Agriculture Reference
In-Depth Information
2
C, an orange-brown colour of the
paper indicating HCN production is observed. For ammonia assessment, the bacte-
rial strains are grown in peptone water (g/l: peptone 10; NaCl 5; pH 7) and
incubated at 28
Parafilm, and after 4 days of incubation at 28
2
C for 4 days. One ml of Nessler's reagent [potassium iodide
50 g; distilled water (ammonia free) 35 ml; add saturated aqueous solution of
mercuric chloride until a slight precipitate persists; potassium hydroxide 400 ml;
dilute the solution to 1,000 ml with ammonia-free distilled water. Allow to stand for
1 week, decant supernatant liquid and store in a tightly capped amber bottle] is
added to each tube and the development of yellow colour indicating ammonia
production is recorded following the method of Dye (
1962
).
1.5.5.5 Bioassay of Exo-Polysaccharides
The exo-polysaccharides (EPS) produced by the PS bacterial strains are determined
under in vitro conditions as suggested by Mody et al. (
1989
). For this, PS bacterial
strains are grown in 100-ml capacity flasks containing basal medium supplemented
with 5 % sucrose. Inoculated flasks are then incubated for 5 days at 28
2
Con
rotary shaker (100 rpm). Culture broth is spun (5,433 g) for 30 min, and EPS is
extracted by adding three volumes of chilled acetone (CH
3
COCH
3
) to one volume
of supernatant. The precipitated EPS is repeatedly washed three times alternately
with distilled water and acetone, transferred to a filter paper and weighed after
overnight drying at room temperature. In a study, Ashraf et al. (
2004
) have shown
that inoculating wheat seedlings with EPS-producing bacteria restricts sodium
uptake and stimulates plant growth under salt stress.
1.5.5.6 Determination of Antifungal Activity
Antifungal activity of the PSB against plant pathogenic fungi, for example,
Rhizo-
ctonia
sp.,
Penicillium
sp. and
Alternaria
sp., can be assessed on agar plates as
described by Weller and Cook (
1986
) and Wong and Baker (
1984
). Fungal patho-
gens maintained on potato dextrose agar (PDA) are transferred to Petri dishes
containing fresh PDA (g/l: potato infusion 4; dextrose 20; agar 15; pH 5.4) to
produce fungal mycelium plugs. The PS bacteria are grown in YEM broth (N
2
PSB), Ashby's broth (
Azotobacter
with PS activity) and Luria-Bertani (other PSB
like
Pseudomonas
/
Bacillus
) broth, respectively. A 1-ml stationary cell of each PSB
(10
8
cells/ml) is inoculated into 100 ml YEM broth, Ashby's broth and Luria-
Bertani broth, for rhizobia,
Azotobacter
and PS bacteria, respectively. The samples
(1.8 ml) of each broth is removed in eppendorf and centrifuged at 3,875
g
for
10 min, and the supernatants are filtered through sterile Millipore filter. A 200-
l
sample of each strain is then placed in an 8-mm well cut into the centre of
pre-inoculated fungal plates. Inoculated plates are incubated at 28
μ
2
C for
2 days (PS bacteria) and 5 days (rhizobia and
Azotobacter
), and the zone of growth
