The Diversity of Legume-Nodulating Bacteria from Several Agroecosystems in Sumberjaya, Lampung

Bacteria that capable of forming root nodules on legumes are known as Rhizobia. They have also known as LegumeNodulating Bacteria (LNB). They can fi x nitrogen from the atmosphere. Diversity of Legume-Nodulating Bacteria is affected by biotic factors (such as their genetic factors, plants, and competition with the other soil microbes) and abiotic factors (such as land use, soil’s temperature, pH, chemistry and soil’s properties). The aim of this experiment is to know the diversity of eleven LegumeNodulating Bacteria based on their phenotypic and genotypic characters. The eleven LNB used in this experiments were isolated from several agroecosystems in Sumberjaya, Lampung. The analysis of these LNB diversity were carried out by characterizing both phenotypic and genotypic properties. The diversity analysis showed that the eleven LNB isolates had high diversity, based on nodule formation, and classifi ed into two groups of cross inoculation group.

Diversity of Rhizobia can be analyzed by characterizing their phenotypic and genotypic properties (Naz et al., 2009).Phenotypic characterization of Rhizobia can be carried out by identifying their cell's morphology, colony's morphology, cell's biochemical properties, their ability to form root nodules (cross inoculation group) and nitrogen fi xation (Boncher, 2009;De Bruijn 1992).Although phenotypic characterization is helpful in the identifi cation of soil rhizobia diversity, the identification based on the molecular approaches believed to be more accurate (Naz et al., 2009).One of the molecular techniques that can be used to quickly identify the microbial diversity is by using Repetitive Extragenic Palindromic Polymerase Chain Reaction (REP-PCR) (Beyer et al., 1998).Diversity of Legume-Nodulating Bacteria in the soil is affected by biotic factors such as cross inoculation group and abiotic factors such as land management (Madigan et al., 2003;Purwaningsih, 2009).

Cells and colony's morphology
Characterization of cell morphology include Gram properties, shape and size of the cells, and cell's motility.Bacterial culture in Yeast Mannitol Broth (YMB) (24-48 h) were used for testing Gram staining, shape and size of cells.Cell's motility was examined by using cultures that were grown on semi solid Yeast Mannitol Agar (YMA).Colony's morphology was examined by using cultures that were grown for 24-48 h on YMA.

Cell's biochemical properties
Biochemical properties were examined by testing isolates growth in several media, namely: Congo Red Yeast Mannitol Agar (CRYMA), Bromothymol Blue Yeast Mannitol Agar (BBYMA), and Glucose Pepton Agar (GPA).Bacterial isolates were also tested in the formation of 3-ketolactose, catalase activity, and the use of multiple sources of C and N.
The ability of nitrogen fixation was examined by using Acetylene Reduction Assay (ARA) (Halbleib and Ludden, 2000).

DNA isolation.
1,5 ml bacterial culture in Nutrient Broth (NB) medium (18-24 h) were transferred into microtube, then centrifuged at 12,000 rpm for 5 min.Supernatant was then discarded and the pellets were dissolved with 410 μl TE (10mM Tris-HCl; 1mM EDTA (pH 8) supplemented with 50 μl lysosim (60 mg/ml) and incubated at 37 o C for 30 min.The solution was added with 30 μl SDS 10% (10 gram in 100 ml sterile distilled water) and 10 μl proteinase-K (20 mg/ml) and incubated at 37 o C for 30 min.It was coupled with 100 μl NaCl 5M and 200 μl solution CTAB 10% in NaCl, then incubated at 65 o C for 10-15 min.After incubation, 600 μl chloroform was added and homogenized, then centrifuged at 12.000 rpm for 5 min.It resulted in a solution with 3 layers.Then the top layer was transferred into new microtubes and isopropanol was added to 500 μl.The mixture was then incubated for 24 h in the refrigerator and centrifuged again at 12,000 rpm for 5 min.Supernatant was discarded and the pellet was washed with 100 ml 70% ethanol and centrifuged at 12,000 rpm for 5 min and then air dried for over night and redissolved with 50 ml TE buffer solution.The purity of genomic DNA were examined by electrophoresis on 1% Agarose.

REP-PCR.
The reaction mixture contained 1μl DNA genome, 12.5 μl KAPPA PCR kit, 9 μl nuclease free water and 2.5 μl primer BOX AIR (5'-CTAC GGCAAGGCAAGGCGACGCTGACGCTGA CG-3'), so the total reaction volume was 25 ml.The reaction mix was placed on a termocycler and subjected to PCR cycles: 95 o C for 4 min., followed by 30 cycles of 94 o C for 30 sec, 51 o C for 1 min.and 65 o C for 8 min, and followed by the fi nal elongation at 65 o C for 8 min.PCR amplified fragments were electrophoresed in an agarose gel (1.5%) for 50 min.and were visualized using ethidium bromide staining.

Phenotypic characterization of Legume Nodulating Bacteria (LNB)
Phenotypic characterization of rhizobia was performed by identifying the colony morphology, cell morphology, Gram properties, biochemical properties of cells (Boncher, 2009) and examination of crossinoculation group bacteria (De Bruijn, 1992).Rhizobium is a Gram-negative, rod-shaped, aerobic, motile and does not form spores. Colonies usually white or cream with circulair shape, convex, semi-transculent or dark and low convex with diameter 2-4 mm at the age of 3-5 days in medium Yeast Mannitol Agar (YMA).The genus Rhizobium bacteria grow at optimum temperature of 25-30 o C at pH 6-7.Almost all bacteria of the genus Rhizobium are able to form root nodules on legume whether or not they perform nitrogen fi xation (Kuykendall et al., 1889).Both morphological observations of the cells or colonies, indicating that overall the tested bacteria are Gram-negative bacteria to form colonies almost entirely circular and forms the majority of the cells are rod (Table 1).
Biochemical data indicated that all of bacterial isolates tested were from different groups (Figure 2).This was indicated by the low coeffi cient of similarity.Only isolates UGM26 and UGM27 that indicated a close relationship with similarity coeffi cient was 1.This indicates that the two isolates were from the same group.
Biochemical data indicated that there was a high diversity of bacterial isolates tested.Judging from the origin of the isolates tested, these isolates came from different agroecosystems.This proved that environmental factors, especially soil tillage systems and different types of crop diversity affect rhizobia in the soil.The evaluation on the ability of root nodule formation showed that there was low cross-inoculation group.This was indicated by the uniformity of the results shown in the fi ve plants used in testing (Table 2).

Genotypic characterization of Legume-Nodulating Bacteria (LNB).
R E P -P C R c a n b e u s e d i n t h e identifi cation and classifi cation of bacterial strains (De Bruijn 1992;Xue-Xian et al.,1999).Techniques Repetitive Extragenic Palindromic Polymerase Chain Reaction (REP-PCR) can be used to distinguish microbial diversity based on the number and spacing of its repetitive sequences.REP-PCR data indicated that eleven bacteria tested had a high diversity (Figure 3 and 4).

Figure 1 .
Figure 1.Plant growth scheme with minirhizotron methods A) plants B) Dispossable petridish covered with aluminum foil, C) root nodules, and D) zeolite

Table 1 .
The observation of colony and cell morphology

Table 2 .
Ability to form root nodules (cross inoculation group) test Figure 2. Dendogram of similarity relationship between tested isolates by biochemical tests