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Isolation and Screening of Multifunctional Rhizobacteria from the Selected Sites of Madhupur, NARShingdi and Mymensingh, Bangladesh

Moonmoon Nahar Asha
Department of Agricultural Chemistry, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Atiqur Rahman
Department of Agricultural Chemistry, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Quazi Forhad Quadir
Department of Agricultural Chemistry, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Shahinur Islam
Department of Agricultural Chemistry, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

A laboratory experiment was performed to isolate some native rhizobacteria that could be used as bioinoculants for sustainable crop production. A total of 43 rhizobacteria were isolated from undisturbed plant rhizosphere soils of three different locations of Bangladesh and evaluated their plant growth promoting traits, both direct and indirect. The study has screened out isolates on the basis of their phosphorous solubilization and nitrogen (N) fixation. The phosphate solubilization assay in National Botanical Research Institute of Phosphate (N BRIP) medium revealed that 12 bacterial isolates were able to solubilize trical cium phosphate and the rhizobacteria M25 showed best performance with a PSI of 3.33 at 5 day. Exactly 47% (20 isolates) of the isolated rhizobacteria were able to grow in N-free Winogradsky’s medium, which is an indication of potential N₂ -fixers. Among the 20 potential N-fixers, 15 were able to grow within 24 hours of incubation indi cating that they are more efficient in N-fixation. The present study successfully isolated and characterized 43 rhizobacteria. Some of these isolated rhizobacteria have potential plant growth promoting traits and are potential plant growth promoting rhizob acteria (PGPR) candidate. Considering all plant growth promoting traits, the isolate F37 was the best followed by M6. However, further experiments are needed to determine the effectiveness of these isolates under in vitro and different field conditions to understand the nature of interaction with the plant and environment.

Keywords: Rhizobacteria, Bioinoculants, Phosphorus, Solubilization, N-fixation, Plant growth

Location: Shivpur Upazilla, Narshingdi and Botanical Garden of Bangladesh Agricultural University, Mymensingh

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Bio fertilizer

Objectives:

To isolate and purify the rhizobacteria from the forest flora of Madhupur, Narshingdi and Mymensigh and to screen for P-solubilizing and N-fixing bacteria.

Methodology:

Fern (Pteris spp.) was collected from acidic soils of Madhupur forest, Cyperus spp. was collected from Rasulpur during the month of September 2013 and Melastroma samples were collected from red soils of Shivpur Upazilla, Narshingdi and Botanical Garden of Bangladesh Agricultural University, Mymensingh during January 2013. Plant samples were collected along with their root and carefully kept in plastic bags, labeled and sealed and stored in a refrigerator at 4°C. A total of four plant samples were collected for the isolation of rhizosphere bacterial isolates. All the glassware including petridishes were sterilized for 20 minutes at 120°C by autoclave (Model: JSAC-80 JSR). A small part of roots from each sample were separately taken in a test tube. After pouring 10 mL of sterilized distilled water, vigourous shaking was done in bio-safety cabinet (Model: JSCB-900SB JSR). At the end of shaking, the samples were serially diluted upto 10^-1 and 10 ^-2 with sterilized distilled water. Then, 2-3 drops from dilutions and originals were placed on nutrient broth agar (NBA) by Spread Plate Technique and incubated at 28°C for 48h in a microbial incubator. Several screenings were done to isolate pure cultures. Pure colonies were isolated and maintained on NBA plates at 4°C for further studies. For long term storage, the pure isolates were preserved in eppendorf tubes and stored in 10% glycerol solution at -20°C.The bacterial isolates were grown in NBA medium (containing nutrient broth 1%, sucrose (1%, agar 1.5% and pH=6.5) and incubated in microbial incubator at 28°C for 24 hours to study their colony structure, shape, color, elevation and pigmentation. All the glassware and media were sterilized before incubation. To study the biochemical characteristics, all the bacterial isolates were grown in NBA medium and incubated in microbial incubator at 28°C for 48 hours. On glass slide a loopful of bacteria from a well grown colony was mixed with a drop of 3% KOH aqueous solution. Mixing was continued for less than l0 seconds. A toothpick was used for picking as well as mixing bacteria from a colony. The toothpick was raised a few centimeters from the glass slide. Strands of viscid material confirmed that the bacterium was gram-negative. Producing thread with a toothpick indicates that it was gram (+ve) bacteria. A small amount of bacterial isolate was placed from culture onto a clean microscope slide. A few drops of H₂O₂ were added onto the smear. A positive result is the rapid evolution of O₂ as evidenced by bubbling. A negative result is no bubbles or only a few scattered bubbles. All bacterial strains were tested by an agar assay using National Botanical Research Institute Phosphate (NBRIP) containing sucrose = 10 gL ^-1, Ca₃ (PO₄ )₂ =5 g L ^-1, MgCl₂ .6H₂O = 5 g L ^-1, MgSO₄.7H₂O = 0.25 g L ^-1, KCl = 0.2 g L ^-1, (NH₄)₂ SO₄ = 0.1 g L ^-1, pH=7.0 medium supplemented with 1.5% agar. Six strains per plate were stabbed in triplicate using sterile toothpicks. The halo and colony diameters were measured after 5 day of incubation of the plates at 25°C. To study nitrogen fixation ability of the bacteria, isolates were grown in modified Winogradsky’s medium which is an N-free medium and kept for 48 hours in microbial incubator at 28°C.

Source of Information: Agric. Livest. Fish. Vol. 2, No. 1, April 2015: 01-08

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Further study is needed to evaluate microbe-microbe and plant-microbe-environment interactions and their plant growth-promoting activities to ensure their use in a sustainable manner in crop production system.

Knowledge Management: Journal