Rust samples must be collected and prepared before using them to inoculate the seedlings of the susceptible host or the differential lines. For the survey, required materials are GPS, paper bags, pencil, and alcohol. Samples of stem rust infected stems/leaves are collected at 5-10 km intervals from wheat fields. Separate the infected leaf sheath from the stem and cut the infected stems into small pieces of 5-10 cm length (Fig. 6) and place them in paper bags — this allows the viability of spores to stay reasonably high. Label the paper bags with the name of the zone, district, variety, GPS data, name of collector and date of collection (Fig. 7) and then transport them to the laboratory to be prepared for race analysis.
After collecting and bringing the infected stem rust samples from the wheat field, the rust spores must be collected into gelatin capsules using a vacuum pump in the cubicle (Fig. 8). If a vacuum pump is not available, spores may be collected on clean glasses/petri dishes by scraping the infected stem with a scalpel. When spore collection of the sample is finished, sterilize hands and other materials with 70% solution of alcohol. Collect the spores from the next sample using a new clean compartment. The same precaution is followed for each sample.
Rust inoculations are performed in an independent room that consists of two inoculation chambers. The pieces of equipment necessary for inoculation are: the inoculation chamber, rotating table, vacuum pump, inoculator, pipette, mineral oil such as Soltrol, gelatin capsules, test tubes, hand-sprayer, distilled water, watch glass, scalpel, cryovial, Tween 20, and labels.
Inoculations in the greenhouse are done either to revitalize the field spores, multiply the isolate, or inoculate the differential lines sets for race identification. Inoculation of the susceptible check McNair, or stem rust differentials, is usually done late in the afternoon when the ambient temperature is low and cool. Cool temperatures help moisture to stay longer on the leaves, thus facilitating the germination of spores resulting in infection.
The equipment/materials are organized to do the inoculation by bringing the wheat seedlings from the seedling room into a clean inoculation chamber. The seedlings, on a tray, are placed on a rotating table. To prepare the inoculum, Soltrol oil is added to the spores in the gelatin capsule and mixed by inversion. The solution should be light brown/tea color (Fig. 9). When you suspend the spores with Soltrol in the capsule, pipette the Soltrol not directly on the spore mass, but on the internal wall of the capsule in order to reduce contamination. Tips should be changed between samples or rinsed and dried with alcohol for future use.
For inoculation, the gelatin capsule containing the inoculum is attached to the inoculator, which is attached to the hose of the electric vacuum pump. During inoculation, the rotating table is rotated clockwise as well as counter-clockwise to ensure the inoculum spray covers all the leaves of the seedlings (Fig. 10). During inoculation with the vacuum pump, do not get too close to the seedlings, but inoculate from a given distance away (6-8 inches/15-20 cm) so that the spread of inoculum covers the whole seedlings. After each inoculation, the pump is turned off and the persons working close to the samples should sterilize their hands with 70% alcohol.
If a vacuum pump is not available, you can inoculate the seedlings using a scalpel, after mixing the spores with Soltrol oil or distilled water with a droplet of Tween 20 surfactant on a watch glass. Or, turn-by-turn, you can deeply rub the seedlings on the watch glass with the spore suspension.
In order to clean and wash down the suspending spores after inoculation, spray/shower the chamber with water (Fig. 11). After each inoculation, and before doing the next one, technicians should sterilize their hands and other necessary materials with alcohol. The inoculation of another isolate continues in a second chamber and then come back to the first chamber for the third isolate. After all isolates are inoculated, clean the chambers with moistened clothes. The equipment and materials used during inoculation must be sterilized and dried with clean cloths. Participants in this activity should wear gowns.
Upon completion, the inoculated seedlings are placed on a table for 30 minutes until the Soltrol evaporates and leaves have dried out. Following this, the seedlings are moistened by spraying with distilled water and placed in the incubation chamber.
Inoculators and collectors have to be autoclaved periodically in an oven at 120°C for an hour in order to burn the spores that clog the tubes.