Two compartments in the incubation room have light, dew chambers, and humidifiers. The incubation/dew chambers are wooden boxes covered with white polyethylene sheets (Fig. 12). The dew chamber must be cleaned with water before and after the incubation process.
A black polyethylene sheet should cover the white sheet in order to create darkness in the box; this condition facilitates the initial infection process of stem rust. After drying the inoculated seedlings for 30 minutes on a table, moisten them by spraying with distilled water. Then place them in the dew chamber for a longer period (overnight) to create high relative humidity. Leave the humidifier on for about 1-1:30 hours so the seedlings have enough moisture for the whole dark period, making the infection process successful (Fig. 13). The dew facilitates the germination of the spores on the leaves. If dew is not abundant during the whole dark period, the spores on the seedlings will not germinate and cause infection.
For infection, stem rust requires dark period of 16-17 hours, so cover the chamber with the black polyethylene sheet for that period (Fig. 14). After the sheet is removed, the seedlings must be exposed to light for about 4 hours. The light is again turned off and the door of the dew chamber opened so that the seedlings dry gradually. This hardens the seedlings for when they are taken to the outside environment. Once leaves are dried, the seedlings are taken to the growth room/greenhouse until disease develops.
In addition to preparing seedlings of the differential lines, it is important to grow seedlings of a known susceptible variety—usually this susceptible check variety is McNair. McNair is planted for three purposes: to revitalize the rust spores collected from the field for further investigation; to multiply the rust races; and to be part of the identification set (Fig. 15). Whenever McNair is used as a susceptible check and for multiplying inoculum, it is advisable to use Maleic Hydrazide 99% (1,2 Didydropyridazib-3-6-dion 99% ) C4H4N2O2 at the concentration of 0.3g/l, applied when the emerging coleoptile is visible. This Maleic Hydrazide is used as growth regulating agent on the check and to make seedlings more susceptible. To prepare the Maleic Hydrazide solution, allow 0.3g of the chemical to dissolve in 1 litre of water at least for one hour on a stirrer. The solution then is poured on the coleoptile at 10-20 ml per pot. Never apply this chemical on the differential lines.
After inoculation and incubation, the seedlings are taken to the growth room where they are kept on a table for 14-15 days until disease reaction can be evaluated (Fig. 16). If the season is cold (7-12°C night and morning), the evaluation date can be extended from 14 days to 17 days provided that the pustules have developed well and the leaves of the seedlings are green.
When placed in the greenhouse, the seedlings are kept at the temperature of 18-25°C and relative humidity of 60-70%. Each set of inoculated differentials plus one pot of McNair are placed on a table separated from each other (Fig. 17). On the 6th or 7th day after inoculation, flecks/chlorosis appear on the leaves. The inoculated McNair pot and the differential set are removed and separately placed in a clean cubicle. This is done to reduce contamination of the isolate from the other pots. If the isolate is to be repeated on the differential, the rust isolates from McNair are used for re-inoculation.
When the flecks appear on the leaves, a small amount of urea is applied on the seedlings so that infection continues successfully (Fig. 18). It is also important to place isolates inoculated the same day together, but not touching each other, on a bench in the growth room.