SRI bacterial isolates
A total of seven bacteria isolated from rhizosphere of a SRI fields, SRI-156 (Pseudomonas plecoglossicida; NCBI Accession Number: JQ247008), SRI-158 (Brevibacterium antiquum; NCBI Accession Number: JQ247009), SRI-178 (Bacillus altitudinis; NCBI Accession Number: JQ247010), SRI-211 (Enterobacter ludwigii; NCBI Accession Number: JQ247011), SRI-229 (E. ludwigii; NCBI Accession Number: JQ247012), SRI-305 (Acinetobacter tandoii; NCBI Accession Number: JQ247013) and SRI-360 (P. monteilii; NCBI Accession number: JQ247014), reported earlier by us as potential for biocontrol and PGP traits in sorghum (Gopalakrishnan et al. 2011a), were further studied in this investigation.
Evaluation of SRI isolates for their physiological traits
SRI isolates were streaked on Luria Bertani (LB) agar with various concentrations of NaCl ranging from 0% to 10% at the interval of 2% and incubated at 28°C for 72 h.
The seven SRI isolates were streaked on LB agar, adjusted to pH 5, 7, 9, 11 and 13 and incubated at 28°C for three days. For pH 3, LB broth was inoculated with the seven SRI strains and at the end of 72 h incubation the intensity of growth was measured at 600 nm in a spectrophotometer.
The seven SRI isolates were streaked on LB agar and incubated at 20, 30 and 40°C for 72 h. For 50°C, the LB broth was inoculated with the seven SRI strains, and at the end of 72 h incubation, the intensity of growth was measured at 600 nm in a spectrophotometer.
Antibiotic resistance/susceptible pattern
A total of six antibiotics viz. ampicillin, chloramphenicol, kanamycin, nalidixic acid, streptomycin and tetracycline were studied for their resistance/susceptible pattern against the seven SRI isolates. The required quantities of antibiotics were dissolved in sterilized Milli Q water and mixed into Nutrient agar just before pouring into the Petri plates (when the temperature of the media was about 50°C). Upon solidification, the SRI isolates were streaked and incubated at 28°C for 72 h.
SRI isolates were evaluated for their tolerance to fungicides at field application level. A total of four fungicides viz. benlate (benomyl 50%; methyl [1-[(butylamino) carbonyl]-1H-benzimidazol-2-yl] carbamate), captan (captan 50%; N-trichloromethylthio-4-cyclohexene-1, 2-dicarboximide), bavistin (carbindozim 50%; methyl benzimidazol-2-ylcarbamate) and thiram (dimethylcarbamothioylsulfanyl N, N-dimethylcarbamodithioate) were evaluated at field application levels at 3000, 2500, 4000, 3000 and 3000 ppm concentrations, respectively. The required quantities of fungicides were dissolved in sterilized Milli Q water and mixed into LB agar just before pouring into the Petri plates. Upon solidification, the SRI isolates were streaked and incubated at 28°C for 72 h.
Responses of the seven SRI isolates to salinity, pH, temperature, antibiotics and fungicide tolerance were recorded as follows: - = no growth; + = slight growth; ++ = moderate growth and +++ = good growth.
Evaluation of SRI isolates for PGP traits on rice under field conditions
The experiment was conducted in Kharif 2011 (wet season) at ICRISAT, Patancheru, Andhra Pradesh, India (17o53”N latitude, 78o27”E longitude and 545 m altitude) with a medium duration rice variety, Sampada (135 days).. Soils at the experimental site are classified as sandy loam in texture (55% sand, 17% silt and 28% clay) with alkaline pH of 8.5 − 9.0. The mineral content of the top 15 cm layer were as follows: available nitrogen 292 kg ha-1, available phosphorus 26 kg ha-1, available potassium 527 kg ha-1 and organic carbon 0.76 − 1.27%.
Design and treatments
The experiment was laid out in a randomized complete block design (RCBD) with three replicates and 10 m × 7.5 m subplots. Rice was grown by the system of rice intensification (SRI) method proposed by the Central Rice Research Institute (http://crri.nic.in). The seven SRI isolates were grown on a LB broth at 28°C for three days and further evaluated for their PGP traits. The control contained no SRI isolates.
Protocol for field experiment
Eleven-day-old single seedlings were uprooted from the nursery, their roots dipped in the respective SRI isolates broth (containing 108 CFU ml-1) for 45 min and transplanted on 4th August 2011 at a spacing of 25 × 25 cm. Rice plants were inoculated with the SRI strains (1000 ml; 108 CFU ml-1) once in 15 days until the flowering stage along with the irrigation water. The recommended dose of NPK (120, 60 and 40 kg ha-1, respectively) was supplied through compost and organic manures mixed with farm yard manure and rice straw. Water management was done as recommended for the SRI method, i.e. the alternate wetting and drying method of irrigation. After panicle initiation, all the plots were kept flooded with a thin layer of water (1–2 cm), and all were drained at 15 days before harvest. The crop was harvested manually on 23rd November 2011 and observed for plant height, tiller number, primary and secondary panicle number, panicle length, test seed weight, stover and grain yields and total dry matter. Root samples were collected from 0 to 30 cm soil profile and analyzed for root length density (EPSON expression1640×, Japan), volume and dry weight (dried in an oven at 70°C for 48 h). Soil samples were collected from 0 to 15 cm soil profile at harvest. These were analyzed for soil chemical analysis (% organic carbon, available phosphorous and total nitrogen as per the protocols of Nelson and Sommers 1982; Olsen and Sommers 1982 and Novozamsky et al. 1983, respectively) and biological analysis (dehydrogenase activity, microbial biomass nitrogen and microbial biomass carbon as per Casida 1977, Brooks et al. 1985 and Anderson and Domsch 1989, respectively).
For each trait, data were analyzed by using Analysis of Variance (ANOVA) technique, by SAS GLM (General Linear Model) procedure (SAS Inst. 2002–08, SAS V9.3) considering replication and isolates as fixed in RCBD. Depth wise ANOVA was performed for the traits root length, root volume and root dry weight. Isolate means were tested for significance and compared using fishers protected least significant difference (lsd).