2.9. In vivo challenge experiment in greenhouse condition
In vivo challenge experiment was performed in greenhouse condition.
Approximately two months old A. vera plants were taken and
grouped into six sets with four replicates, five plants being taken in
each set. Except for the first set the leaf surfaces of all the plants were
initially sterilized with ethanol (70%). While the first set was kept
untreated, the second set was treated with VBK1 (1.8 108 CFU/ml)
and the third set was treated with VBC7 (2.3 108 CFU/ml). In the
fourth, fifth and sixth sets the leaves were rubbed mildly but
uniformly with sterile sand papers and were sprayed with conidial
suspension (3.1 104 conidia/ml) of A. alternata VBAV007 at the
time of sunset during the months of March–April. A. alternata
VBAV007 was grown on ME agar plates for 5 days at 28 C, scrapped
and suspended in sterilized distilled water to prepare the conidial
suspension. In the fifth and the sixth sets rhizobacteria VBK1 and
VBC7 were applied immediately after applying pathogenic spores.
Biocontrol strains were grown in NB at 28 C and each plant was
sprayed with approximately 10 ml of early stationary phase cultures
at intervals of 15 days, using a hand sprayer. A sticker of CMcellulose
(0.5% W/V)was used in culture suspension before spraying.
Plotted plants were kept in the experimental garden for maintenance
and were watered once in every two days. The disease severity
of the leaf spot disease was calculated over a period of the next three
months. To examine the physical presence of the biocontrol agents
on leaves surfaces, they were re-isolated after 15 days of application
on nutrient agar plates supplemented with two antibiotics, to which
they were found to be resistant.