4.4. In Vivo Antifungal Activity Evaluation

The artificial inoculation trials were conducted in a glass house located of the University of Natural Resources and Life Sciences, Vienna, Department of Agrobiotechnology Tulln, Austria. Seeds of the durum wheat genotypes Marco Aurelio and DBC480 were germinated into pots (17 cm diameter, 20 cm height) containing a mixture of peat, compost, and sand and placed in the greenhouse. Temperature in the greenhouse was on average 18/12 °C (day/night) from tillering to heading with 12–14 h of light. During flowering time, the conditions in the greenhouse were controlled and set at 21 °C, 55% relative humidity during daytime and 17 °C, 55% humidity during night with a 16 h photoperiod. Chitosan hydrochloride at 0.5%, tebuconazole at 0.06%, azoxystrobin at 0.08%, and the mixture of tebuconazole 0.06% and azoxystrobin 0.08% were prepared in distilled water as previously described. Macroconidia of the single-spore F. graminearum IFA66 were produced in liquid mungbean medium as described by Buerstmayr et al. (2000, 2002) [105,106]. The mungbean medium was removed from the conidial suspension by multiple centrifugation steps in double-distilled water. Aliquots of conidia stock solutions were stored at −80 °C then thawed at 37 °C and diluted with distilled water to achieve the desired final spore concentration just prior to inoculation. The antifungal compounds were sprayed on plants (wetted until runoff) 48 h before inoculation in two different ways: On the spikes, to test the effective antifungal efficacy of the compounds, or on the flag leaves, to test the ability of boosting the SAR on durum plants. Plants subjected only to artificial inoculation and mock treatment were sprayed with distilled water. Plants were subjected to artificial inoculation at anthesis (Zadok stage 69) [107] by homogenously spraying a conidial suspension of 25,000 conidia/mL, while mock plants were sprayed with distilled water (wetted until runoff). The wheat heads were covered with plastic bags for 48 h to maintain high humidity levels (>80%). The disease incidence and severity of the FHB (%) were determined by counting the number of diseased spikes and the number of bleached spikelets in diseased spikes as well as the total number of spikes and spikelets, respectively, from 3 to 21 dpi. Data were obtained from three independent experiments organized as a complete randomized block, each one consisting of 10 plants for each experimental group (wheat genotype × compound × mode of application).