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Isolation, Identification and Characterization of Rhizobium from Lentil Root Nodule

Most. Farhana Begom
Department of Agronomy and Agricultural Extension, University of Rajshahi, Rajshahi, Bangladesh

Md. Giush Uddin Ahmed
Department of Agronomy and Agricultural Extension, University of Rajshahi, Rajshahi, Bangladesh

Rebeka Sultana
Department of Agronomy and Agricultural Extension, University of Rajshahi, Rajshahi, Bangladesh

Md. Zahurul Islam
Horticulture Center, Chapainawabgonj, Bangladesh

Abstract/ Executive Summary:

The use of some microorganisms that are capable of fixing atmospheric nitrogen can reduce chemical (nitrogen) contamination in the soil. The main purpose of this research paper is to isolate Rhizobium as a nitrogen-fixing bacteria from root nodules of lentil (Lens culinaris) and its identification, characterization, and hence the production of biofertilizer. In culture initiation, survival rates were higher using 2-3% chlorox with 70% ethanol as a disinfection medium and the contamination rate was also lower. The primary inoculation of Rhizobium was effected by cutting and smashed nodules at 30-40 days after sowing of lentil plant. Gram staining, biological microscope and scanned electron microscope observations showed that the bacteria were gram-negative, rod-shaped, the length to width ratio is 3:1, and even smooth edges. Rhizobium was also positive in all the biochemical tests. In vitro culture initiation, microscopic reviews, and biochemical testing for specific microbial isolates are essential for microbial fertilizer production.

Keywords: Rhizobium, Root nodules, Red gram, Nitrogen fixation, Biofertilizer.

Location: Agronomy and Agricultural Extension, Rajshahi University, Rajshahi.

Start Date: 00-00-2018

End Date: 00-00-2019

Program Area: Variety and Species

Commodity: Lentil

Objectives:

The objectives of this experiment were isolation of Rhizobium strain from lentil (Lens culinaris) root nodule, identification and characterization of isolated strain and optimization of culture conditions.

Methodology:

Isolation of Rhizobium: Collection of root nodules- Lentil plants were collected from the experiment field of the Department of Agronomy and Agricultural Extension, Rajshahi University, Rajshahi. 45 days old lentil plants were carefully uprooted with the help of grubber for the isolation of bacteria (Rhizobium). The roots were first washed thoroughly under running tap water to remove soil particles and soaked into blotting paper. Surface sterilization of root nodules For the experiment healthy unbroken brown nodules was selected. Guidelines were followed for collecting nodules and preserving according to Date and Halliday (1987) and Somasegaran and Hoben (1994). The nodules were immersed in sterilizing agent Sodium hypochlorite (1%, 2%, 3% and 4%) for 2 minutes and then washed repeatedly with sterile distilled water. After that sterilized in ethanol of 70% and again washed in sterile distilled water for 2 minutes for about 7 times to remove all sterilants. The nodules were cultured by three types; (i) whole nodule (ii) small piece of the nodule (iii) Smashed nodule (crushed each nodule with a sterile glass rod in a test tube and added sterile water). The sterilized nodules were cultured in YEMA plate and incubated at 25⁰C for 2-3 days and checked for possible microbial growth. Preparation of culture media- Yeast Extract Mannitol (Vincent, 1970) Agar plates were prepared and All culture media were sterilized at 121⁰C for 20 minutes by autoclaving. Prepared nodules were cultured in YEMA plates. The samples were inoculated throughout the YEMA plates and were incubated for 2-3 days at 25^oC. Then isolated Rhizobium was sub-cultured in YEMA plates for further experiments. Identification and External morphology study of isolated bacterial strains by Gram staining- The pure cultures of isolated bacterial strains were taken for gram staining for more specific identification of the colonies. The gram staining was done in a laminar airflow hood. For this purpose, the six slides were taken from the slide rack. The slides were washed with ethanol. Then each colony was marked on the slides. Then with the help of inoculating needle, the loopful strains were picked from each test tube and made a smear on the slides and heated to fix. Then the slides were taken in the staining room for staining the smears. Then smears were stained in the following steps (i) First applied crystal violet on every six slides and has kept for 30 seconds. (ii) Washed using sterile distilled water. (iii) Put Iodine on the slides as mordant (1 min) then 95% alcohol washed and then washed with sterile distilled water. (iv) Safranin was applied on the slides and then washed with distilled water. (v) Finally air-dried the slides. The entire gram staining technique was done following the Christian Gram technique and Collee, Mackie (1989). Biochemical characteristics of isolated bacteria Catalase test- This test was performed to study the presence of enzyme Catalase which hydrolyzes H₂O₂ into H₂O and O₂ in bacterial strain. Rhizobial colonies (2-3 days old) were taken on a glass slide and one drop of H₂O₂ (30%) was added. The appearance of a gas bubble indicated Catalase enzyme presence. Lipase test- Lipase presence around bacterial colonies was detected by supplementing YEM with 1% (w/v) Tween 80. Bromothymol blue test- It selectively identifies fast and slow-growing isolates of Rhizobium. In this test, samples were allowed to grow YEMA media contains BTB. After incubation for 48 hours at 25⁰C positive sample showed yellow color due to acid production. Starch hydrolysis- This test was performed to determine the capability of Rhizobium to use starch as a carbon source19. Starch Agar Medium was inoculated with Rhizobium and analyzed for starch utilization. The iodine test was used to determine the capability of microbes to use starch. A drop of iodine (0.1N) was spread on 24-hour old culture and a clear zone of inhibition was formed. Oxidase test- Oxidase test was performed to determine the presence of oxidase enzyme in different isolates of Rhizobium spp. Kovac’s reagent (1% N, N, N.N-tetramethyl- p-phenylenediamine) was dissolved in warm water and stored in a dark bottle. A strip of filter paper was dipped in this reagent and air-dried and put into one-day-old Rhizobial colonies from agar plates. Citrate utilization test- Citrate utilization as a carbon source was examined by adding sodium citrate and Bromothymol blue (25 mg/L) instead of mannitol in YEMA medium. Isolates of Rhizobium spp. were streaked in sodium citrate added YEMA medium plates with bromothymol blue as an indicator. Then plates were incubated for 24 - 48 hours.

Source of Information: International Journal of Biosciences; Vol. 18, No. 4, p. 22-28, 2021

Article Link (Online Journal): http://dx.doi.org/10.12692/ijb/18.4.22-28

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The isolated bacterial culture appeared as Gram-negative rods and they are motile. It gives negative results to methyl red, voges-proskauer, citrate utilization, urease, Catalase, gelatin hydrolysis, and ketolactose test. It gives a positive result to starch hydrolysis and acid from the glucose test. All this biochemical tests confirmed that the isolated bacterial culture is Rhizobium. In this experiment, we used a rapid, inexpensive method for isolating and identifying Rhizobium leguminosarum strains from lentil root nodules. The method can be used for other small or medium-sized nodules producing legume plants.

Knowledge Management: Journal