Microb Ecol (2012) 64:450–460 DOI 10.1007/s00248-012-0025-y
SOIL MICROBIOLOGY
Changes in Bacterial Community Structure of Agricultural Land Due to Long-Term Organic and Chemical Amendments Vasvi Chaudhry & Ateequr Rehman & Aradhana Mishra & Puneet Singh Chauhan & Chandra Shekhar Nautiyal
Received: 3 October 2011 / Accepted: 3 February 2012 / Published online: 15 March 2012 # Springer Science+Business Media, LLC 2012
Abstract Community level physiological profiling and pyrosequencing-based analysis of the V1-V2 16S rRNA gene region were used to characterize and compare microbial community structure, diversity, and bacterial phylogeny from soils of chemically cultivated land (CCL), organically cultivated land (OCL), and fallow grass land (FGL) for 16 years and were under three different land use types. The entire dataset comprised of 16,608 good-quality sequences (CCL, 6,379; OCL, 4,835; FGL, 5,394); among them 12,606 sequences could be classified in 15 known phylum. The most abundant phylum were Proteobacteria (29.8%), Acidobacteria (22.6%), Actinobacteria (11.1%), and Bacteroidetes (4.7%), while 24.3% of the sequences were from bacterial domain but could not be further classified to any known phylum. Proteobacteria, Bacteroidetes, and Gemmatimonadetes were found to be significantly abundant in OCL soil. On the contrary, Actinobacteria and Acidobacteria were significantly abundant in CCL and FGL, respectively. Our findings supported the view that organic compost amendment (OCL) activates diverse group of microorganisms as compared with conventionally used synthetic chemical fertilizers. Functional diversity and evenness based on carbon source utilization pattern was significantly higher in OCL as compared to CCL and FGL, suggesting an improvement in soil quality. This abundance of microbes possibly leads to the enhanced level of soil organic carbon, soil organic nitrogen, and microbial biomass in OCL and FGL soils as collated with CCL. This work increases our current understanding on the effect of long-term organic and V. Chaudhry : A. Rehman : A. Mishra : P. S. Chauhan : C. S. Nautiyal (*) Division of Plant Microbe Interactions, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India e-mail:
[email protected]
chemical amendment applications on abundance, diversity, and composition of bacterial community inhabiting the soil for the prospects of agricultural yield and quantity of soil.
Introduction Soil microorganisms are highly diverse and abundant organisms on earth; 1 g of soil may contain billions of microbes with thousand of different species [48]. These microbes play a pivotal role in the decomposition of plant and animal organic matter for plant growth and soil structure and fertility. Several biotic or abiotic factors lead to the alteration of microbial community structure and composition which may directly or indirectly influence the soil ecosystem, nutrient cycle activity, and crop production [7, 32, 43, 52]. In addition, anthropogenic intervention for the management and treatment of soil via pesticide [17], chitin [16], compost, manure [14, 41], or genetically modified microorganism and plant [5] also influence microbial diversity. Thus maintenance of microbial diversity and composition is very important for the sustainable agricultural production. Soils under organic farming (compost and green manure) have better quality and microbial activity [32] due to crop rotation and reduced application of synthetic nutrients and pesticides in organically managed soil [42, 51]. Chemical fertilizers (nitrogen, phosphorus, and potassium) enhance crop yield but also bring alteration in soil properties, functional diversity in microbial population, and their enzymatic activities [19, 20]. The long-term experiments demonstrated that initial restoration successes may be transient, so monitoring after long term is crucial for restoration experiments, especially when results can be influenced strongly by time effects [24]. Several studies have been made to evaluate how the application of fresh and composted organic wastes modifies the structure, size, and activity of soil microbial
Impact of Chemical and Organic Amendments on Soil
community [2]. Although long-term impact of chemical fertilizers on soil microbial biomass and diversity is not well documented, it has been shown that chemical fertilizers could increase the soil microbial biomass C and N [15, 45], as well as no significant change in the microbial characteristics of the soil [24]. Zhong and Cai [53] suggested that changes in microbial parameters are correlated with the soil organic carbon content and not to the application of P and N. Evidences linking direct impact of chemical fertilizers on microbial diversity function and phylogeny are not so evident [53]. In the current study, we have compared microbial community structure, diversity, and bacterial phylogeny in chemically cultivated land (CCL), organically cultivated land (OCL), and an adjacent land left as fallow grassland (FGL), where no cultivation have been practiced in last 16 years. OCL under organic farming was fortified annually with composted organic manure (primarily of cow dung) and no tillage and no crop residues were removed for the same period of time, whereas in CCL, chemical fertilizers in form of nitrogen, phosphorus, and potassium were applied. State of the art technology, community level physiological profiling (CLPP), and recently developed 16S rRNA gene-based high throughput sequencing (454 pyrosequencing) were employed to understand the functional microbial diversity and underlying phylogenetic changes in response to long-term use of chemical and organic fertilizers into the farm soil.
Materials and Methods Site Description and Soil Sampling Soil samples were collected from three different fields: (1) treated with chemical fertilizers (CCL); (2) OCL; and (3) untreated for past 16 years (FGL), of Dorli village of Yavatmal District (19.26′ and 20.42′ N, 77.18′ and 79.9′ E) of Maharashtra State of India. Maximum and minimum temperature ranges between 45.6°C and 5.6°C, with mean annual rainfall of 10,566 mm. Due to soil degradation processes such as wind and water erosion, organic matter decline, soil compaction, and soil fertility loss occur rapidly in these areas because of high temperatures and low and irregular precipitation. The region is prone to recurring droughts. Crops grown in the Yavatmal District depend on natural precipitation and therefore, mainly grain legumes are cultivated. OCL received about 20 tons of composted cow manure per hectare. Thus natural grassland was converted to cropland growing grain legume crops under organic management. No tillage and crop residue removal been practiced since the past 16 years. Adjacent land of 1- ha size was left uncultivated for permanent FGL, as described earlier [32]. In CCL, chemical fertilizers were applied in form of elements of nitrogen, phosphorus, and potassium in 60:30:30 kg/ha ratio, respectively.
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The fertilizers were applied annually for the past 16 years. Within each from CCL, FGL, and OCL, four 10×10-m areas were selected for uniform topology and soil type. Soil samples were collected randomly from three places, at the depth of 15 cm, in each of the four selected areas in October 2010. Three samples collected from four areas were randomly mixed, and the composite was considered as the three replicates for analysis. Soil samples were kept in plastic bags on ice and transported to the laboratory and stored at 4°C. All the biological assays were conducted within 48 h. Soil samples were sieved to pass 2-mm round-holed sieve. Visible organic matter was removed before analysis, and the residual soil was air-dried for chemical analysis. Soil Analysis Physical and chemical properties of soil were determined as described by Marinari et al. [29]. Organic carbon was determined by dichromate oxidation [33]. Total available nitrogen was determined by Kjeldahl method [3]. Total available P (w/v) and K (w/v) were analyzed by extracting 5 g soil with 50 ml 2 M KCl, 100 ml 0.5 M NaHCO3, and 50 ml 1 M NH4OAc, respectively [35]. Microbial biomass C was determined by chloroform fumigation extraction method, using 0.5 M K2SO4 as extractant [22]. Soil was preconditioned by spreading between the two polythene sheets for an overnight period. It was then transferred to polythene bags and incubated for 7 days at 25°C in an air-tight container which contained two vials, one with 20 ml distilled water to maintain 100% relative humidity and other with soda lime to absorb CO2. The cover of the container was opened for a few minutes every day for the aeration. The soil was taken out after 1 week and mixed thoroughly for analysis of microbial biomass C by the fumigation extraction method as described above. Cultivable Microbial Population The microbiota in CCL, OCL, and FGL soils was determined by the culture enrichment technique [32]. The total microbial counts were determined using nutrient agar for bacteria, Kenknight and Munaier’s medium for Actinomycetes spp., and Rose Bengal Chloramphenicol agar (HIMEDIA Laboratories Pvt., Ltd., Bombay, India) for selective isolation of fungi. Microbial Metabolic Diversity Using Biolog Biolog ECO GN2 and MT plates (Biolog, Inc., Hayward, CA, USA) were used to determine the community level functional profiling based on carbon source utilization pattern for CCL, FGL, and OCL soil samples. The Biolog MT plates were prepared using the manufacturer’s instructions (Biolog Inc., Hayward, CA 94545, USA). Individual 1-g soil samples were
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Table 1 Physicochemical and microbial properties of soils samples from chemical cultivated land (CCL), fallow grass land (FGL), and organically cultivated land (OCL) CCL
FGL
OCL
pH
8.00
6.50
7.80
Texture Soil EC
Deep black granular sandy loam 0.23
Dark brown granular sandy loam 0.80
Black granular sandy loam 0.40
Total available potassium (kg/ha)
134.97
158.32
848.78
Total available phosphorus (kg/ha) Total available nitrogen (kg/ha)
7.53 188.16
18.28 200.70
40.23 288.51
Organic carbon (%)
0.38±0.05 146.47±8.39 106.87±2.45
0.47±0.02 149.36±10.07 108.37±5.36
1.03±0.02 228.16±3.65 184.24±1.67
4.66 2.26
4.37 2.30
5.16 2.49
3.28
4.22
4.82
−1
Microbial biomass carbon (μg gm of dry soil) Readily mineralizable carbon (μg gm−1 of dry soil) Microbial population (log CFU g−1 soil) Bacteria Fungi Actinomycetes
shaken in 9 ml of sterile saline MQW (0.85% NaCl) for 2 h and then made up to a final dilution of 10−2. After incubation, 150 μl of the sample was inoculated in each well of Biolog Eco and MT plates and incubated at 30°C. The rate of utilization is indicated by the reduction of tetrazolium, a redox indicator dye, which changes from colorless to purple. Data were recorded for day 1–14 at 590 nm. Microbial activity in each microplate expressed as average well color development (AWCD) was determined as described by Garland [12]. Diversity and evenness indexes and principal component analysis (PCA) was performed on the seventh day data divided by the AWCD [11]. Statistical analyses were performed using SPSS 16.0 and Statistica 7.0.
Table 2 Diversity and related evenness indices based on carbon substrate utilization pattern for soils samples from chemical cultivated land (CCL); fallow grass land (FGL) and organically cultivated land (OCL) Diversity measures
CCL
FGL
OCL
Shannon diversity index Shannon evenness McIntosh diversity index McIntosh evenness Simpson diversity index
4.308±0.027b
4.090±0.050a
4.545±0.001c
0.940±0.004b
0.921±0.003a
0.981±0.000c
0.973±0.003b
0.948±0.006a
0.991±0.000c
0.972±0.002b
0.957±0.004a
0.992±0.000c
0.994±0.001b
0.988±0.002a
0.998±0.000c
Different letters showing significant difference at P00.05 using Waller Duncan test CCL chemically cultivated land, FGL furrow grassland, OCL organically cultivated land
DNA Extraction and Pyrosequencing Ultra Clean™ Soil DNA Isolation Kit (MoBio, Solana Beach, CA, USA) was used to isolate the DNA from CCL, FGL, and OCL soil samples according to the manufacturer’s instructions. Variable region V1-V2 for 16S rRNA gene was amplified by using 27 F and 338R composite primers [8, 48]. Unique 10 base identifiers (AGACTATACT for CCL, AGCGTCGTCT for FGL and AGTACGCTAT for OCL, respectively) were used to tag PCR product. Replicate PCR reactions were performed for each sample; reaction mixture consists of 1 μl (10 pmol/μl) of each primer, 40 ng of DNA, 1 μl of dNTPs mix (10 mM stock), 1 unit of Phusion hot start II high-fidelity DNA polymerase, and 10 μl of 5× Phusion HF buffer. Final volume of reaction was adjusted to 50 μl by adding PCR-grade water. Amplification was performed using an initial denaturation of 3 min at 98°C followed by 30 cycles, denaturation at 98°C for 10 s, annealing at 55°C for 20 s, and elongation at 72°C for 30 s. Final extension was at 72°C for 10 min. Amplified products were run on agarose gel; specific bands were excised and amplicons were purified by Qiagen gel purification kit. Concentrations of eluted and purified amplicons were measured by Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). Equal amount of PCR products were mixed in a single tube and sequenced using Roche 454 titanium chemistry. Analysis of Sequences After pyrosequencing, all sequence data were screened and filtered for quality and length using PERL script [13]. Sequences were trimmed and binned by samples using the unique 10 base identifiers as stated above. Sequences failing following
Impact of Chemical and Organic Amendments on Soil 1.8
b CCL
1.6
Absorbance at 590 nm
Figure 1 Categorized carbon substrate utilization pattern of soil samples from chemical cultivated (CCL), fallow grass land (FGL), and organically cultivated land (OCL) using Biolog Eco and GN2 plates after 7-day incubation at 28°C. Bars with the same letter are not significantly different (at P
