Molecular Detection of RNA Viruses in Agaricus Bisporus on Mushroom Farms in Ukraine

Abstract

Aim. To study the prevalence and identify viral agents affecting cultivated button mushrooms (Agaricus bisporus) on mushroom farms in Ukraine, using molecular biological methods, and to assess the relative viral load. Methods. Visual assessment of symptoms was conducted on samples of mushroom fruiting bodies and mycelia from farms in the Kyiv, Ternopil, Rivne, Lviv, Dnipropetrovsk, and Odesa regions. Total RNA was isolated using a GeneJET Plant RNA Purification Kit. Identification of viral agents (La France disease virus – LFDV; Mushroom bacilliform virus – MBV; Agaricus bisporus virus 6 – AbV6; Agaricus bisporus virus 16 – AbV16; Agaricus bisporus virus X – AbVX) was performed using two-step Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) on a Rotor-Gene 6000 amplifier. Glyceraldehyde-3-phosphate dehydrogenase II (GPDII) gene of A. bisporus was used as an internal control for normalization and assessment of RNA/cDNA quality. Melt curve analysis was performed, and the relative amount of viral RNA was calculated using the ΔCt method. The study used a straightforward estimation of viral load based solely on the GPDII reference gene. It was used as a reference gene. This method serves as a preliminary step, acknowledging its limitations in precision. Results. Six A. bisporus samples with various morphological changes were studied: a small cap and a strongly curved stipe (Ternopil), a thickened stipe (Kyiv), a small cap and an elongated stipe (Rivne), a dense stipe and a flat cap with pink gills (Dnipro), browning of fruiting bodies (Lviv), and an abnormal cotton-like mycelium (Odesa). The performed RT-qPCR analyses identified various combinations of LFDV, MBV, AbV6, AbV16, and AbVX. Noteworthy, mushroom virus X (MVX), associated with the "bare patch" disease, was not detected in any of the samples. The most common viruses were MBV (detected in all 6 samples) and AbVX (in 5 samples). AbV6 and AbV16 viruses were detected in 4 samples, and LFDV in 3 samples. Mixed infections by more than four viruses were detected in four samples. ΔCt calculation showed a significant relative level of MBV viral RNA in most samples (especially in Dnipro and Lviv), as well as LFDV, AbV6, and AbVX in the Lviv sample, compared to the expression level of GPDII. Melt curve analysis confirmed the specificity of the PCR products. Conclusions. RNA viruses were detected and identified in the culture of A. bisporus, and their quantitative representation was assessed. On the surveyed mushroom farms, viruses LFDV, MBV, AbV6, AbV16, and AbVX are prevalent, often as mixed infections with different levels of relative viral load. The obtained results emphasize the need for implementing systematic molecular monitoring of mushroom viral diseases in Ukraine to ensure phytosanitary safety and production stability.