Morpho-Physiological Diversity of Root Nodule Rhizobia from Mimosa (Mimosa pudica L) and Water Mimosa (<em>Neptunia oleracea</em> L)

Research Article

J Bacteriol Mycol. 2018; 5(1): 1061.

Morpho-Physiological Diversity of Root Nodule Rhizobia from Mimosa (Mimosa pudica L) and Water Mimosa (Neptunia oleracea L)

Rahman MH1, Khatun S1, Ali SR1, Yasmin S1, Kamruzzaman M2 and Rashid MH3*

¹Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh

²Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh, Bangladesh

³Department of Biology, Bangladesh Institute of Nuclear Agriculture, Mymensingh, Bangladesh

*Corresponding author: Rashid MH, Senior Scientific Officer, Biotechnology Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh, Bangladesh

Received: January 22, 2018; Accepted: February 16, 2018; Published: February 23, 2018


Legume genera Mimosa and its root nodulating rhizobia are studied recently by many researchers around the world due to its unique symbiotic relationship with nitrogen fixing bacteria which reduces the requirements of nitrogen during their growth. Considering the scarcity of information on rhizobia from mimosa from Bangladesh, we studied diversity of rhizobia associated with two mimosa species growing at dry land and wet land in Bangladesh. Twenty two (22) strains from root nodules of dry land mimosa and water mimosa from five different districts of Bangladesh and two strains from Malaysia were isolated and characterized on the basis of their growth, morphological and biochemical properties. A diverse characteristics were found in morphological (colony size, shape, color) and physiological (response to BTB reaction, salt, pH and temperature tolerance) characteristics. Their colony size was 1.9-3.5mm and color varied from transparent to milky white; produced green, light blue and blue color in response to BTB reaction; salinity (NaCl) tolerance up to 2.5% (MB-17, MB-22, MB-26, MB-32 and MB-33); growth at highly acidic (pH 4.0; MB-17, MB-32, MB-33, MB-43, MB-47, MB-48, MB-49) medium. The study showed that rhizobial strains from dry land mimosa grown well at highly acidic and saline condition while rhizobia from water mimosa grown well at more alkaline condition. Among 24 strains, MB-17, MB-32, MB-33 and MM- 54 showed resistance to high temperature (43oC). However, rhizobia from dry land mimosa were very specific for nodule formation only with their original host while rhizobia from water mimosa have broader host range to nodulate water mimosa and giant mimosa. Interestingly, two strains (MB-64 and MB-65) from water mimosa were availed to form nodules with three types of mimosa and might have diverse nodulation genes for nodulating a wide range of mimosa. Moreover, in pot experiment the strains MB-49 and MB-66 produced the highest nodules and dry matter weight in dry land and wet land mimosa over control treatment, respectively. This study helps us to know the diversity of mimosa nodulating rhizobia which will extend their application in agriculture practices, biotechnological application and alleviation of salt stress affected soils.

Keywords: Rhizobia; Mimosa; Stress Tolerance; Diversity


Nitrogen (N2) fixation is an ancient prokaryotic trait that predates plant evolution [30]. Some bacteria contains nitrogenase enzyme to assimilate atmospheric nitrogen. The rhizobial enzyme system supplies a constant source of reduced N2 to the host plant and plant furnishes nutrients and energy for the activities of the bacterium. The malate (a breakdown product of sucrose) is the direct carbon source for the bacteroid. The legume plants are capable to produce root nodules which make them an ideal agricultural organism. Within legume nodules, atmospheric N2 is converted into ammonia, which is then assimilated into amino acids, nucleotides, and other cellular constituents. Mimosa poses a unique symbiotic relationship with nitrogen fixing bacteria which reduces the requirements of nitrogen during their growth [8].

Mimosa (Mimosa pudica L.) is a creeping annual or perennial herb, commonly found on cultivated lands or road side of Bangladesh.

It contains alkaloids, non-protein amino acid (mimosine), flavonoids, C-glycosides, sterols, terpenoids, tannins, fatty acids, a-spinasterol and phenyl ethylamine derivatives [17]. Mimosa exhibits various anti-bacterial, anti-depressant, anti-estrogenic, anti-implantation, vibriocidal, aphrodisiac, sinus, urolithiasis and hypolipidemic activity [11,20,31,32]. Mimosa root symbionts are also well-known metal-resistant bacteria [7,28]. Another leguminous plant, water mimosa (Neptunia oleracea L.) has been extensively used in phytoremediation to treat heavy metals (Cd, Cu, Zn, Pb, and Mn), and soluble solids [15,27,33]. Moreover, it efficiently reduces the biological and chemical oxygen demands [33]. Water mimosa is a good source of minerals and vitamins [34]. It exhibits antimicrobial and anticancer properties [4].

Specific strains of rhizobia are required to make functional nodules on the roots of legumes as they can promote growth of the host plants by increasing supply or availability of the primary nutrients, through the natural processes by fixing N2 or via solubilizing phosphorus [29]. Rhizobia are now important component of integrated nutrient management. They are relatively safer, environmentally friendly and cost-effective and an alternative to reduce chemical fertilizer. The rhizobial strains from mimosa have significant effects to increase ‘seedling height’ and ‘total dry weight’ at the later stage of rice [29]. But the selection of bacterial strains with multiple beneficial characteristics is important to maximize the effectiveness on the host plant [16]. It’s important to study the diversity of root nodulating rhizobia of mimosa and water mimosa from Bangladesh to get maximum benefit. Their isolation and characterization might provide a platform for their use in agricultural practice, crop improvement and future research. Therefore, present study was undertaken to assess the morpho-physiological diversity of mimosa and water mimosa root nodulating rhizobia and to identify potential strains for agricultural and industrial use.

Materials and Methods

Nodule collection

The dry land mimosa root nodules were collected from naturally grown mimosa plants from fallow-lands and road sides of four different districts of Bangladesh (Khulna, Magura, Mymensingh and Satkhira). Water mimosa root nodules were collected from naturally grown water mimosa on pond at ‘Sherpur’ districts of Bangladesh. Two strains were isolated from Bangi, Malaysia. Nodules from five plants from each site were collected and preserved in silica gel for further use. Well developed, uninjured, round shape and pink colored healthy nodules were chosen for bacterial isolation purposes.

Surface sterilization of nodules

Collected nodules from silica gel were soaked in sterile water for 5 hours at room temperature before processing for rhizobial isolation purpose. Then nodules were washed with 70% ethanol for 1 minute followed by 3min washing with 2% NaOCl. Then, nodules were washed 7 times with sterile distilled water (dH2O) to remove extra surface disinfectant.

Isolation of rhizobia from nodules

After surface sterilizing, nodules were crushed individually in eppendorf tubes containing 50µL sterile dH2O by sterile micro homogenizer. Subsequently, one full loop of suspension was picked from crashed nodule containing bacteria, and spread smoothly on Congo red yeast extract mannitol agar (CRYEMA) media in Petridish. Then inoculated plates were incubated at 280C for 72 hours. Cultures were purified by repeated streaking on CRYEMA plates to get pure single colony.

Preservation of isolated rhizobial strains

Single colony was preserved in agar slant at 40C for working sample. For long term preservation of the isolates, 5mL of fresh liquid culture of bacteria was taken into the equal volume of 50% glycerol and stored at -800C in 2mL eppendorf tube.

Colony size measurements of isolated strains

Colony size of the strains was measured using millimeter graph paper. A single loop of bacterial culture was streaked on a CRYEMA containing plate sequentially like cord of circle from one edge to another and incubated at 280C. After 3 days, using millimeter graph paper on plates keeping just opposite site of colonies, the diameters of them were measured. Mean value was taken from each strain of five colonies.

Nodulation and cross inoculation test

Mimosa, water mimosa and giant mimosa seeds were collected from field grown plant for nodulation and cross inoculation tests. Collected seeds were taken in 15mL Falcon tubes and washed with flooded sulfuric acids (H2SO4, 98%) for 3 minutes and then the seeds were washed with sterile water. Afterward, 2% NaOCl were added to the seeds and shaking for 10 minutes. Later, the seeds were washed for seven times with dH2O via vortex to remove excess surface disinfectant. Finally, these seeds were immersed overnight in sterilized dH2O in that tube. Overnight water-immersed sterilized seeds were transferred to water-Agar medium in a Petridis for germination in dark for three days at 370C. Germinated seedlings were then transferred to 150mL conical flasks/test tubes containing Fahraeus-nitrogen free medium. After two weeks of plant transfer at plant growth room, three types of mimosa (dry land mimosa, water mimosa and giant mimosa) plants were inoculated with 2mL of each overnight growth bacterial culture. To know the host range, each strain was used to inoculate three types of mimosa (dry land, water and giant). Three replications for each strain were maintained and every flask was labeled according to strain name and date of inoculation. Nodulations were observed after four to five weeks of inoculation. A 16/8 hrs light/dark photoperiods were maintained at growth chamber and plants were irrigated with 0.5X Fahraeus N-free nutrients medium at 7 days intervals for five weeks.

Morpho-physiological characterization of isolated rhizobial strains

In case of bromo-thymol blue (BTB) test, YEMA medium were prepared containing 10mL/L BTB solution in YEMA. For salt tolerance test, media were prepared with 0.5%, 1.0%, 1.5%, 2.0%, 2.5% and 3.0% (w/v) NaCl containing YEMA, in conical flax. For pH tolerance test, four different levels of pH (pH4.0, pH5.0, pH9.0 and pH10.0) were made where the pH was controlled either by adding 1M acetic acid or 1M sodium hydroxide to make YEMA media acidic or basic. For temperature tolerance test, only YEMA media were used but inoculated plates were incubated at different temperature. One µL of each overnight grown rhizobial culture was dropped on labeled squares in petri-plates by micropipette. Three replications were maintained for accurate result. Until the drops were absorbed by the solid media, the petri-dishes wrapped with paraflim. Finally, plates were incubated at 280C for 3 days except culture plates for temperature tolerance test were incubated at 10C, 40C, 310C, 340C, 370C, 400C, 430C and 460C.

Collection and preservation of mimosa seeds for plant infection and symbiotic affectivity test

Mature, healthy and fresh seeds of mimosa and water mimosa were collected from naturally grown plants during October to December in 2015 to conduct plant infection and pot experiment. The collected seeds were dried under sun-light, and then stored in screw caped glass bottle in room temperature for further use.

Symbiotic effectiveness of isolated rhizobia on host plant growth

Each pot contained 2.00Kg soil, mixed with some chemical fertilizer, with TSP (12.38gm), MoP (9.52gm), ZYP (7.73gm), ZnSO4 (0.42gm), H3BO3 (0.45gm). Mimosa seeds were prepared by 10 minutes acidic treatment (98% H2SO4), then 7 times washing with water. Followed by overnight soaked in water; moisture containing viable seeds (4 seeds per pot) were sowed in pots and cultivated as usual. After 2 weeks of sowing, plants were inoculated with bacterial cultures (3mL per plant). Beside bacterial treatment a treatment of urea and a control were kept in the study. Each treatment implemented with 5 replications.

Plant harvesting, dry weight measurement and data analysis

After six weeks of inoculation, the plants were harvested, washed and nodules were separated from the roots. The nodules were separated from plants, counted and data were recorded. Then nodules were preserved in paper packets and plants were kept in brown paper packets and both plants and nodules were dried at 650C for 72 hours in hot air oven, slightly modified from Laguerre et al. [13]. After drying properly the weight of dry mass was measured by electric balance. The data were analyzed using MSTAT-Program and the means were compared using ANOVA at 1% level of statistical significance. The compared effectiveness of each treatment were evaluated through Duncan’s, Multiple Range Test (DMRT) by comparing the mean values of different characters [9] and the results are shown in (Table 5,6).

Results and Discussion

Nodulation and cross inoculation test

In vitro nodulation test was performed on respective hosts (dry land mimosa and water mimosa) to confirm the nodulation nature of our collected strains. We evaluated 24 strains of which 21 strains produced nodule in vitro in their respective hosts (Figure 1). All strains were availed to form nodules with their respective host at laboratory conditions except the strain MB-22 and MB-59 (Table 1). These are possible for opportunistic bacteria which enter insides the nodules with the help of other nodule forming bacteria at field conditions. Thus, the strains MB-22 and MB-59 might be opportunistic bacteria entered into the nodules of mimosa at field conditions with nodule forming rhizobia [24]. Thus, they were unable to form nodules at laboratory conditions. Interestingly, two strains MB-64 and MB-65 were availed to form nodules with dry land and giant mimosa in additions to their respective host suggesting that they had broad host range for nodulation and might have different nodulation genes than other strains from water mimosa.