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Effects of Plasmid Curing on Rhizobium Spp.

Mukitu Nahar
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh

Hasan M. Zahid
Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission, Dhaka 1000, Bangladesh

Khadiza Zaman
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh

Fahmida Jahan
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh

M Majibur Rahman
Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission, Dhaka 1000, Bangladesh

Rashed Noor
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh

Zinat Mahal
Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission, Dhaka 1000, Bangladesh

Abstract/ Executive Summary:

Present study was carried out to determine the difference between the wild type rhizobial strains and plasmid cured strains for some important characteristics such as antibiotic resistance. The rhizobial strains were isolated from locally produced legumes of different varieties such as Pisum sativum, Sesbania aculeata, Vigna mungo, Phaseolus vulgaris, Lens culinaris and Arachis hypogea.They were purified and subjected to various morphological, cultural and biochemical analysis, and also to antibiogram. All the strains showed resistance against amoxicillin and bacitracin, and exhibited higher (50% or more) resistance against ampicillin, erythromycin, nalidixic acid and strptomycin. The isolates were then subjected to plasmid profile analysis. To determine appropriate dose for plasmid curing, Sesbania nodulating rhizobial strain was treated with different concentrations of acridine orange (plasmid curing agent). Rhizobial growth was found in yeast mannitol broth after curing with 30 μg/ml of acridine orrange. The rhizobial strain was found to be sensitive against amoxicillin, streptomycin and ampicillin after plasmid curing and no exopolysaccharides were found. The present study reveals that the plasmid in rhizobial species might confer the antibiotic resistance and possibly involve in maintaining symbiotic relationships.

Keywords: Rhizobium species; legumes; nitrogen fixation; plasmid

Location: Department of Microbiology, Stamford University Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Pulses

Objectives:

1. In present study, rhizobial species from different leguminous plants were detected and plasmids were cured from the cell using acridine orange to determine their effect on Rhizobium spp. for antibiotic resistance.

Methodology:

The study was carried out within the time frame of January 09 to April 09. Six different types of legume plants were collected from rural areas of Dhaka and surroundings. Fresh and healthy nodules were selected from each plant. The selected nodules were usually light brown or grayish in color, which indicated the establishment of an active nitrogen fixation between the nodule bacteria and the legume plant. Sample Processing. Nodules were thoroughly washed under tap water and then separated from the root. Intact, undamaged nodules were immersed in 95% ethanol for 5-10 seconds, transferred to a 3% solution of H₂O₂ and soaked for 2-3 minutes, following rinsing with sterile distilled water for five times. The primary step of the isolation process was to crush the sterile nodules with a blunt tipped forcep in a large drop of sterile water in a Petri dish. One loopful of nodule suspension was streaked on yeast mannitol agar (YMA) plates. Identification of the isolates. The colony characteristics were determined by observing the colonies on YMA plates. The purity of the rhizobial isolates was detected by adding Congo red (0.25g/100 ml of EtOH; 10 ml stock/liter of YMA) in YMA media. Glucose- peptone medium was used to differentiate rhizobia, which usually shows little or no growth on the media. The presence of the enzyme catalase in the rhizobial isolates was examined by suspending one loopful of organism in a drop of 3% H₂O₂ on a glass slide and formation of bubbles would indicate a positive result. Citrate utilization test was performed using Simmon’s Citrate agar and blue coloration of the media would refer to as positive reaction. Study of antibiogram. Resistance of the test strains against different antibiotics were determined by Kirby-Bauer Method. A suspension of the test organism organism was prepared by adjusting the turbidity, which was then adjusted to a 0.5 McFarland standard. With the help of a sterile cotton swab, a uniform lawn of bacterial growth was prepared on Muller-Hinton agar plates (pH 7.0). Before streaking, the swab was pressed against the wall of the tube containing the suspension to drain out the excess fluid. Antibiotic discs such as ampicillin (10 μg), amoxicillin (25 μg), bacitracin (10 μg), erythromycin (15 μg), streptomycin (10 μg), tetracycline (30 μg), ciprofloxacin (30 μg) were placed aseptically on the surface of the inoculated plates by a sterile needle. The plates were then incubated at 30⁰C for 48-60 hours. After incubation, the plates were observed for the presence of zones of inhibition. The zone diameters for individual antimicrobial agent were translated into susceptible, intermediate and resistant categories by referring to an interpreting table.

Source of Information: Stamford Journal of Microbiology, December 2012. Vol. 2, Issue 1; ISSN: 2074-5346

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

It is well known that acridine orange can also act as a mutagen. However, due to time constraint, it was not possible to study further whether the cured (or mutated) strains had lost its nitrogen fixing or nodulation property. In spite of the diversity of the root nodulating bacteria, present study has opened large prospectives which would make rhizobial biology even more fascinating as loss of plasmids from rhizobia has often been reported and found to be linked to antibiotic resistance. Future study will be needed to determine the mutagenic effects on diverse rhizobial strains and plant infection tests with the wild type and cured strains to compare their nodulation and nitrogen fixing trait.

Knowledge Management: Journal