Agriculture Reference
In-Depth Information
microbial populations; (c) bioassay of P-solubilizing activity of the microbial
strains; (d) characterization and identification of PSM; (e) bioassay of plant
growth-promoting activities; (f) selection of suitable carriers, mixing of inocula
with selected carriers and development of microbial inoculants; and (g) pot/field
trials of prepared microphos before commercial recommendation for agricultural
practices.
1.5.1 Collection of Samples and Assessment of Microbial
Diversity
The soil samples are collected generally in sterile polythene bags from a depth of
15-12 cm
2
from conventional/polluted non-rhizosphere and rhizosphere soils,
mixed thoroughly and are used for determining microbial diversity. The total
bacterial, fungal, actinomycetal populations, phosphate-solubilizing micro-
organisms (PSM) and asymbiotic nitrogen fixers, for example,
Azotobacter
, can be
isolated using standard media and microbiological methods (Holt et al.
1994
). For
this, soil samples are serially diluted in sterile normal saline solutions (NSS), and
100
l of diluted suspension is spread plated (Buck and Cleverdon
1960
) on nutrient
agar [g/l:beef extract 3; peptone 5; agar 15; pH 7], Martin's medium [g/l: dextrose 5;
potassium dihydrogen orthophosphate 1; magnesium sulphate 0.5; streptomycin
0.006; Rose Bengal 2 part in 3,000 part of medium; 1 g of chloramphenicol/nalidixic
acid can be dissolved in 100 ml of sterile water and 0.3 ml of this solution is added to
100 ml of Rose Bengal medium after it is cooled to 45
C], Kenknight's medium [g/l:
dextrose 1; potassium dihydrogen phosphate 0.1; sodium nitrate 0.1; potassium
chloride 0.1; magnesium sulphate 1.50] or starch casein agar (SCA) medium [g/l:
starch 10; casein 0.3; KNO
3
2; NaCl 2; K
2
HPO
4
2; MgSO
4
·7H
2
O 0.05; CaCO
3
0.02;
FeSO
4
·7H
2
O 0.01 agar 18; pH 7.2; tetracycline (100
μ
μ
g/ml) and amphotericin B
(50
g/ml) are added to medium after autoclaving to prevent bacterial growth and
fungal growth, respectively (Williams and Davies
1965
; Porter and Tresner
1960
)],
Pikovskaya (Table
1.1
) medium, Ashby's medium (Table
1.1
) and yeast extract
mannitol (YEM) agar medium (Table
1.1
) for total bacterial counts, fungal
populations, actinomycetes, phosphate solubilizers,
Azotobacter
and rhizobia,
respectively.
Each sample should be replicated at least three times and incubated at 28
μ
2
C
for 2, 3, 5, 5 and 5 to 7 days for quantifying the populations of bacteria, fungi,
actinomycetes, PSM and
Azotobacter
, respectively. Where microbiological assay is
not done immediately, the samples are kept in sterile polythene bags and stored at
4
C for a short period of time. Standard culture medium and growth conditions
should be used for isolation and enumeration of microbial populations as given in
Table
1.2
.
