Response of bacterial community composition to long-term applications of different composts in agricultural soils

Differences in the bacterial community composition of agricultural soils caused by a long-term (12 year) application of different composts were identified by cultivation-dependent and -independent methods (PCR-DGGE and 16S rRNA clone libraries). The number of colony forming units indicated that the successive incorporation of organic amendments increased the bacterial abundance (6.41-5.66 log10 cfu g dry soil) compared to control and mineral soils (5.54-3.74 log10 cfu g dry soil). Isolated bacteria were dominated by Actinobacteria, whereby compost-amended soils and green compost-amended soils showed, respectively, higher number of members of Actinobacteria (100% and 64%) than control and mineral soils (50% and 40%). The 16S rRNA clone libraries were dominated by Proteobacteria (43%), Acidobacteria (21%) and Actinobacteria (13%). Proteobacteria and Actinobacteria were most abundant in compost amended soils while Acidobacteria were more frequently found in mineral fertilizer and control soils. Partial 16S rRNA gene clone libraries revealed a higher bacterial diversity than cultivation. In conclusion, we found differences of bacterial community composition with a cultivation approach and clone libraries between compost amended soils and control and mineral soil. Additional key words: 16S rRNA clone libraries; fertilizer; isolated bacteria; organic amendments.

Soil application of compost made from organic wastes is gaining importance since integrated and biological agriculture is becoming increasingly popular (Lalande et al., 2000;Masciandaro et al., 2000).Compost is considered to be an environmentally safe, agronomically advantageous, and relatively cheap organic amendment able to stimulate soil microbial activity, improve crop growth (Pascual et al., 1997), avoid organic matter loss (Ros et al., 2003) and increase microbial diversity (Peacock et al., 2001).Composition and activity of soil bacterial communities are key issues due to their fundamental role in biogeochemical cycles and in the formation of soil structure (Lynch et al., 2004).Bacterial communities can be measured by various techniques, e.g.traditional plate counting and molecular based-techniques such as PCR-based approaches.However, each one of them has their particular limitations.For this reason, the best way to study soil bacterial communities is to use a variety of different approaches to obtain the broadest picture possible.
Few studies have focused on the response of bacterial communities to long-term compost applications to agricultural soils (e.g.Saison et al., 2006), some of them have been carried out on basis of the field experiment that is the basis of the present study, involving PCR-DGGE, phospholipid fatty acid fingerprinting, community level physiological profiling and volatile organic compound profiling (Innerebner et al., 2006;Ros et al., 2006a, b;Seewald et al., 2009).The objective of this work was to give additional evidence for compost effects on bacterial community composition in a longterm crop rotation experiment with continuous compost amendments using a cultivation approach and clone libraries.
From 1991 on, a long-term crop rotation [maize (Zea mays L.), summer-wheat (Triticum aestivum L.) and winter-barley (Hordeum vulgare L.)] field experiment was performed near Linz (Austria).The soil was loamy silt (17.4% clay, 69% silt, 13.6% sand) with a pH (H 2 O) of 6.8.The soil contained 1.9% organic matter, and 260 and 300 mg kg -1 available P and K, respectively.The experiment was performed using twelve randomly distributed plots (4 treatments with 3 replicates; 10 × 3 m).Treatments were performed annually in spring time as follows: a) Control, soil without fertilization; b) OWC, annual application of compost from source-separate collection of domestic organic waste corresponding to 175 kg N ha -1 (12.6 10 3 kg compost ha -1 ) plus 80 kg N (NH 4 NO 3 ) ha -1 ; c) GC, annual application of green waste compost from roadside and park leaves corresponding to 175 kg N ha -1 (9.3 10 3 kg compost ha -1 ) plus 80 kg N (NH 4 NO 3 ) ha -1 ; d) Mineral: mineral fertilization treatment corresponding to 80 kg N (NH 4 NO 3 ) ha -1 .The main characteristics of composts are given in Table 1.Soils were sampled on October 15, 2003 after maize harvest.Ten random soil cores (6 cm diameter, depth 0-20 cm) from each treated soil were taken.These samples were pooled to reduce spatial heterogeneity and sieved (< 2 mm).
Isolation of bacterial cultures was performed from the higher dilutions of each sample; the most frequently occurring bacteria were selected, and picked according to morphological characteristics such as colour, shape and type growth.They were subcultured to assume that they were pure and preserved on a Standard I agar as described by Mayrhofer et al. (2006), but omitting cycloheximide.After 7 d at 25°C, colony forming units (CFUs) of each sample were counted and DNA from the isolated bacteria was extracted using the GenElute Bacterial Genomic DNA kit (Sigma).Bacterial DNA was amplified with primers 338f-GC/907r as described in Ros et al. (2006b).Microbial community DNA was extracted from the bulk soils (0.4 g) using the Fast DNA Spin Kit for soil (BIO 101, USA).The extracted DNA was subjected to PCR amplif ication with the 63f/1378r primer set, to generate nearly full-length 16S rRNA clones (Marchesi et al., 1998).Three independent PCR reactions from each soil sample were mixed and submitted to cloning with the pCR ® 4-TOPO ® TA cloning ® kit for sequencing (Invitrogen TM life technologies).The presence of inserts was determi- The number of CFUs (log 10 cfu g -1 dry soil) was signif icantly higher (p ≤ 0.05) in soils treated with OWC (6.41 ± 0.06), GC (5.66 ± 0.05), or mineral fertilizer (5.54 ± 0.03) as compared to control soil (3.74 ± 0.06) due to the incorporation of organic matter and nutrients that activate the autochthonous microorganisms of the soil (Pascual et al., 1997).The highest number of CFUs in OWC may be attributed to the easy degradability of its organic matter compared to the recalcitrant material of the green waste compost (more bulky cellulose and lignin-rich materials) (Pascual et al., 2000).This increase in CFUs supports earlier data on positive effects of compost amendments on microbial biomass (Ros et al., 2006a) probably due to the increased availability of substrate C that stimulates microbial growth, but a direct effect from microorganisms added with the compost is also possible (García-Gil et al., 2000;Ros et al., 2003).
A total of 24 different isolates were selected based on their colony morphology.Sequences were distributed among three phyla, Actinobacteria (62.5%),Firmicutes (25.0%) and Proteobacteria (12.5%).GC and OWC isolates mainly belonged to the phylum Actinobacteria (100% and 63.7% respectively), being assigned to the genera Microbacterium, Arthrobacter, Streptomyces, Mycobacterium, Rhodococcus and Micromonospora (Table 2).In control and mineral fertilizer soils the distribution of Actinobacteria, Firmicutes (Bacillus, Staphylococcus) and γ-Proteobacteria (Pseudomonas and Lysobacter) was balanced (Table 2).The low diversity of cultivated bacteria may be attributed to the simple cultivation approach chosen and a short incubation time.Most of the isolates were identified as members of the phylum Actinobacteria that includes some of the most common soil microorganisms, playing important roles in decomposition and humus formation (Felske  , 1997;Rheims et al., 1999).Due to the difficult lysis of Actinobacteria cells, DNA extraction from members of this phylum is hampered (Feinstein et al., 2009), which is one possible explanation why Actinobacteria typically only display 10-16% in clone libraries from soil ecosystems, while they represent 40-100% of cultured soil bacteria (Kaiser et al., 2001).A total of 122 clones from 200 were selected from the clone libraries of the different treatments.Clones were affiliated to nine phyla generally found in soils (Janssen, 2006), the predominant being Proteobacteria (43%), Acidobacteria (21%) and Actinobacteria (13%).Members of the phyla Firmicutes, Bacteroidetes, Gemmatimonadetes, Chloroflexi, Nitrospirae and Cyanobacteria as well as unclassified bacteria were also found (Fig. 1).Proteobacteria encompass an enormous morphological, physiological and metabolic diversity, and are of great importance to global carbon, nitrogen and sulfur cycling (Kersters et al., 2006).The percentage of Proteobacteria was higher in OWC and GC (50 and 48% respectively) than in the control and mineral fertilizer treatments (40 and 32% respectively).In the OWC and GC treatments α-Proteobacteria were predominant, while in the control and mineral fertilizer treatments γ-Proteobacteria dominated.The percentage of Actinobacteria members was higher in OWC (20%) and GC (13%) than in mineral and control soil (10%).However, the percentage of sequences belonging to Acidobacteria was higher in control and mineral soil (27 and 26%, respectively) than in OWC and GC amended soil (17 and 16%) (Fig. 1).Similar results were observed by other authors (e.g.Sessitsch et al., 2001;Valinsky et al., 2002;Sun et al., 2004;Toyota and Kuninaga, 2006) where variations in the composition of bacterial community also depended on the soil type, sample origin and plant growth stage.Smit et al. (2001) suggested that the ratio between Proteobacteria and Acidobacteria frequency can be indicative of the soil nutrient status when calculated as % Proteobacteria/(sum of % Proteobacteria and % Acidobacteria).We found a higher ratio for OWC (0.82) and GC (0.75) compared to control (0.60) and mineral soil (0.64).This indicated a higher nutrient supply in the compost amended samples (Ros et al., 2006a).In comparison, a ratio of 0.87 was observed by McCaig et al. (1999) in high-input agricultural soil.
The Sorensen's similarity coefficients among treatments were very low; the highest similarity was observed between GC and OWC (0.14).It could indicate that the incorporation of both organic amendments changed the microbial community composition in a similar way.This was supporting earlier findings by Innerebner et al. (2006) andRos et al. (2006a), who found a greater microbial diversity in the compost amended samples than in the control and mineral fertilizer samples.A very low species overlap ranged (0.09-0.00) was detected for the rest of overlapping between the treatments.A Sorensen's similarity coefficient of 0.15 reflects the low overlap between taxa detected by cloning and isolation techniques.This supports earlier findings by Suzuki et al. (1997) and Kaiser et al. (2001).In contrast, Chandler et al. (1997) showed an overlap of 41%.The amount of overlap between cultured organisms and clone libraries depends on several factors, such as the complexity of the environment, the discrepancy between plate counts and direct counts, the media and culture conditions and the sample size of 16S rRNA clones (Dunbar et al., 1999).However, despite the biases Bacterial community in amended agricultural soils 341  inherent in each method, both methods also identified compost amended soils to be similar in terms of phylotype composition compared to control and mineral soils.
In conclusion, we found differences of bacterial community composition with a cultivation approach and clone libraries between compost amended soils and control and mineral soil.Compost amended soils are dominated by Proteobacteria and Actinobacteria and control and mineral soil by Acidobacteria.

Table 1 .
Average chemical properties of the different composts.Data on a dry mass basis and an average of chemical properties obtained during 12 years for each compost.Numbers in parenthesis indicate standard deviation (n = 12)

Table 2 .
Phylogenetic assignments of control, mineral, organic waste compost (OWC) and green compost (GC) amended soil isolates