, Ma K, Okonski A, Levin S, McCreath D. 2003. Characterising biocomplexity and soil microbial dynamics along a smelter-damaged landscape gradient. The Science of the Total Environment 311: 247–259. Anonymous 2000. 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