Optimization of an in vitro bioassay for wheat diseases

Abstract

Alternaria triticina, Fusarium graminearum, Pyrenophora tritici-repentis, Blpolaris sorokinisna (Helminthosporium sativum); Pythium sp., and Rhizoctonia sp. were tested on the basis of being a representative group of important fungal pathogens in order to establish a reliable bloassay system to test eventual fungal resistance in transgenic plants. The inoculum was prepared on V-8 agar medium (in petri dishes) for P. tritici-repents and Pythlum sp., and on PDA (potato dextrose agar) medium for the other fungi. The cultures were incubated at room temperature for 7- 10 days in a culture chamber with a constant standard illumination. The suspensions of conidia or micella were prepared on sterile distilled water with 1 few drops of tween 20 and scrapings of the fungi culture. The inoculum was homogenized by vorlexing the suspension for a few seconds. The concentration used for the conidia solution was adjusted to 106 conidia/ml. Fresh leaf samples from adult plants (heading stage) were sterilized and then dipped In the inoculum suspension. The inoculated leaves were then transferred to water agar medium (1% agar), in 8-well rectangular multi-dishes, at room temperature. The level of resistance of the material to these pathogens was evaluated 4-7 days after inoculation. We believe that the routine use of this protocol (fast, reliable, and inexpensive) could allow the easy identification of resistant/tolerant plants to these diseases. In addition, because of the simplicity of this test, it can be useful for early screening of a large number of individuals, as required for transgenic plant screenings.