Multiplex Detection of Pleurotus ostreatus Mycoviruses

Area of Science:

• Mycology
• Virology
• Molecular Biology

Background:

• Mycoviruses infect fungi, with four known types in *Pleurotus ostreatus*: oyster mushroom spherical virus (OMSV), oyster mushroom isometric virus (OMIV), *Pleurotus ostreatus* spherical virus (POSV), and *Pleurotus ostreatus* virus 1 (PoV1).
• Accurate detection of these mycoviruses is crucial for understanding their impact and managing diseases in oyster mushroom cultivation.

Purpose of the Study:

• To develop a multiplex RT-PCR assay for the simultaneous detection and differentiation of four major mycoviruses in *Pleurotus ostreatus*.
• To establish a reliable diagnostic tool for epidemiological studies and disease control in oyster mushroom farming.

Main Methods:

• Design of four primer pairs targeting conserved regions of OMSV, OMIV, POSV, and PoV1 sequences.
• Optimization of multiplex RT-PCR conditions, including an annealing temperature of 62 °C.
• Determination of assay sensitivity, with detection limits of 100 fg for OMSV/OMIV and 1 pg for POSV/PoV1.

Main Results:

• The multiplex RT-PCR successfully amplified specific products for each mycovirus: 672 bp (OMSV), 540 bp (OMIV), 310 bp (POSV), and 200 bp (PoV1).
• The assay demonstrated high sensitivity and was effectively applied to detect OMSV, OMIV, and POSV in various *P. ostreatus* strains.
• The method also successfully detected the PoV1 sequence from a plasmid, confirming its utility.

Conclusions:

• The developed multiplex RT-PCR is a powerful and efficient diagnostic tool for surveying the incidence and epidemiology of four key *P. ostreatus* mycoviruses.
• This methodology will contribute to the prevention and treatment strategies for mycoviral diseases affecting oyster mushrooms.