ISOLATION AND IDENTIFICATION OF SOME RHIZOBIAL BACTERIA FROM FABA BEAN PLANTS UNDER EGYPTIAN SOIL CONDITIONS.

Zeinat M. Kamel 1 , Mahmoud M. Nassef 2 and Kholod I. Khalifa 3 . 1. Microbiolody department, Faculty of science, Cairo university, Giza, Egypt. 2. Microbiolody department, Soil, Water and Environment Research Institute, Agricultural Research Center, Giza, Egypt. 3. Microbiolody department, Faculty of science, Cairo university, Giza, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 05 December 2018 Final Accepted: 07 January 2019 Published: February 2019

Five Petri dishes were used as replicates for each particular treatment as well as the control. All dishes were incubated at 28°C for 72 hr then examined for bacterial growth.
Screening for most potent isolates:-A pot experiment was carried out under given conditions of greenhouse to study the ability of tested 16 isolates to form nodules on faba bean compared with the three reference rhizobial strains (ICARDA 441, ICARDA 481 and ARC 202).
Two faba bean varieties [Giza 843 (V1) and Sakha 1 (V2)] were used. The two faba bean varieties were kindly obtained from department of legume crops, field crops research institute, ARC, Giza, Egypt.
Pure cultures of 16 rhizobial isolates and 3 reference strains were prepared by inoculating YEMA broth medium from 24 hours old slant. Liquid cultures were incubated on a rotary shaker at 28 o C for 48 hours. 13 Kalubia K-ISORH13 14 Giza K-ISORH14 15 Baniswaif K-ISORH15 16 Assiut K-ISORH16 The soil was sterilized by NaOCl 2.5%, and then washed several times with distilled water, air dried and distributed into 10 Kg portions in plastic pots 10 Cm diameter. Five seeds from each faba bean variety were sown in each pot. Table (3): Physical and chemical properties of the soil used Genotypic characterization: Isolation of genomic DNA from rhizobial isolates: Genomic DNA of rhizobial cells was isolated and purified using GeneJetTM Genomic DNA purification Kit (Fermentas ® , Thermo Scientific, EU). The procedures were carried out according to manufacture instructions: Rhizobial cells up to 1x10 9 were harvested and resuspended in 180 µl digestion solution. Twenty microliters of proteinase K was added and incubated at 56 o C for 30 min. Twenty microliters of ribonuclease (RNase) solution was added and incubated for 10 min at room temperature. Two hundreds microliters of lysis solution were added and mixed well by vortex for 20 sec. Four hundreds microliters of 50% ethanol were added, transferred to GeneJet TM Genomic DNA purification column and was centrifuged for 1 min at 8000 xg. The column was washed twice by washing buffer I and washing buffer II, separately. Finally purified genomic DNA was eluted by elution buffer. Genomic DNA was electrophorised on 0.7% agarose gel, stained by ethidium bromide and visualized by gel documentation system (BIO-RAD ® , USA).

Amplification and sequencing of 16S Rrna gene:
In the present study, universal primers 27 F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') (Lane, 1991) were used to amplify nearly full-length 16S rRNA gene (1500 bp  Results and discussion:-Isolation and characterization of rhizobia:-Sixteen isolates of rhizobia were recovered from the root nodules of faba bean (Vicia faba L.) grown in different soil locations and properties as well as various climatic conditions in Egypt. Pure colonies of rhizobial isolates were microscopically examined to determine cell shape, size, Gram stain reaction and motility in liquid culture. Colony morphology of rhizobial isolates was studied to determine the transparency and viscosity by using binuclear microscope. Data in table (4) clearly showed that all rhizobial isolates tested were fast growing on YEMA medium, non capsulated, short rod, Gram negative and motile. Cell size ranged from 1 to 4 mm among all tested isolates. The most isolates colonies are opaque (10 isolates) while (6) isolates were translucent. Six of the tested isolates recorded in low viscosity and four isolates recorded in very high viscosity while the other isolates gave high viscosity. In this respect the obtained data are in harmony with data reported by Bergey's Manual of systematic Bacteriology (2005), Garrity, (2005), who reported that the characteristics of rhizobia were short rod (0.5-1.0 x 1.2-3.0 µm), non spore forming, Gram negative reaction and motile by 1-6 peritrichous flagella. Colonies are usually white or beige, crawlers, convex, semi-translucent or opaque raised and mucilaginous usually 2-4 mm in diameter.    , who found that rhizobial isolates were able to grow up to 5% NaCl concentration and not completely inhibited. They added the rhizobial growth was not affected by low and medium level as salinity (NaCl% concentration).

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Data in table (5) clearly showed that both of the isolates K-ISORH7 and K-ISORH15 succeeded to grow in elevated salt concentrations where they showed observed growth till NaCl concentration of 6.0%. So they are considered the most potent and tolerant isolated.

Isolates and nodulation status as well as plant growth: Isolates and nodulation status:
Nodules number (no plant -1 ), nodules dry weight (mg plant -1 ) and nitrogenase activity (ml mole plant -1 hour -1 ) of 16 rhizobial isolates as well as 3 reference rhizobial strains results are presents in table (6). The obtained data illustrated that the all tested rhizobial isolates had nodules formation on the roots of the two faba bean varieties used and scored nodules number values ranged from 10 to 188 nodules per plant and recorded significant differences between the various rhizobial isolates namely K-ISORH13 recorded the lowest nodules number per plant (10 nodules Data present in table (6) indicated that the nodules dry weight of the obtained rhizobial isolates and / or rhizobial references strains had the same trend which obtained at nodules number. The highest nodule dry weight were found at K-ISORH7, 11 and 15 and these values were 597, 421 and 607 mg per plant respectively for faba bean variety G-843 (V1) and the corresponding values for faba bean variety Sakha-1 (V2) were 395, 297 and 404 in the same order.
The lowest values were found at K-ISORH13 (63 mg/plant) at variety G-843 and K-ISORH9 (82 mg / plant) at variety Sakha-1. For reference rhizobial strains tested, the ICARDA-441 gave the highest nodule dry weight and recorded 396 and 330 mg / plant for G-843 and Sakha-1 faba bean varieties respectively, as compared to other tested reference strains. Nitrogenase activity, the all tested rhizobial isolates and strains had a positive effect and scored nitrogenase activity (N-ase) among isolates these values ranged from 1.03 to 81.1, while at reference strains were 1.1 to 10.36 ml mole plant -1 hour -1 . The highest nitrogenase values were found at K-ISORH15 (81.1) followed K-ISORH7 (45.6) with G-843 faba bean variety. In general with regardless the varieties used the highest nitrogenase (N-ase) values scored were 50.1 and 39.8 ml mole plane -1 hour -1 at K-ISORH15 and K-ISORH7 respectively, As compared to other tested rhizobial isolates and strain. From the above mentioned data, faba bean variety G-843 was superior as compared to variety Sakha-1 in the all tested parameters where recorded 69 and 230 for nodule number and nodule dry weight respectively, and the corresponding values at variety Sakha-1 were 59 and 185 in the same order. In this respect, the obtained results are in harmony with (Tsyganov, et al., 2002;Sprent, 2007;Peter, et al., 2013 andMona, 2017) who reported that the legume-rhizobia symbiosis is well known as a highly specific mutually beneficial plant-microbe interaction, which means that certain legume species can only successfully interact with certain strains of rhizobia and they added that the inoculated legume plants with different rhizobial isolates and strains led to scoring the higher values of nodule number and dry weight per plant as well as nitrogenase activity for the nodules formed. Also, Digvijay, et al., (2011), reported that the native rhizobial strains gave a positive response between plant-bacteria relationship as well as eco-soil system and they added to get new rhizobial strains.

Nodules dry weight (mg) Number of nodules Parameters
Isolates  Table 6:-Nodules number, dry weight and nitrogenase activity of 16 various rhizobial isolates and 3 reference rhizobial strains at 45 DAP Isolates and plant growth:-Data of shoot and root dry weight of faba bean plants (gm plant -1 ) as affected by application of 16 rhizobial isolates as well as 3 reference rhizobial strains are shown in table (7). The obtained results illustrated that the all tested rhizobial isolates and/or strains gave significant differences at shoots and roots dry weight. Faba bean plants recorded shoot dry weight ranged from 1.7 to 3.3 gm plant -1 and root dry weight from 0.5 to 1.0 gm plant -1 among the all tested isolates and strains with the two faba bean varieties {G843 (V1) and Sakha1 (V2)} tested. The highest shoot dry weights were recorded at rhizobial isolates namely K-ISORH5, K-ISORH7 and K-ISORH15 and these values were 3.0, 3.1and 3.3 gm plant -1 respectively for faba bean variety G843 and the corresponding values for the same isolates for faba bean variety Sakha1 were 2.1, 2.5 and 2.7 gm plant -1 in the same order.
The results of faba bean root dry weight at table (7) had the same trend which obtained at shoot dry weight and rhizobial isolates namely K-ISORH5, K-ISORH7 and K-ISORH15 scored the highest root dry weight (gm plant -1 ) and these values were 0.9, 0.9 and 1.0 gm plant -1 respectively at V1 and the corresponding values at V2 were 0.7, 1.0 and 1.0 gm plant -1 in the same order.
From the above mentioned data, faba bean variety G843 was superior as compared to variety Sakha1 in both shoot and root dry weight of faba bean plants.
Generally, irrespective to the two faba bean varieties tested, the two rhizobial K-ISORH7 and K-ISORH15 gave the highest shoot and root plants dry weights as compared to other tested rhizobial isolates and strains and recorded 2.8 and 3.0 gm plant -1 for shoot dry weight respectively, and for roots dry weight were 1.0 and 1.0 gm plant -1 in the same order.
In this respect, the obtained results are in agreement with (EL-Zanaty, et al., 2005;Workalemahu, 2009;Digvijay, et al., (2011), Zahran, et al., 2012and Mona, et al., 2016. Who reported that the isolated native rhizobial strains are of good traits also tolerant to high salt levels, have the ability to form root nodules in high number as well as to induce host plant to give good vegetative growth ( shoot and root dry weight).
From the above mentioned data it is clearly shown that the both two rhizobial isolates K-ISORH7 and K-ISORH15 as well as faba bean variety G843 were superior as compared to other tested rhizobial isolates and faba bean varieties at all nodulation and vegetative growth tested parameters.