Agriculture Reference
In-Depth Information
hydrochloride, erythromycin, etc.) disc using a sterile forceps. Each antibiotic-
mounted plate is incubated at 28
2
C for 24-48 h. After incubation, the zone
of inhibition is measured, and the strains are scored as resistant (R) and susceptible
(S). Following the standard antibiotic disc sensitivity testing method (Margalejo
et al.
1984
), the plates are recorded for comparing the zone of inhibition (diameter
in mm) with chart provided by the disc manufacturers.
1.5.4.2
Identification of Phosphate-Solubilizing Organisms
Microbial cultures showing greater P-solubilizing activity in vitro, when grown on
Pikovskaya medium, and exhibiting optimum solubilization of insoluble P in liquid
culture medium are selected and presumptively identified to the genus level using
morphological and biochemical test. Such organisms are then identified to the
species level using whole-cell fatty acid methyl ester (FAME) profile and 16S
rDNA sequence analysis (Chung et al.
2005
; Chen et al.
2006
). For 16S rDNA
sequence analysis, partial 16S rRNA gene sequences of selected strains are done
using universal primers, 518 F (5
0
CCAGCAGCCGCGGTAATACG3
0
) and 800R
(5
0
TACCAGGGTATCTAATCC3
0
). All nucleotide sequence data should then be
deposited in the public domain (e.g. GenBank sequence database). There are
various agencies which are providing molecular sequencing for identifying bacte-
rial cultures to species level, for example, Macrogen Inc., Seoul, South Korea. The
online programme BLASTn is then used to find related sequences with known
taxonomic information in the databank at the NCBI website (
http://www.ncbi.nml.
nih.gov/BLAST
)
to accurately identify and compare the isolates with nearest
neighbour sequence available in the NCBI database.
1.5.4.3 Construction of Phylogenetic Tree
The sequence obtained from nucleotide-sequencing agencies is initially estimated
by the BLASTn online programme facility of NCBI (
http://www.ncbi.nml.nih.gov/
ClustalW (Thompson
1994
). Phylogenetic tree is then reconstructed by neighbour-
joining method (Saitou and Nei
1987
). Bootstrapped neighbour-joining relation-
ships are estimated with MEGA4 software (Tamura et al.
2007
).
