Age of leaf and plant material treatment
Age of leaf was determined form the day of seed sowing. Three stages of leaves were used to determine the
effect of leaf age on detached leaf bioassay I) Leaves were isolated form 40 day old portray seedlings. II) Terminal
leaflet from 2 month (60 day) old plant and III) The third oldest leaf from 3 month (90 day) old plant were used for
detached leaf bioassay. All the leaf samples were collected, the leaves were treated with 70% ethanol followed by 1%
sodium hypochlorite wash for 2 mins and the samples were washed with sterile distilled water to remove all the traces
of sodium hypochlorite and blotted on a tissue paper. The petiole of the all the leaves were covered with moist cotton to
avoid leaf drying. Sterile blotting paper was cut into the required size of petridishes and moistened with sterile distilled
water. Fungal plug containing Alternariasolani were placed in centre on the adaxial side of leaf along with control leaf
(devoid of fungal plug). Petridishes were moistened with sterile distilled water every 48 hrs. The plates were incubated
in room temperature and all the experiment was carried out in three replications. Observations were taken from third
day and lesion diameter was recorded. In the end of observation a post hoc analysis was carried out by inoculating the
infected leaf on PDA media.