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DNA Isolation, Molecular Characterization and Gene Sequences of Salt Tolerant Rhizobial Strains and Their Symbiotic Effect on Groundnut at Different Salinity Level Under Pot Culture (2018–2019)

F. Alam
Soil Science Division, BARI, Joydebpur, Gazipur, Bangladesh

M. Rahman
Soil Science Division, BARI, Joydebpur, Gazipur Bangladesh

M.F.A. Anik
Soil Science Division, BARI, Joydebpur, Gazipur, Bangladesh

M.E. Ali
Soil Science Division, BARI, Joydebpur, Gazipur, Bangladesh

S. Akhter
Soil Science Division, BARI, Joydebpur, Gazipur, Bangladesh

Abstract/ Executive Summary:

A pot experiment was carried out at the net house of Soil Science Division, Bangladesh Agricultural Research Institute, Joydebpur, Gazipur (24.00^o N latitude, 90.25^o E longitude and 8.4 m elevation) during the rabi season of 2018 with the objective to symbiotic and molecular characteristics of salt-tolerant rhizobial strains and to evaluate the performance of salt-tolerant bacterial trails on growth and yield of groundnut at different salinity level under pot culture. The pot soil belongs to the Chiata series of Grey Terrace Soil. The experiment was laid out in Complete Randomized Design (CRD) with three replications. There were 12 treatments viz. T₁: Rhizobium GR1, T₂: Rhizobium GR2, T₃: Rhizobium GR3, T₄: Rhizobium GR4, T₅: Rhizobium GR5, T₆: Rhizobium GR6, T₇: Rhizobium GR7, T₈: Rhizobium GR8, T₉: Rhizobium GR9, T₁₀: Rhizobium GR10, T₁₁: Rhizobium BARI RAh 801, T₁₂: Control. The pot soils were modified with 0, 4, 8, 12, 16 dS m^-1 salinity level by adding market salt solution on to the pot. The tested crop was groundnut (cv. BARI Chinabadam-8). Seeds were shown on 05 December 2018. Peat based rhizobial inoculum containing 10⁸ cells g^-1 inoculum was used. Seeds were mixed thoroughly with inoculum (20:1 ratio) before sowing. Six seeds were sown in each pot at 1 cm soil depth. Application of salt tolerance Rhizobium biofertilizer on groundnut, the inoculated plants exhibited better performance in nodule numbers, nodule weights, aboveground biomass, root biomass, and plant height than non-inoculated plants, indicating that all Rhizobium sp. effectively enhanced nodulation and growth parameters on groundnut plant than non-inoculated plants in all salinity levels. In pot trials, plants inoculated with Rhizobium sp. showed higher pod yield, stover yield and seed yield than non-inoculated plants, revealing the positive impact of Rhizobium sp. on yield parameters of groundnut. Groundnut plants inoculated with Rhizobium sp.GR9 showed the highest nut yield of groundnut than other strains inoculated and non-inoculated plants at different salinity levels.

Keywords: Salt tolerant, Pot culture, Salinity, Performance, Symbiotic

Location: Soil Science Division, Bangladesh Agricultural Research Institute, Joydebpur, Gazipur, Bangladesh

Start Date: 00-00-2018

End Date: 00-00-2019

Program Area: Crop-Soil-Water Management

Commodity: Soil salinity, Bio fertilizer

Objectives:

The objective to symbiotic and molecular characterize of salt-tolerant rhizobial strains and to evaluate the performance of salt tolerant bacterial trails on growth and yield of groundnut at different salinity level under pot culture.

Methodology:

The experiment was conducted at the net house of Soil Science Division, Bangladesh Agricultural Research Institute, Joydebpur, Gazipur (24.00^o N latitude, 90.25^o E longitude and 8.4 m elevation) during the rabi season of 2018. The experiment was laid out in Complete Randomized Design (RCD) with three replications. There were 12 treatments viz. T₁: Rhizobium GR1, T₂: Rhizobium GR2, T₃: Rhizobium GR3, T₄: Rhizobium GR4, T₅: Rhizobium GR5, T₆: Rhizobium GR6, T₇: Rhizobium GR7, T₈: Rhizobium GR8, T₉: Rhizobium GR9, T₁₀: Rhizobium GR10, T₁₁: Rhizobium RAh 801, T₁₂: Control. The pot soils were modified with 0, 4, 8, 12, 16 dS m^-1 salinity level by adding market salt solution on to the pot. The tested crop was groundnut (cv. BARI Chinabadam-8). Seeds were sown on 07 December 2018. The pot soil belongs to the Chiata series of grey terrace soil. Peat-based rhizobial inoculum containing 10⁸ cells g^-1 inoculum was used. Seeds were mixed thoroughly with inoculum (20:1 ratio) before sowing. Six seeds were sown in each pot in 1 cm soil depth. Phosphorus, potassium, sulphur, zinc and boron @ P₄₂K₄₀S₄₀Zn₅B₁ kg ha^-1 were used in the form of TSP, MoP, gypsum, zinc sulphate and boric acid, respectively. All P, K, S, Zn, B were applied at the time of pot preparation. All the intercultural operations such as irrigation, weeding, insect control etc. were done as and when necessary. Nodules were collected by uprooting carefully and five sample plants were selected randomly from each unit plot at the 50 percent flowering stage. Nodules were separated from the roots, counted and then oven-dried and weighed. Data on yield and yield components were recorded at maturity. The crop was harvested on 02 April 2018. The initial soil samples at a depth of 0-15 cm from the experimental fields were collected and analyzed following standard methods. Organic carbon was determined by the wet oxidation method. Total N was determined by the modified Kjeldahl method. Soil pH was measured by a combined glass calomel electrode. Calcium, K and Mg were determined by NH₄OAc extraction method. Copper, Fe, Mn and Zn were determined by DTPA extraction followed by AAS reading. Boron was determined by CaCl₂ extraction method. Sulphur was determined by CaH₄(PO₄)₂.H₂O extraction followed by turbidimetric turbidity method with BaCl_2.Phosphorus was determined by Bray and Kurtz method (Acid soils) and the Modified Olsen method (Neutral + Calcareous soils). DNA sequencing was performed using an Applied Biosystems 3730 automated sequencer and the M13 primer to obtain nearly full-length bacterial 16S rDNA sequences (approximately 1,500 bp). The bidirectional gene sequences were compiled using DNAMAN software (DNAMAN version 4.11, Lynnon Biosoft, San, Ramon, CA, USA) and the sequences were analyzed with MEGA 5.2 software. The sequences were searched in the NCBI GenBank database using BLAST analysis. Phylogenetic analysis was conducted using MEGA version 5.2 and a neighbor-joining tree is constructed using Kimura 2-parameter distances with 1,000 replicates to estimate bootstrap support. All data were statistically analyzed using two-factor analysis. Treatment effects on measured variables were tested by analysis of variance (ANOVA), and comparisons among treatment means were made using the least significant difference (LSD) multiple range tests calculated at a 5% level of probability (≤ 0.05) (Gomez and Gomez, 1984). Statistical procedures were carried out with the software program Statistix 10™.

Source of Information: Annual Research Report 2018–2019, Soil Science Division, BARI, Joydebpur, Gazipur, Bangladesh

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Achievement/Findings:

Rhizobium biofertilizer applied plants exhibited better performance in nodule numbers, nodule weights, aboveground biomass, root biomass, and plant height than non-inoculated plants, representing that all Rhizobium sp. effectively enhanced nodulation and growth parameters on groundnut plant than non-inoculated groundnut in all salinity level. Rhizobium inoculated groundnut plants showed higher pod yield, stover yield, and seed yield than non-inoculated plants, revealing the positive impact of Rhizobium sp. on yield parameters of groundnut. Groundnut plants inoculated with Rhizobium sp. showed better performance comparatively with Rhizobium sp.GR9 and Rhizobium sp.GR4 considering the average nut yield of groundnut at different salinity levels than non-inoculated plants. Therefore, Rhizobium sp. established an effective symbiotic association with groundnut and was responsible for increased growth and biomass, and improved yield characteristics of groundnut at different salinity levels. Rhizobium sp. GR4 and Rhizobium sp. GR9 bacterial sequences were found, and phylogenetic analysis was revealed that all Rhizobium strains were belonging to bacteria kingdom, proteobacteria phylum, alpha proteobacteria class, rhizobiales order, rhizobiaceae family, and genus Rhizobium.

Knowledge Management: Report/Proceedings