ISSN 10623590, Biology Bulletin, 2013, Vol. 40, No. 3, pp. 266–274. © Pleiades Publishing, Inc., 2013. Original Russian Text © M.V. Semenov, E.V. Stolnikova, N.D. Ananyeva, K.V. Ivashchenko, 2013, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2013, No. 3, pp. 299–308.
MICROBIOLOGY
Structure of the Microbial Community in Soil Catena of the Right Bank of the Oka River M. V. Semenova, E. V. Stolnikovab, N. D. Ananyevab, and K. V. Ivashchenkoc a
Moscow State University, Department of Soil Science, Moscow, 119991 Russia Institute of Physicochemical and Biological Problems of Soil Science, Russian Academy of Sciences, ul. Institutskaya 2, Pushchino, Moscow oblast, 142290 Russia c Pushchino State Institute of Natural Sciences, pr. Nauki 3, Pushchino, Moscow oblast, 142292 Russia email:
[email protected] b
Received June 5, 2012
Abstract—The structure of the microbial community (the fungitobacteria ratio) has been assessed by selec tive inhibition of the substrateinduced respiration (SIR) using streptomycin sulfate and cycloheximide anti biotics in the gray forest soil of eluvial, transite, transite–accumulative, and accumulative (meadow alluvial) parts of slope landscape on the right bank of the Oka River (near Pushchino, Moscow oblast) which repre sents an fallow, smallleaved wood, spruce forest, and meadow. The concentrations of bactericide and fun gicide were selected experimentally for each landscape parts which provide the greatest SIR inhibition of the soil upon their individual application and in combination. Fungi were established to be predominant in the contribution to the total SIR which was found to be 82–97%. A dependence between the structure of the microbial community and the C/N ratio and pH of the soil was shown. DOI: 10.1134/S1062359013030084
The differentiation of soil microbial communities into ecologically significant groups is one of the approaches to getting knowledge about their function ing, i.e., copyotrophes and olygotrophes, autochtho nous and zymogenic groupings, and r and kstrate gies, as well as eukaryotes (fungi) and prokaryotes (bacteria) (Strickland and Rousk, 2010). The fungi tobacteria ratio in ecosystems is a good indicator of decomposition processes, changes in nutrient ele ments cycles, and as a consequence, the ability of self regulation of ecosystems (Bailey et al., 2002; van der Heiden et al., 2008). In order to determine the percentage of fungi and bacteria in the biomass of soils, the approach of selec tive inhibition (SI) of substrateinduced respiration (SIR) with antibiotics is used, in addition to micros copy technique and determination of the specific components of the cell wall and membranes (Ander son and Domsch, 1973, 1975). One of the most important aspects about SI is the selection of antibi otic concentration in order to avoid side effects (Ander son and Domsch, 1973, 1975; Rousk et al., 2009). Another aspect of this approach is related to the degree of respiration inhibition by biocides (West, 1986; Velvis, 1997). The fungitobacteria ratio is especially sensitive to disturbances in the soil. A low fungitobacteria ratio is associated with intense cultivation of the soil (Beare et al., 1990; Frey et al., 1999; Bailey et al., 2002), cat tle pasturing (Bardgett et al., 1996, 1998), and the
introduction of nitrogen mineral fertilizers (Bedgett et al., 1996; 1998; Frey et al., 2004). It was also shown that the relative biomass of fungi in natural ecosystems is higher compared to that in arable soils (Beare et al., 1990; Frey et al., 1999; Susyan et al., 2005; Ananyeva et al., 2006). There is limited information on the spatial distri bution of bacteria and fungi in soils of terrestrial eco systems, especially in natural undisturbed ecosystems, including at the local level (Parkin, 1993). There is hardly information available on the changes in the structure of microbial communities in interfaced soils of natural ecosystems. The question also remains open on the relationship between the chemical properties of the soil (plant substrate) and the fungitobacteria ratio at the local level. The soil catena gradient may serve as a natural model for this kind of study. The aim of the present study is to determine the structure of the microbial community (fungi/bacteria) of the soil from different landscape parts (the length of the catena is ~1000 m), which include diverse ecosystems (fallow, forest, and meadow) with different soil physicochem ical properties. Special attention is paid to optimiza tion of the procedure for optimization of the fungal and bacterial contribution to the total microbial bio mass of the soil. MATERIALS AND METHODS The object of this study is soils (points 1–5) of the catena located near the city of Pushchino (latitude
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STRUCTURE OF THE MICROBIAL COMMUNITY IN SOIL CATENA
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250 1 Height, m
54°50′13′′ N; longitude 37°34′44′′ E), Moscow oblast, at a slope in the western exposition of the right bank of the Lyubozhikha River in the Oka River basin (Semenov et al., 2010). The length of the catena is 960 m, and the height difference between extreme points is 80 m (Fig. 1). In the eluvial, transite, transit– accumulative, and accumulative parts of slope land scape, gray forest soil was identified with varying degrees of soil erosion, in the accumulative parts— meadow–alluvial soil. The ecosystems of the catena are the following: fallow (15 years), secondary wood (75 years) including smallleaved species, spruce for est, and meadow (Table 1). Soils (upper mineral 10 cm layer) were collected in June 2011 from five points of the plane surface (25 m2, the “envelope” method) for each type of ecosystem which were then mixed. Plant litter was not included in the analysis. A mixed sample from each point of the catena was dried at room temperature to an air–dry state sifted through a 2mm sieve, and stored until used in the experiments. The SIR method is based on measuring the initial maximal release of CO2 from soil saturated with glu cose (Anderson and Domsch, 1978; Ananyeva et al., 2008; Ananyeva et al., 2011). A weighted soil sample (1 g) was placed into a vial (of 15 mL in volume), and an water solution of glucose was added (0.2 mL; to a concentration of 10 mg/g soil). The vial was then her metically sealed, and the time was recorded. The sam ple of soil with glucose (3–5 h, 22°C) was incubated; a sample of air was then collected using a syringe (the time was also fixed) and analyzed with a KristalLyuks 4000M gas chromatograph (thermal conductivity detector) (Russia). The SIR rate was expressed in μL of C–CO2 / (g soil h). The microbial biomass carbon content was calcu lated according to the formula Cmic (μg C/g soil) = SIR (μL C–CO2 / (g soil h) × 40.04 + 0.37) (Anderson and Domsch, 1978). Basal respiration (BR) was determined like SIR, with the only difference that water was added into the soil instead of a glucose solution. The time of incuba tion of vials with soils comprised 24 h. The rate of BR was expressed in μg C–CO2 / (g soil h). The specific respiration activity of the microbial biomass (microbial metabolic quotient, qCO2) was assessed as the ratio of the rate of the basal respiration to the microbial biomass carbon content: BR / Cmic = qCO2 (μg C–CO2 / (mg Cmic h)). Antibiotics for assessment of the fungal and bacte rial contributions in SIR of soil, i.e., streptomycin sul fate (0.1 mL, water solution) (С21Н39N7O12 ⋅ 3H2SO4, AppliChem, CASNo 3810740) + 0.1 mL of glucose and cycloheximide (powder) (С15Н23NО4, Appli Chem, CASNo 66819) + 0.2 mL of glucose, were introduced into the soil individually, and SIR was measured. Cycloheximide was added into the soil 4 h
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E (East)
W (West)
2
200
3 4
150
5
100 0
200
400
600
800
1000
Distance, m Fig. 1. The location of objects under study (right bank of the Lyubozhikha River, Oka River basin). 1 is eluvial, 2 and 3 are transite, 4 is transite–accumulative, and 5 is alluvial parts of landscape.
before glucose was introduced, and streptomycin— 0.5 h before (Susyan et al., 2005). For a better distri bution of cycloheximide in soils (points 3 and 5), inert material talcum was used, the mass of which was equal to the mass of the inhibitor. A sample of soil with glu cose (10 mg/g soil) was used as the control for SIR inhibition by antibiotics. The coefficient of antibiotic activity overlap, or the inhibitor additivity ratio (IAR), was calculated from the equation IAR= [(A – B) + (A – C)] / (A – D), in which A is respiration (СО2 production) of soil with glu cose, B is respiration of soil with glucose and fungicide, C is respiration of soil with glucose and bactericide, and D is respiration of soil with glucose, bactericide, and fungicide (Bailey et al., 2002). The ratio of the fungal (F) and bacterial (B) contri butions in SIR was assessed using formulas F = [(A – B) / (A –D)] × 100%, B = [(A –C) / (A –D)] × 100% provided that A – [(A –B) + (A – C)] = D ± 5–10% (Lin and Brookes, 1999). SIR, BR, and the fungitobacteria ratio were assessed in soil samples from each point of the catena (~0.5 kg) after preliminary sample incubation at a humidity of 50–55% of the total water holding capac ity and 22°С in the dark for 7 days in a polyethylene bag with air exchange. The content of the total organic carbon (Corg) was determined by the dichromate oxidation method, the total nitrogen (Ntotal) by the indophenols test, pH by the potentiometric approach (soil : water = 1 : 2.5), and the granulometric composition by the gravimetric method (with sodium pyrophosphate). All the measurements were done three replicates and calculated into dried soil (105°С, 8 h); the data are given as the average ± standard deviation. The results were statistically processed using the Statistica 7.0
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3.44
1.93
1.96
2
1.7
Corg, %
7.00
5.05
5.19
5.51
6.24
pH H2 O
0.337
0.173
0.199
0.222
0.162
Ntotal, %
10.2
11.2
9.8
9.0
10.5
C/N
282 ± 39 a
192 ± 4 b
175 ± 34 b
0.56 ± 0.06 bc
1.05 ± 0.14 d
0.51 ± 0.05 ab
0.67 ± 0.08 c
0.37 ± 0.01 a
262 ± 1 a
277 ± 15 a
BR, µg C–CO2/g h
Cmic, µg/g
2.00 ± 0.47
5.48 ± 0.86
2.92 ± 0.72
2.44 ± 0.19
1.43 ± 0.03
qCO2, µg C–CO2/(mg Cmic h)
3.7
0.41
0.74
0.48
0.14
1–0.25
15.34
12.11
13.5
8.32
8.78
0.25–0.05
sand, mm
11.88
11.12
11.36
13.56
14.6
silt, mm (1 indicates the induction of the overlapping antibiotic effect, and
