PHILIPP AGRIC SCIENTIST ISSN 0031-7454 Vol. 98 No. 3, 270–278 September 2015 Evaluation of Broad-Spectrum Streptomyces sp. for Plant Growth Promotion Traits in Chickpea (Cicer arietinum L.) Subramaniam Gopalakrishnan*, Vadlamudi Srinivas, Gottumukkala Alekhya, Bandikinda Prakash, Himabindu Kudapa and Rajeev Kumar Varshney International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Telangana, India *Author for correspondence; e-mail: [email protected]; Phone: +91 40 3071 3610; Fax: +91 40 3071 3074 The study evaluated three strains of Streptomyces (CAI-21, CAI-26 and MMA-32) for their plant growth promoting (PGP) traits in chickpea (Cicer arietinum L.) under field conditions in two consecutive post-rainy seasons, 2012–2013 and 2013–2014, to confirm colonizing ability in chickpea and to demonstrate gene expression profiles of indoleacetic acid, siderophore and β- 1,3-glucanase genes. The Streptomyces enhanced nodule number, nodule weight, root weight and shoot weight at 30 d after sowing (DAS) and plant height, pod number, pod weight, leaf area, leaf weight and stem weight at 60 DAS in both seasons over the uninoculated control. At crop maturity, the Streptomyces enhanced stover yield, grain yield and total dry matter in both seasons over the uninoculated control. In the rhizosphere, the biological and mineral nutrient activities, such as microbial biomass carbon, dehydrogenase activity, total nitrogen, available phosphorus and organic carbon were also found higher in plots inoculated with Streptomyces in both seasons over the uninoculated control plots. Scanning electron microscopy analysis revealed that the Streptomyces colonized the roots of chickpea. Quantitative real time PCR analysis on selected PGP genes of Streptomyces revealed up-regulation of β-1,3-glucanase gene in CAI-26, β-1,3-glucanase and siderophore genes (siderophore synthetase) in CAI-21 and β-1,3-glucanase, siderophore (siderophore synthetase) and indole-3-acetic acid (IAAH and IAAM) genes in MMA-32. The three Streptomyces demonstrated broad-spectrum PGP activity. Key Words: plant growth promotion, Streptomyces sp., field demonstration, scanning electron microscopy, qRT-PCR analysis Abbreviations: IAA – indoleacetic acid, PGP – plant growth promoting/promotion, qRT-PCR – quantitative real time polymerase chain reaction, SEM – scanning electron microscopy INTRODUCTION 2006; Gopalakrishnan et al. 2011a, b). Streptomyces, Gram-positive bacteria with high In recent years, application of plant growth G + C content, are abundantly found in the promoting (PGP) bacteria is gaining importance in rhizosphere of agriculturally important crops and sustainable agricultural systems because of their low compost. They are known for their PGP traits, the production cost, consumption of less non-renewable breakdown of carbohydrates such as chitin and resources and eco-friendliness in nature. The cellulose and for degradation of soils contaminated mechanisms involved in plant growth promotion by with pesticides and heavy metals. PGP potential of PGP bacteria include secretion of PGP hormones, Streptomyces was reported on cereals such as wheat chelation of iron, nitrogen fixation, solubilization of (Sadeghi et al. 2012) and rice (Gopalakrishnan et al. phosphorus and inhibition of plant pathogens 2013, 2014), legumes such as bean (Nassar et al. (Tokala et al. 2002; Soares et al. 2006; Cheng et al. 2003) and pea (Tokala et al. 2002) and vegetables 2007; Hao et al. 2011; Panhwar et al. 2012). such as tomato (El-Tarabily 2008). Streptomyces Inoculation of PGP bacteria on the rhizosphere had promote plant growth by producing indole-3-acetic been reported to enhance root and shoot growth, acid (IAA) (Aldesuquy et al. 1998) or siderophores nitrogen fixation and solubilization of minerals (Tokala et al. 2002). Besides, Streptomyces have (Shaukat et al. 2006; Richardson et al. 2009). been extensively used for biocontrol of soil-borne Strains of Pseudomonas, Bacillus and Streptomyces fungal pathogens (Mahadevan and Crawford 1997; were reported to help the plants not only by Trejo-Estrada et al. 1998; Macagnan et al. 2008; mobilizing the nutrients but also by controlling Gopalakrishnan et al. 2011b). plant pathogens (Postma et al. 2003; Perner et al. 270 The Philippine Agricultural Scientist Vol. 98 No. 3 (September 2015) Evaluation of Broad-Spectrum Streptomyces sp. in Chickpea Subramaniam Gopalakrishnan et al. Chickpea (Cicer arietinum L.) is the third most 200 mm of plant-available water. The experimental important food legume crop in the world, after bean field was kept fallow except for the post-rainy and soybean, with a total production of 11.6 million season crop. The field was prepared into broad beds tons from an area of 13.2 million ha and a and furrows with 1.2-m wide beds flanked by 0.3-m productivity of 880 kg ha-1 (FAOSTAT 2011). furrows in both seasons (2012−2013 and 2013– Global yields of chickpea have been relatively 2014). stagnant for the last two decades in spite of the use Surface application and incorporation of 18 and of various conventional and molecular breeding 20 kg of N and P ha-1, respectively, as di- approaches. Hence, in the present study, we ammonium phosphate (DAP), were applied 3 d proposed the use of PGP Streptomyces as a tool to before sowing in both seasons. The experiment was enhance the plant growth and yield of chickpea. laid out with three replicates and subplot sizes of 4 Previously, we demonstrated a set of three m × 3 ridges in a randomized complete block design Streptomyces strains (CAI-21, CAI-26 and MMA- (RCBD). 32) isolated from herbal vermicompost, with the potential for bio-control of charcoal rot disease, Evaluation of Streptomyces Strains on Chickpea caused by Macrophomina phaseolina (Tassi) Goid., under Field Conditions in sorghum (Gopalakrishnan et al. 2011a) and for The test Streptomyces (CAI-21, CAI-26 and MMA- PGP in rice (Gopalakrishnan et al. 2012). The 32) were grown individually on a starch casein objectives of this investigation were to further broth at 28 °C for 4–5 d. Seeds of chickpea (variety demonstrate the three Streptomyces strains for their ICCV2) were treated individually with a PGP traits in chickpea under field conditions, to Streptomyces strain (108 CFU ml-1) for 45 min and confirm colonizing ability in chickpea by scanning sown by hand planting on 10 October 2012 in the 1st electron microscopy (SEM) analysis and to year and on 2 November 2013 in the 2nd year in demonstrate gene expression profiling studies by rows 30 cm apart at a depth of 5 cm to have an quantitative real time PCR (qRT-PCR) analysis. estimated plant stand of at least 26 plants m-2. Streptomyces strain (1000 mL; 108 CFU mL-1) was applied once in 15 d, from the day of sowing, on the MATERIALS AND METHODS soil close to the plant until the flowering stage. Control plots contained no strains of Streptomyces. Streptomyces Strains Weeding was performed as and when required Three strains of Streptomyces, CAI-21 (NCBI while the plots were irrigated at 21 and 49 d after accession: JQ682620), CAI-26 (NCBI accession: sowing. No serious plant pathogens or insect pest JQ682621) and MMA-32 (NCBI accession: attacks were observed during the cropping period. JQ682626) reported earlier by us as having potential The crop was harvested manually on 17 January for bio-control of charcoal rot in sorghum caused by 2013 in the 1st year and on 7 Feb 2014 in the 2nd Macrophomina phaseolina (Gopalakrishnan et al. year. 2011a) and PGP in rice (Gopalakrishnan et al. Samplings were done at 30 d after sowing 2012), were further studied. (DAS), 60 DAS and at crop maturity. On the day of The experiment was done for two consecutive sampling, plant aerial and underground parts were post-rainy cropping seasons in 2012−13 and 2013– harvested separately with care to eliminate border 14 at the International Crops Research Institute for effects in each plot, and dried to constant weight in the Semi-arid Tropics (ICRISAT), Patancheru dryers equipped with high air draught at 45 °C for (17o30ꞌ N; 78o16ꞌ E; altitude 549 m) in Telangana 72 h; total dry weights were recorded. State of India. Rhizosphere soils (0–15 cm) at the At 30 DAS, nodule number, nodule weight, root experimental field are classified as Vertisols (fine weight and shoot weight and at 60 DAS, plant montmorillonitic isohyperthermic typicpallustert) height, pod number, pod weight, leaf area, leaf having 53% clay, 21% silt and 25% sand with an weight and stem weight were recorded in both alkaline pH of 7.6−8.3 and an organic carbon seasons (2012–2013 and 2013–2014). At crop content of 0.4−0.6%. maturity, stover yield, grain yield, total dry matter The mineral content of the rhizosphere soil (TDM), 1000-seed weight, pod weight, seed number include 24.7, 8.6 and 298 mg kg-1 soil of available and seed weight were recorded in both seasons. nitrogen, available phosphorus and available Rhizosphere soil samples were collected from potassium, respectively. The soil depth of the 0–15 cm soil profile at crop maturity and analyzed experimental site was ≥ 1.2 m and this soil retained for soil mineral nutrients (total nitrogen, available The Philippine Agricultural Scientist Vol. 98 No. 3 (September 2015) 271 Evaluation of Broad-Spectrum Streptomyces sp. in Chickpea Subramaniam Gopalakrishnan et al. phosphorus and organic carbon per the protocols of The quality and quantity of RNA was estimated by Novozamsky et al. 1983, Olsen and Sommers 1982 a Nanodrop (Thermo Scientific, USA) and RNA and Nelson and Sommers 1982, respectively) and integrity was estimated by a 2100 Bioanalyzer soil biological activity (microbial biomass carbon (Agilent, USA). by the fumigation method and dehydrogenase Quantitative real time PCR (qRT-PCR) was activity by the triphenyl formazan production performed using the Applied Biosystems 7500 Real method per the protocols of Anderson and Domsch Time PCR System with the SYBR green chemistry 1989 and Casida 1977, respectively).