INTRODUCTION
The irrational and excessive use of chemical fertilizers and pesticides has led to pollution of water, air and soil, which is proving to be a major environmental problem and concern. In this context, deployment of cyanobacteria (blue–green algae), many of which are not only photosynthetic but also nitrogen fixers, can be a promising option. Cyanobacteria comprise a morphologically and physiologically diverse group within the domain Prokarya, which proliferate in a wide range of habitats, including terrestrial, freshwater, marine and saline environments, exhibiting several diverse metabolic activities (Prasanna et al., Reference Prasanna, Singh, Joshi, Madhan, Pal and Nain2010; Whitton, Reference Whitton, Whitton and Potts2000). In the 1970s, algalization or the enrichment of soil via inoculation of selected cyanobacterial strains led to the promotion of these biofertilizers among the farming community of South East Asia (Kaushik, Reference Kaushik, Subramanian, Kaushik and Venkataraman1998; Roger and Kulasooriya Reference Roger and Kulasooriya1980; Venkataraman, Reference Venkataraman1972). In paddy fields, their relative occurrence varies within large limits, ranging from 0 to 85%; and limited systematic analyses on their distribution has been undertaken in relation to major environmental factors (Kaushik, Reference Kaushik, Subramanian, Kaushik and Venkataraman1998; Roger et al., Reference Roger, Zimmerman, Lumpkin and Metting1993; Singh and Bisoyi, Reference Singh and Bisoyi1989; Venkataraman, Reference Venkataraman1972; Whitton, Reference Whitton, Whitton and Potts2000). Analyses of the abundance and genera-wise diversity of cyanobacteria isolated from the rice-based cropping systems of North and Eastern India revealed the dominance of heterocystous forms, with Nostoc and Anabaena comprising 40–90% of isolates (Nayak and Prasanna, Reference Nayak and Prasanna2007; Prasanna and Nayak, Reference Prasanna and Nayak2007; Prasanna et al., Reference Prasanna, Jaiswal, Nayak, Sood and Kaushik2009a). However, the poor establishment of cyanobacteria in soil as a result of predators/grazers and competing native flora have led to inconsistent effects on soil health and crop productivity. This has urged researchers to develop methods to understand their ecological behaviour and establishment using modern tools.
In our earlier studies, a set of cyanobacterial strains isolated from the rhizosphere of rice were evaluated for their establishment dynamics and plant growth-promoting ability in pot experiments, which revealed the persistence of strains on roots up to the harvest stage, entry into roots and enhancement in plant growth and yield attributes, besides improved soil fertility/microbiological parameters (Prasanna et al., Reference Prasanna, Jaiswal, Nayak, Sood and Kaushik2009a, Reference Prasanna, Nain, Ancha, Shrikrishna, Joshi and Kaushikb, 2011a, Reference Prasanna, Sharma, Sharma, Kumar, Kumar, Gupta, Pal, Shivay and Nainb, Reference Prasanna, Joshi, Rana, Shivay and Nain2012). Cyanobacterial inoculation also brings about significant changes in the nutrient status of soil (Ghosh and Saha, Reference Ghosh and Saha1997; Jaiswal et al., Reference Jaiswal, Kashyap, Prasanna and Singh2010; Prasanna et al., Reference Prasanna, Nain, Pandey and Saxena2012). Our present investigation was aimed at analyzing the colonization ability of a set of cyanobacterial isolates from this available germplasm at field level using DNA-based markers and correlating with soil microbiological attributes, plant growth and yields in wheat–rice cropping sequence under field conditions. This can provide an organic option for integrated nutrient management of the rice–wheat cropping system.
MATERIALS AND METHODS
Growth and maintenance of strains
A set of four cyanobacterial strains isolated from paddy rhizosphere of diverse agro-ecologies of India, namely BF1 Anabaena sp. (Aduthurai, Tamil Nadu); BF2 Nostoc sp. (Shivri, Lucknow, Uttar Pradesh); BF3 Nostoc sp. (Faizabad, Uttar Pradesh) and BF4 Anabaena sp. (IARI, New Delhi) were utilized in this investigation. The details regarding these isolates and their characteristics, including 16S rDNA cataloguing (GU396091-94), have been described earlier (Prasanna et al., Reference Prasanna, Singh, Joshi, Madhan, Pal and Nain2010, 2011, Reference Prasanna, Joshi, Rana, Shivay and Nain2012a, Reference Prasanna, Nain, Pandey and Saxenab). Antibiotic sensitivity profiles and carbohydrate utilization patterns have been generated for these strains besides selection of primers and polymerase chain reaction (PCR)-based amplification protocols optimized for distinguishing among the strains under laboratory conditions and in soil microcosm experiments (Prasanna et al., Reference Prasanna, Singh, Joshi, Madhan, Pal and Nain2010). The strains were axenized by standard procedures (Kaushik, Reference Kaushik1987). Routinely, cultures were grown under light:dark cycles (L:D = 16:8), white light (50–55 μmol photons m−2 s−1) and 27 ± 1 °C in nitrogen-free BG-11 medium (Stanier et al., Reference Stanier, Kunisawa, Mandel and Cohen-Bazare1971).
Evaluation of strains in wheat–rice cropping sequence under field conditions
The cyanobacterial cultures were applied at a rate of 100 μg chlorophyll/g of paddy straw compost as a carrier (Rana et al., Reference Rana, Joshi, Prasanna, Shivay and Nain2012). For each treatment, in triplicate, 100 g of cyanobacteria-inoculated formulation was mixed with carboxymethyl cellulose (as a sticker) such that 75% water holding capacity (WHC) was maintained. The seedlings were dipped in this mixture for 1 h before transplanting in fields. Irrigation was done such that the WHC of soil was maintained at 60% as per routine agronomic practices. The effect of selected combinations of four cyanobacterial strains was investigated with wheat and rice crop (2010–2011). The details of treatments for wheat crop are as follows: T1: absolute control; T2: Full dose (FD) N+P+K (120, 60 and 60 kg/ha respectively); T3: 50% N + Full dose of PK; T4: 75% N + Full dose of PK; T5: 75% N + Full dose of PK+BF1+BF2+BF4; T6: 75% N + Full dose of PK+BF1+BF2+BF3. Treatments without cyanobacteria (T2 and T3) represented positive controls, and T1 (without N+P+K or cyanobacteria) served as absolute control. A similar set of treatments with cyanobacterial combinations were taken for rice crop, in which T1, T2, T3, T4, T5 and T6 were same as in the wheat crop treatments, along with an additional treatment T7, i.e. 75% N + Full dose of PK+BF1+BF2+BF3+BF4. The sources of N+P+K fertilizers were prilled urea, single super phosphate and muriate of potash respectively. All treatments included three replicates, in the plot size of 4 m × 3 m = 12 m2. The electrical conductivity (EC) and pH at the initial stage of crop and the harvest stage of wheat and rice crops are given as Supplementary Table 1 available online at http://dx.doi.org/10.1017/S001447971200107X.
Soil core samples for assessing microbiological parameters were collected from root region (0–15 cm) using an auger/soil coring device, in triplicates from each plot. A minimum of three plants were taken for analyzing the plant-related parameters.
Chlorophyll estimation of photosynthetic biomass
Cyanobacterial chlorophyll a was estimated by Mackinney's (Reference Mackinney1941) procedure. Soil chlorophyll was assayed placing the collected soil cores in test tubes after extraction with 4 mL g−1 soil of dimethyl sulfoxide (DMSO) and acetone (1:1). The contents were thoroughly shaken and incubated for 48–96 h in dark at room temperature. Intermittent shaking was done after every 24 h to completely extract the chlorophyll. The spectrophotometric readings were taken and recorded at 663, 645, 630, 775 nm, and chlorophyll a concentration was determined in the soil as described earlier (Nayak et al., Reference Nayak, Prasanna, Pabby, Dominic and Singh2004).
Acetylene reducing activity (ARA) in soil cores
Gas chromatographic estimation of ethylene (as an index of nitrogenase activity and expressed as ARA) in the soil cores placed in the tubes was quantified (Prasanna et al., Reference Prasanna, Tripathi, Dominic, Singh, Yadav and Singh2003). The samples were incubated under a gas mixture, which was substituted with 10% acetylene under standard growth conditions for 3 h. Commercially available standard ethylene was used for calibration and quantification, and vials with equivalent volume of water served as controls. The ARA values are expressed as n moles ethylene produced g−1 soil h−1. Gas samples were analyzed on Hewlett Packard 5890 series II Gas Chromatograph, using a 2-m-long Porapak R stainless steel column and flame ionization detector. The column temperature was maintained at 100 °C, and injector and detector at 110 °C. A flow rate of 35 mL min−1 of N2 served as carrier gas. All values presented are means of triplicate measurements.
Microbiological parameters and yield analyses
The fluorescein diacetate (FDA) hydrolysis assay was carried out using fluorescein standard and the method outlined by Green et al. (Reference Green, Stott and Diack2006). The values are presented as μg of fluorescein released g−1 soil h−1. Dehydrogenase activity was assayed using the method of Casida et al. (Reference Casida, Klein and Santoro1964). The values were expressed as μg of triphenyl formazon (TPF) g−1 soil d−1, and the β-glucosidase activity was measured as given in Prasanna et al. (Reference Prasanna, Sharma, Sharma, Kumar, Kumar, Gupta, Pal, Shivay and Nain2011b). The acid and alkaline phosphatase activity was assayed in soil (1 g) suspended in modified universal buffer (pH 11) along with 1-mL p-nitro phenyl phosphate (Tabatabai and Bremner, Reference Tabatabai and Bremner1969). The methodology of Hesse (Reference Hesse1971) was employed for measuring the organic carbon of the soil and expressed as percentage of carbon. Total polysaccharides and total glomalin were estimated by the methods given by Liu et al. (Reference Liu, Ma and Bamke2005) and Wright and Upadhyaya (Reference Wright and Upadhyaya1996) respectively.
Microbial biomass carbon (C) was estimated by the method of Nunan et al. (Reference Nunan, Morgan and Herlihy1998), using aliquots of K2SO4 extracts through dichromate digestion. Microbial biomass carbon was calculated after back titration with ferrous ammonium sulphate and calculated using the following equation:
\[
{\rm Biomass}\ {\rm C} = 2.64 \times {\rm CE},
\]%
After threshing, cleaning and drying, the grain yield was recorded at 14% moisture. Yield was expressed in t ha−1.
DNA isolation and fingerprinting
Total DNA was isolated using the power soil DNA isolation kit (MP fast DNA kit for soil). Three primer sequences (based on cyanobacteria-specific sequences; Nayak et al., Reference Nayak, Prasanna, Prasanna and Sahoo2009) 5′–3′-short tandemly repetitive repeat (STRR 1A; CCAATCCCCAATCCCC) and palindromic sequences (HIPTG; GCGATCACT) were employed for PCR amplification (Mazel et al., Reference Mazel, Houmard, Castets and Tandeau de Marsac1990; Nayak et al., Reference Nayak, Prasanna, Prasanna and Sahoo2009; Prasanna et al., Reference Prasanna, Singh, Joshi, Madhan, Pal and Nain2010, 2011a; Smith et al., Reference Smith, Parry, Day and Smith1998). Primers were synthesized by Bangalore Genei (India). Total PCR volume was 25 μL, containing 50 pmol of each primer, 2 mM dNTP, 2.5% (v/w) BSA, 10% (v/v) DMSO, 1 U of Taq DNA polymerase, Taq buffer and 20–30 ng DNA. The amplifications were performed in a DNA Engine DYAD™ (MJ Research, USA) as follows: One cycle at 95 °C for 6 min, 35 cycles at 94 °C for 1 min, 56 °C for 1 min, 65 °C for 5 min and 65 °C for 16 min, with the final step at 4 °C. The PCR products were resolved on 1.5% agarose gel with TAE (1×) buffer, and electrophoresis was done at 80 V for 2 h. The PCR reactions were repeated for at least three times with each primer to ascertain the reproducibility of band profiles. The DNA profiles generated in cyanobacteria-inoculated treatments T5 and T6 were compared with pure cultures along with fertilizer controls T2 and T3 from wheat soil samples. The DNA profiles generated in cyanobacteria-inoculated treatments, T5, T6 and T7, were compared with pure cultures along with fertilizer controls T2 and T4 from rice soil samples.
Statistical analyses
Triplicate sets of data for various parameters were analyzed by ANOVA (completely randomized design) using MSTAT-C statistical package, and critical difference values were calculated at p = 0.05. Standard deviation (SD) values are given in graphs as error bars.
RESULTS
Analyses of samples from wheat field samples
Nitrogen fixation, measured as ARA, was statistically at par in all treatments, except absolute control (T1) at the mid crop stage. However, at the harvest stage, the highest values were recorded in 75% N+FD PK+BF1+BF2+BF3, which was statistically at par with T2 Full dose N+P+K (Figure 1a). Chlorophyll accumulation exhibited significantly higher values at the mid stage of crop in T6 (75% N+FD PK+BF1+BF2+BF3) as compared with T2 (Full dose N+P+K). The 75% N+FD PK+BF1+BF2+BF3 treatment led to significantly higher chlorophyll accumulation at the harvest stage (Figure 1b). The values of acid phosphatase, FDA and β-glucosidase were not significantly influenced by cyanobacterial inoculation (Table 1); however, dehydrogenase activity at the mid crop stage was highest in 75% N+FD PK+BF1+BF2+BF3). Organic carbon was significantly higher in both cyanobacteria-inoculated treatments (T6 and T5). Microbial biomass carbon was significantly higher in the 75% N+FD PK+BF1+BF2+BF4 treatment at both stages of crop growth (Figure 2a). The highest values of alkaline phosphatase activity were recorded in both cyanobacteria-inoculated treatments (Figure 2b), involving cyanobacterial inoculation, i.e. BF1+BF2+BF4 and BF1+BF2+BF3.
Influence of different treatments on soil biological parameters in field experiment on (a) ARA; (b) soil chlorophyll, at mid and harvest stages (M/H) of wheat crop. T1: absolute control; T2: M/H, full dose N+PK (120, 60 and 60 kg/ha); T3: M/H, 50% N + full dose PK; T4: M/H, 75% N + full dose PK; T5: M/H, 75% N + full dose PK+BF1+BF2+BF4; T6: M/H, 75% N+PK+BF1+BF2+BF3; BF1: Anabaena torulosa; BF2: Nostoc carneum; BF3: Nostoc piscinale and BF4: Anabaena doliolum.
Influence of different treatments on soil biological parameters: (a) MBC; (b) alkaline phosphatase activity; (c) 1000 grain weight at mid and harvest stages (M/H) of wheat crop. T1: absolute control; T2: M/H, full dose N+PK (120, 60 and 60 kg/ha); T3: M/H, 50% N+PK; T4: M/H, 75% N + full dose PK; T5: M/H,75% N+PK+BF1+BF2+BF4; T6: M/H, 75% N+PK+BF1+BF2+BF3. BF1: Anabaena torulosa; BF2: Nostoc carneum; BF3: Nostoc piscinale and BF4: Anabaena doliolum.
Soil microbiological parameters in soil samples from wheat field, as influenced by various chemical and biological treatments at mid crop (M) and harvest stage (H).
Note. In a column, means followed by a common letter are not significantly different at 5% level.
CD: Critical difference among different treatments.
Analyses of plant parameters revealed that cyanobacterial inoculation with 75% N+FD PK led to statistically at par values of 1000 grain weight, plant height and panicle length with fertilizer controls (Figure 2c; Table 2). Micronutrient uptake, especially Fe, was significantly higher in grain and plants in T6, while for Zn, T6 was statistically at par with T2 (Table 3); however, uptake of Mn was not influenced by inoculation with cyanobacteria.
Influence of various chemical and biological treatments on plant parameters at harvest stage of wheat.
Note. In a column, means followed by a common letter are not significantly different at 5% level.
CD: Critical difference among means at different treatments.
Micronutrient analysis of samples from wheat field at harvest stage as influenced by various chemical and biological treatments.
Note. In a column, means followed by a common letter are not significantly different at 5% level.
CD: Critical difference among means at different treatments.
DNA fingerprinting of soil samples from wheat field
The PCR-based amplification generated bands in the range of 200–3000 bp, with maximum number of bands in the 500–2000 bp region. The comparative analyses of the profiles generated from pure culture vis-a-vis soil samples revealed that amplification with HIP-TG produced distinct and sharp bands, which were present both in DNA profiles of pure cultures and soil from the various cyanobacteria-inoculated treatments (Figure 3a). Among the bands present in the fingerprints of pure culture, which were present in the treatment when amplified with HIP-TG, were bands of 700 bp for BF1; 2600 and 1100 bp for BF2; 2600 bp for BF3 and bands of 3100 and 2100 bp for BF4. Amplification profiles generated using STRR1A with pure cultures and soil from various cyanobacteria-inoculated treatments revealed common bands of 1100 and 800 bp for BF1; 2300, 1800 and 1100 bp for BF2; 2200 bp for BF3 and bands of 2300 and 1800 bp for BF4 (Figure 3b).
Comparative DNA profiles of soil samples collected at mid and harvest stages of wheat crop generated using (a) STRR 1A-based amplification of the soil DNA samples vis-a-vis pure cultures (mid/harvest stages: M/H). T2: M/H, full dose N+PK (120, 60 and 60 kg/ha); T3: M/H, 50% N+PK; T4: M/H, 75% N + full dose PK; T5: M/H, 75% N+PK+BF1+BF2+BF4; T6: M/H, 75% N+PK+BF1+BF2+BF3; (b) HIPTG-based amplification of the soil DNA samples vis-a-vis pure cultures (mid/harvest stages). BF1: Anabaena torulosa; BF2: Nostoc carneum; BF3: Nostoc piscinale and BF4: Anabaena doliolum.
Comparison of the profiles generated using soil DNA samples from treatments at both mid and harvest stages provided interesting observations. Common bands of 2300, 1200 and 800 bp were present in both mid and harvest stages samples, but unique bands of 1800 bp for STRR1A and 2600 bp in HIP-TG were present in sample from treatment involving strain combination, i.e. BF1+2+4 and BF1+2+3, which matched with those present in DNA fingerprints of pure cultures of BF2 and BF3. This shows that BF2 and BF3 strains were present in the soil up to the harvest stage of crop.
Analyses of samples from rice field samples
Nitrogen fixation, measured as ARA, was significantly higher in T6 75% N+FD PK+BF1+BF2+BF3 at the mid crop stage, and T7 75% N+FD PK+BF1+BF2+BF3+BF4 recorded statistically at par values with T4 75% N+FD PK (Figure 4a). Chlorophyll accumulation exhibited significantly higher values at the mid crop stage in 75% N+FD PK+BF1+BF2+BF4 and 75% N+FD PK+BF1+BF2+BF3 as compared with fertilizer control. The 75% N+FD PK+BF1+BF2+BF3 treatment recorded the highest values in term of soil chlorophyll at the harvest stage, while all values were statistically at par at the mid crop stage (Figure 4b). Microbial biomass carbon was significantly higher in the 75% N+FD PK+BF1+BF2+BF4 treatment at both stages of crop growth (Figure 4c). FDA values were highest in T7, 75% N+FD PK+BF1+BF2+BF3+BF4 treatment at both stages of crop growth (Figure 4d). The highest values of alkaline phosphatase activity were recorded in T5, 75% N+FD PK+BF1+BF2+BF4 treatment, which was statistically at par with the T2 FD NPK treatment (Figure 5a). β-glucosidase activity was statistically at par in all treatments at the mid crop stage, but significantly higher in T7 and T6 at the harvest stage (Figure 5b). The values of acid phosphatase in all the cyanobacteria-inoculated treatments were statistically at par with fertilizer controls (Table 4). Dehydrogenase activity (Figure 5c) and organic carbon were not significantly influenced by cyanobacterial inoculation. Total glomalin was highest in T7, 75% N+FD PK+BF1+BF2+BF3+BF4 treatment, while total polysaccharides were statistically at par in all the treatments.
Soil microbiological parameters in soil samples from rice field as influenced by various chemical and biological treatments at midcrop (M) and harvest stage (H), full dose (FD).
Note. In a column, means followed by a common letter are not significantly different at 5% level.
CD: Critical difference among means at different treatments.
Plant parameters from rice field as influenced by various chemical and biological treatments at harvest stage.
Note. In a column, means followed by a common letter are not significantly different at 5% level.
CD: Critical difference among means at different treatments.
Evaluation of different treatments on soil biological parameters in field experiment at mid crop and harvest stages of rice: (a) ARA, (b) chlorophyll accumulation, (c) MBC; (d) FDA. T1: absolute control; T2: M/H, full dose N+PK (120, 60 and 60 kg/ha); T3: M/H, 50% N+PK; T4: M/H, 75% N + full dose PK; T5: M/H, 75% N+PK+BF1+BF2+BF4; T6: M/H, 75% N+PK+BF1+BF2+BF3; T7: 75% N+PK+BF1+BF2+BF3+BF4. BF1: Anabaena torulosa; BF2: Nostoc carneum; BF3: Nostoc piscinale; BF4: Anabaena doliolum.
Evaluation of different chemical and biological treatments in field experiment at mid crop and harvest stages of rice: (a) alkaline phosphatase and (b) β-D glucosidase. T1: absolute control; T2: M/H, full dose N+PK (120, 60 and 60 kg/ha); T3: M/H, 50% N+PK; T4: M/H, 75% N + full dose PK; T5: M/H, 75% N+PK+BF1+BF2+BF4; T6: M/H, 75% N+PK+BF1+BF2+BF3; T7: 75% N+PK+BF1+BF2+BF3+BF4. BF1: Anabaena torulosa; BF2: Nostoc carneum; BF3: Nostoc piscinale; BF4: Anabaena doliolum.
Biomass and crop yield parameters in wheat–rice cropping sequence
Analyses of plant parameters in wheat crop revealed that grain yield recorded in the 75% N+FD PK+BF1+BF2+BF3 treatment was statistically at par with the 50% N+FD PK treatment (Figure 6a). In rice crop, the 75% N+FD PK+BF1+BF2+BF3+BF4 treatment recorded significantly higher grain yield and total biomass (Figure 6b). The other plant parameters (Table 5) revealed that cyanobacterial-inoculated treatments recorded statistically at par values with fertilizer controls.
Effect of different chemical and biological treatments at mid crop and harvest stages of rice on total dry biomass and yield of wheat and rice respectively. T1: absolute control; T2: full dose N+PK (120, 60 and 60 kg/ha); T3: 50% N+PK; T4: 75% N + full dose PK; T5: 75% N+PK+BF1+BF2+BF4; T6: 75% N+PK+BF1+BF2+BF3; (for rice crop only)T7: 75% N+PK+BF1+BF2+BF3+BF4. BF1: Anabaena torulosa; BF2: Nostoc carneum; BF3: Nostoc piscinale; BF4: Anabaena doliolum.
DNA fingerprinting of soil samples from rice field
The comparison between the DNA profiles generated from pure cultures vis-a-vis soil samples from various treatments revealed that both HIP-TG and STRR1A were discriminative in generating pronounced and sharp bands, which were reflective of cyanobacterial colonization in the field (Figures 7a and 7b). The common bands which were present both in treatment and pure culture profiles when amplified with HIP-TG and STRR1A were 3100, 2100, 1500, 900 bp for BF1; 1500, 1100, 900 bp for BF2; 3100, 2600, 1800, 1600, 1500 bp for BF3; 1400 and 1000 bp for BF4; 1200, 800, 650 bp for BF1; 1800 bp for BF2; 1200, 900 bp for BF3 and 1200, 1000 bp for BF4. It is observed that the band of 600 bp was dominant in the profiles of all the treatments, i.e. BF1+2+3, BF1+2+4 and BF1+2+3+4. A dominant band of 600 bp was present in DNA profiles of pure cultures – BF1 and BF2. In the case of HIP-TG, two common bands of 2100 and 1500 bp were prominent in the profiles (Figures 8a and 8b) generated from amplified soil DNA samples from both mid and harvest stages. These bands were also present in pure culture profiles of BF1, BF2 and BF3.
Comparative analyses of DNA fingerprints generated using STRR 1A-based amplification of the soil DNA samples vis-a-vis pure cultures at (a) mid stage samples – T2: full dose N+PK (120, 60 and 60 kg/ha); T4: 75% N+PK; T5: 75% N+PK+BF1+BF2+BF4; T6: 75% N+PK+BF1+BF2+BF3; T7: 75% N+PK+BF1+BF2+BF3+BF4; (b) harvest stage samples – T2: full dose NPK (120, 60 and 60 kg/ha); T4: 75% N+PK; T5: 75% N+PK+BF1+BF2+BF4; T6: 75% N+PK+BF1+BF2+BF3; T7: 75% N+PK+BF1+BF2+BF3+BF4; BF1: Anabaena torulosa; BF2: Nostoc carneum; BF3: Nostoc piscinale; BF4: Anabaena doliolum.
Comparative analyses of DNA fingerprints generated using HIPTG-based amplification of the soil DNA samples vis-a-vis pure cultures: (a) mid stage samples – T2: full dose N+PK (120, 60 and 60 kg/ha); T4: 75% N+PK; T5: 75% N+PK+BF1+BF2+BF4; T6: 75% N+PK+BF1+BF2+BF3; T7: 75% N+PK+BF1+BF2+BF3+BF4; (b) harvest stage samples – T2: full dose NPK (120, 60 and 60 kg/ha); T4: 75% N+PK;T5: 75% N+PK+BF1+BF2+BF4; T6: 75% N+PK+BF1+BF2+BF3; T7: 75% N+PK+BF1+BF2+BF3+BF4. BF1: Anabaena torulosa; BF2: Nostoc carneum; BF3: Nostoc piscinale; BF4: Anabaena doliolum.
DISCUSSION
The continued and indiscriminate use of chemical fertilizers has often led to negative impacts on geochemical cycles, which are integral for sustained soil health and crop productivity (Adesemoye and Kloepper, Reference Adesemoye and Kloepper2009; Berg, Reference Berg2009). In this context microbial inoculants could be useful supplements as components of integrated nutrient management strategies in agriculture (Ashrafuzzaman et al., Reference Ashrafuzzaman, Hossen, Razi, Hoque, Islam, Shahidullah and Meon2009; Mader et al., Reference Mader, Kaiser, Adholeya, Singh, Uppal, Sharma, Srivastava, Sahai, Aragno, Wiemken, Johri and Fried2011).
Cyanobacteria are known to play diverse roles as nutrient supplements (inoculants) in the environment and soil compaction agents in agriculture, besides having tremendous ecological significance as carbon sequestering and bioremediation agents (Irisarri et al., Reference Irisarri, Gonnet and Monza2001; Prasanna et al., Reference Prasanna, Singh, Joshi, Madhan, Pal and Nain2010; Roychoudhury et al., Reference Roychoudhury, Kaushik, Krishna Murty and Venkataraman1979). These are most relevant in wetland rice fields, which supply 86% of the world's requirement for rice, wherein their importance as nitrogen fixers is well documented (Kaushik Reference Kaushik, Subramanian, Kaushik and Venkataraman1998; Mandal et al., Reference Mandal, Vlek and Mandal1998; Singh and Bisoyi Reference Singh and Bisoyi1989); however, the use of molecular tools is quite limited, especially at field level to understand their colonization (Prasanna et al., 2011, Reference Prasanna, Joshi, Rana, Shivay and Nain2012a, Reference Prasanna, Nain, Pandey and Saxenab) as has been done with other bacterial inoculants (Frank et al., Reference Frank, Ryan, Abbas, Mark, Gara, Cooper and Rao2006). It is well known that besides bringing about an improvement in rice yield (ranging from 5 to 25%), they also produce indirect or direct beneficial changes in the physical, chemical and biological properties of soil and soil–water interface in rice fields. Among the several genera of cyanobacteria found in rice fields, Nostoc and Anabaena have been recorded predominantly in most rice field soils from many localities (Nayak and Prasanna, Reference Nayak and Prasanna2007; Prasanna and Nayak, Reference Prasanna and Nayak2007). Despite the fact that the rhizosphere, which is intimately related to the successful production of crops and sustenance of soil fertility, is well recognized as the hot spot for microbial abundance, diversity and metabolic activities, very few reports on the role of cyanobacteria are available in this niche (Ahmed et al., Reference Ahmed, Stal and Hasnain2010; Jaiswal et al., Reference Jaiswal, Prasanna, Nayak, Sood and Suseela2008; Prasanna et al., Reference Prasanna, Jaiswal, Nayak, Sood and Kaushik2009a, Reference Prasanna, Joshi, Rana, Shivay and Nain2012a). Our aim was to illustrate and analyze the establishment of inoculated cyanobacterial strains through the wheat–rice sequence in field and correlate with the crop- and soil-related attributes.
In our study, the influence of four rhizosphere cyanobacterial strains, Anabaena sp. BF1, Nostoc sp. BF2, Nostoc sp. BF3 and Anabaena sp. BF4, in wheat, followed by rice crop in terms of their proliferation and diazotrophic potential in the field was evaluated. In addition, efforts were undertaken to correlate with the fingerprints generated from soil samples at mid and harvest stages of wheat and rice crops, grown in sequence, using PCR-based markers. In the rice field samples, the unique band of 600 bp for STRR1A and two distinct bands of 2100 and 1500 bp for HIP-TG helped to establish the colonization potential of strains used in the rice field, besides highlighting the synergistic interaction among the strains, which were more competitive in soil.
Analyses of samples from wheat field revealed interesting observations. Microbiological parameters, including photosynthetic biomass accretion in terms of soil chlorophyll, alkaline phosphatase activity and carbon (in terms of microbial biomass carbon and organic carbon) were significantly higher in the cyanobacteria-inoculated treatments (75% N+FD PK+BF1+BF2+BF4; 1+2+3). In general, investigations on the role of cyanobacteria in crops other than rice are limited. Karthikeyan et al. (Reference Karthikeyan, Prasanna, Nain and Kaushik2007; Reference Karthikeyan, Prasanna, Sood, Jaiswal, Nayak and Kaushik2009) demonstrated that cyanobacteria excrete indole 3-acetic acid (IAA), amino acids and other growth-promoting compounds into their immediate environment, which in turn can stimulate the growth of microbial populations in soil. This was the first report on the agronomic potential and plant growth promoting effect of these strains in pot experiments, which emphasized their significance as ideal inoculants for wheat crop. Our field experiment results serve to further support these observations.
In the present investigation, samples from rice field also revealed the promise of these strains, which enhanced not only microbiological parameters but also glomalin content, which is an indicator of soil aggregation. Glomalin content was enhanced by 50–90% in the cyanobacteria-inoculated treatments, over the initial pre-transplanting values. The treatments comprising the combination of cyanobacterial strains – 75% N+FD PK+BF1+BF2+BF3 followed by 75% N+FD PK+BF1+BF2+BF4 were the most promising treatments in terms of soil ARA, which was 4–5-fold higher at harvest stage. In our earlier investigations, time course studies undertaken in liquid and soil microcosm experiments inoculated with the same set of four rhizosphere cyanobacterial strains (BF1 Anabaena sp., BF2 Nostoc sp., BF3 Nostoc sp. and BF4 Anabaena sp.) followed by pot experiments with rice crop had revealed similar promising results in terms of soil ARA (Prasanna et al., 2011a, Reference Prasanna, Sharma, Sharma, Kumar, Kumar, Gupta, Pal, Shivay and Nainb).
Nitrogen fixation by cyanobacteria is an economically viable input in rice cultivation and selection, and manipulation of high nitrogen-fixing strains is an ongoing process in most rice growing countries. Nitrogen fixation by blue–green algae is known to vary from a few to 80 kg N ha−1 crop, with a mean of 27 kg N ha−1 (Irisarri et al., Reference Irisarri, Gonnet and Monza2001; Prasanna et al., Reference Prasanna, Jaiswal, Singh and Singh2008; Roger and Kulasooriya, Reference Roger and Kulasooriya1980). In our study, a savings of 25% N could be obtained in both rice and wheat crop at field level, as yield parameters were in general statistically at par with fertilizer controls. Among the several benefits of microbial inoculants, enhanced plant growth and yields are increasingly the focus of most investigations. The increase in percentage of soil carbon through cultivation with cyanobacteria varies with soil type, and in our study an almost two-fold enhancement was recorded in 75% N+FD PK+BF1+BF2+BF3 over Full dose application of NPK in wheat crop. Subhashini and Kaushik (Reference Subhashini and Kaushik1981) observed an increase of 22.1–28.5% in Andhra Pradesh saline soils, 18.75% due to native flora and 32.15% in saline soil of Delhi (Kaushik, Reference Kaushik1989, Reference Kaushik, Subramanian, Kaushik and Venkataraman1998). Micronutrient enhancement can provide an additional benefit to the crop, and in the present study Fe uptake in grain and plants was significantly influenced by application of cyanobacterial consortia, illustrating the significance of cyanobacteria in biofortification.
In an earlier investigation, methods for quantifying the establishment of cyanobacteria using physiological activity (chlorophyll as a growth index and nitrogen-fixing potential as a measure of their biofertilizing capacity), along with DNA fingerprints generated using repeat sequences/palindromes, had provided useful information; however, these were undertaken only in pot experiments (Prasanna et al., 2011). In the present study, DNA fingerprints from field samples revealed that specific bands could be distinguished for monitoring the presence of inoculated strain(s) in various combinations. The presence of unique bands of 1800 and 2600 bp of BF2 and BF3 shows that these strains exhibit better establishment and growth in the field. In rice field samples, the unique bands of 600 bp for STRR1A and 2100 and 1500 bp for HIP-TG show that this strain combination established better in the rice field and there was no competence within the strains. When the comparison between DNA profiles and yield was made, it was observed that the treatment with cyanobacterial combination BF1+BF2+BF4 and BF1+BF2+BF3 showed yields comparable to fertilizer controls. As compared with absolute control, which gave yield of 2.31 t ha−1, treatments 75% N+FD PK+BF1+BF2+BF4 and BF1+BF2+BF3 gave a yield of 3.9 t ha−1, which was statistically at par with all fertilizer controls in wheat crop. In rice crop, the 75% N+FD PK+BF1+BF2+BF3+BF4 treatment was found to record the highest yields, which were 30–40% higher than the samples from plots receiving full dose NPK application. Further, in the present investigation, we used the amplification profiles generated through PCR with STRR 1A and HIP-TG in soil samples and pure cultures, which confirmed the colonization potential and establishment in soil through the wheat–rice-cropping sequence in field. This illustrates that such colonized cyanobacterial strains can lead to definite N savings, which also does not compromise on yields. To the best of our knowledge, this represents the first report on evaluating the establishment of cyanobacterial inoculants in wheat–rice cropping sequence; using a combination of agronomic, microbiological and soil DNA-related attributes. In future, field-level evaluation of these strains under different agro-climatic conditions will help to evaluate their agronomic efficiency and utility in integrated nutrient management of the rice–wheat-cropping system.
Acknowledgements
The study was undertaken as a part of the All India Network Research Project on Biofertilizers (AINPB), funded by the Indian Council of Agricultural Research (ICAR), New Delhi. We thank the Institute (IARI, New Delhi) and the Division of Microbiology and Agronomy for providing necessary facilities involved in undertaking this study.
Supplementary materials. For supplementary material for this article, please visit http://dx.doi.org/10.1017/S001447971200107X.
