Assessment of microbial biota associated with rhizosphere of wheat (Triticum aestivum) during flowering stage and their plant growth promoting traits.
D Sachdev, V Agarwal, P Verma, Y Shouche, P Dhakephalkar, B Chopade
arthrobacter globiformis, diazotrophs, pseudomonas, rhizosphere, wheat
D Sachdev, V Agarwal, P Verma, Y Shouche, P Dhakephalkar, B Chopade. Assessment of microbial biota associated with rhizosphere of wheat (Triticum aestivum) during flowering stage and their plant growth promoting traits.. The Internet Journal of Microbiology. 2008 Volume 7 Number 2.
Microbial biota associated with wheat rhizosphere during flowering-stage and their plant growth promoting traits was investigated. 16S rRNA gene sequencing was performed of the isolates, which were obtained on selective media. Isolates belonged to: alpha-proteobacteria, beta-proteobacteria, gamma-proteobacteria; Actinobacteria; Bacteroidetes
Rhizosphere soil is a “hot-spot” for microbial growth and major microbial activities. The growth of many microorganisms in the rhizospheric region depends on the root exudates released by the plants (Bais et al. 2006). Interactions between plant and microbes are intensely studied and especially those that benefit plant growth. Such free-living soil bacteria isolated from the rhizosphere of plants, which have been shown to be beneficial for plant growth are referred to as plant growth promoting rhizobacteria (PGPR) (Klopper et al. 1980). It has been well established that PGPR enhance plant growth by direct or indirect means. The direct means may includes : Fixation of atmospheric nitrogen (Zehr et al. 2003; Dixon 1984), production of siderophores (Machuca and Milagres 2003; Schwyn and Neilands 1986), solubilization of minerals like phosphorus (Tilak et al. 2005) and synthesis of phytohormones like indole acetic acid (IAA) (Chung and Tzeng 2004; Huddedar
Among all the plant growth promoting properties, N2 fixation is of prime importance for plant growth. N2 fixers, also called ‘diazotrophs’ play a critical role in the plant ecosystem by reducing dinitrogen (N2) to ammonia (NH3) (Dilworth 1974). N2 fixation is carried out by a diverse group of prokaryotes, Bacteria and Archaea (Zehr et al. 2003). These include symbiotic nitrogen fixing forms;
Wheat is one of the major crops cultivated in India and all over the world. The different stages of life cycle of wheat consist of elongation (30days), flowering stage (45days), fruiting stage (60days) and ripened fruiting stage (75days) (Huddedar and Chopade 2000; Huddedar et al. 2002). It is found that rate of root exudates released by the roots of the wheat at flowering stage is higher as compared to other stages, hence greater microbial biota and activity is expected during this stage (Huddedar and Chopade 2000). Thus present study was done to investigate the microbial biota, with special emphasis on diazotrophs, associated with rhizosphere of wheat variety Lokwan during flowering stage and
Materials And Methods
Collection of soil samples
The rhizosphere soil samples of the wheat during the flowering stage were collected from three agricultural fields in triplicates in the winter season. The wheat plants were uprooted from the agricultural fields and the rhizosphere soil was pooled together and immediately microbiological processing was carried out.
Processing of soil samples
Soil samples were processed within 1-2 h of sampling as follows: 10 g of rhizosphere soil was weighed aseptically and added into 100 ml of sterile phosphate buffer saline (PBS), pH 7.0 in a 250 ml flask. Flask was kept in shaking condition at 200 rpm for 15 min and 1 ml of the suspension was diluted up to 10-7 in 9 ml sterile PBS in tubes. 100 µl of each dilutions, 10-1, 10-3, 10-5 and 10-7 were spread on different nitrogen free media like Burk’s medium, Jensen’s medium, yeast extract mannitol agar,
Identification of the bacterial isolates by 16S rRNA gene sequencing
Genomic DNA was extracted from the isolates by the standard method (Sambrook
PCR products were purified using the QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer’s suggested protocol and nearly complete sequences of 16S rRNA genes were obtained using the primers 343r, 27f, 1525r (Rainey
The partial16S rRNA gene nucleotide sequences of all the isolates determined in this study have been deposited in GenBank database (http://www.ncbi.nlm.nih.gov/GenBank/index .html) under accession numbers as mentioned in Table 1.
Screening of plant growth promoting traits
The isolates were grown on Jensen’s nitrogen free medium (Himedia, Mumbai, India) for 48 h. The cotton plug was then replaced with gas tight septa and 2 ml of air was replaced with acetylene gas (10%). The tubes were incubated for 24 h and the production of ethylene was detected using gas chromatography with FID detector (Hardy
Indole acetic acid (IAA) production
Isolates were grown at 28oC in LB broth supplemented with 1mg/ml of tryptophan (LBT). After 48 h of incubation at 150 rpm, cells were harvested by centrifugation at 10000 rpm for 10 min at 4oC. 1 ml of supernatant and 4 ml of Salkowski reagent were mixed and allowed to react in dark at room temperature for 20 min. Development of pink to red color formation was considered positive evidence for IAA production (Huddedar et al. 2002).
10 µl of overnight grown isolates in LB broth were spot inoculated on Pikovskaya’s agar (Pikovskaya 1948) and incubated at 28oC up to 5 days. The bacterial isolates forming clear halos were considered positive for phosphate solubilization.
10 µl of overnight grown isolates in LB broth were spot inoculated on chrome azurol S agar plates and incubated at 28oC up to 5 days. Microorganisms exhibiting an orange halo were considered positive for production of siderophores (Schwyn and Neilands 1987).
Seven fungal phytopathogens viz;
Results And Discussion
Rhizosphere is a rich habitat of microbes and should be explored for obtaining potential PGPR, which can be useful in developing bio-inoculants for enhancement of growth and yield of crop plants. Enumeration of total bacteria population was determined on standard plate count (SPC) agar. Gram-negative bacterial populations were enumerated on violet bile red (VRB) agar. Count of Gram negative population in the rhizosphere during flowering stage was found to be about ca. 107 per g of soil. A total of 69 strains were isolated in this study from rhizosphere of wheat at flowering stage. 16S rRNA gene of these isolates was sequenced using internal primers (≥90% of the
The organisms were identified as
To best of our knowledge the following are the nitrogen fixers reported from the rhizopshere of wheat :
Each of the isolates was investigated for the plant growth promoting traits such as production of IAA and siderophore, as well as ability to inhibit fungal growth and solubilize phosphate. The PGP properties of each isolate tested are mentioned in Table 1. It was observed that 31 isolates from rhizosphere of wheat produced plant growth promoting hormone IAA. Production of IAA by
Seven isolates from rhizosphere demonstrated
Eleven strains isolated from rhizosphere of wheat were able to produce siderophore.
Fifteen strains isolated from rhizosphere exhibited
This research was supported by a grant from the Department of Biotechnology (DBT), Govt. of India, New Delhi (Project Sanction No. BT/PR6454/AGR/05/302/2005) awarded to Professor B.A. Chopade and Ms. Dhara Sachdev was recipient of SRF. We are thankful to Soil Survey and Soil Testing Laboratory, Krushi Bhavan, Govt. of Maharashtra, Shivajinagar, Pune and Soil Chemistry Section, Mahatma Phule Krishi Vidyapeeth, Krishi Mahavidyalaya, Pune for physical and chemical analysis of the soil samples. We are grateful to Professor B.P. Kapadnis, Department of Microbiology, University of Pune for help with the fungal inhibition assay.