Standardization of protocols to test wheat (Triticum aestivum L.) for reaction to blast in a biocontainment laboratory
C. D. CRUZ (1), W. W. Bockus (1), K. Pedley (2), G. Peterson (2), J. Stack (1), X. Tang (1), B. Valent (1)
(1) Kansas State University, Manhattan, KS, U.S.A.; (2) USDA-ARS, Fort Detrick, MD, U.S.A.
Phytopathology 101:S40

Growth medium, spore age, and inoculum density are essential factors for determining host responses to a plant pathogen. The standardization of these factors is important to obtain adequate and reproducible disease assessments. We are testing U.S. wheat cultivars for reaction to the exotic disease blast, caused by the fungus Magnaporthe oryzae, in a Biosafety level 3 lab. Although several protocols have been published, it was necessary to adapt those to a biocontainment environment. Four culture media (potato dextrose, V8, oat meal, and corn meal agar), three spore ages (7, 14, and 21 days), and two levels of spore hydration (non-dried and dried) were used to study their effect on appressoria formation by isolate T-25. Over 99% of hydrated and then dried spores did not germinate regardless of growth medium or age, and spores from 14- and 21-day-old cultures had low germinability. The preferred method for production of large amounts of infective spores was the use of 7-day-old cultures from V8 or oat meal agar. Hydrated conidia and conidia that had been dried for 1 min., 15 min., 30 min., or 1 hr, before being rehydrated, were tested for infectivity. In general, dried, and then rehydrated conidia lost their germinability and did not infect wheat. The optimum inoculum density and volume was then determined on three cultivars showing different levels of susceptibility. The preferred inoculum density was 20,000 spores per ml with an inoculation volume of 1.0 ml per wheat head.