Comparative assessment of spray nozzles efficacy in the control of fusarium head blight in the barley crops using developed quantitative PCR assay

Abstract

Fusarium species infect cereal spikes during anthesis and cause Fusarium head blight (FHB), a destructive disease of cereal crops with worldwide economic relevance. The necessity for these phytopathogenic fungi effective control becomes increasingly important for the production of both cultivated plants and those plants seeds. Fungicide application is a key methodology for controlling the disease development and mycotoxin contamination in cereals. Polymerase chain reaction (PCR) is currently the most commonly admitted DNA-based technology for specific, rapid and precise Fusarium detection. We have developed and patented the method for detection and quantitative determination of phytopathogenic fungi F. avenaceum and F. graminearum in plant seeds using Real-Time PCR with a pair of primers, designed to amplify sequences of the internal transcribed spacer at the ribosomal RNA gene cluster of those phytopathogenic fungi. This study was aimed to perform a comparative assessment of the efficacy of different spray nozzles for antifungal treatment to control F. avenaceum and F. graminearum infection of barley grains using a developed qPCR diagnostic system. A single application of a fungicide (active ingredient's content: 250 g/l propiconazole, 80 g/l cyproconazole) at BBCH 65 (middle of flowering) was carried out. For this purpose, four spray nozzles with different technical characteristics were used: Flat Fan 030, Amistar 030, Defy 3D 030 and Vegetable 060 (Pentair, USA). DNA-based fungi detection and identification was performed using conventional PCR and developed qPCR. The level of mycotoxins in barley grain was determined using enzyme-linked immunosorbent assay (ELISA). Grain count in the ear of barley and thousand seed weight (TSW) were also examined. A single application of the fungicide inhibited the development of FHB and is accompanied by the slight increase of TSW values in treated plants. It was found, that the most effective fungicide was against F. avenaceum and F. graminearum. The inhibitory effect depended on sprayer type. According to qPCR results, the best performance was achieved when using Amistar 030 and Flat Fan (FF) 030 sprayers. The average concentration of deoxynivalenol (DON) content in all barley grain samples were up to 4 times higher than the permissible level. Overall, because of the high contamination levels, found in tested samples, it is possible to state that a single application of the fungicide at the flowering phase was not able to effectively reduce DON contamination in barley samples. The developed test-system for qPCR provides new important information in the study of the effectiveness of fungicides and development of strategies to control FHB in cereals, not achievable with conventional PCR.