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Most previously studies had considered that plant fungal disease spread widely and quickly by airborne fungi spore. However, little is known about the release dynamics, aerodynamic diameter, and pathogenicity threshold of fungi spore in air of the greenhouse environment. Grape gray mold is caused by Botrytis cinerea; the disease spreads in greenhouses by spores in the air and the spore attaches to the leaf and infects plant through the orifice. In this study, 120 μmol/L propidium monoazide (PMA) were suitable for treatment and quantitation viable spore by quantitative real-time PCR, with a limit detection of 8 spores/mL in spore suspension. In total, 93 strains of B. cinerea with high pathogenicity were isolated and identified from the air samples of grapevines greenhouses by a portable sampler. The particle size of B. cinerea aerosol ranged predominately from 0.65–3.3 μm, accounting for 71.77% of the total amount. The B. cinerea spore aerosols were infective to healthy grape plants, with the lowest concentration that could cause disease being 42 spores/m3. Botrytis cinerea spores collected form six greenhouse in Shandong Province were quantified by PMA-qPCR, with a higher concentration (1182.89 spores/m3) in May and June and a lower concentration in July and August (6.30 spores/m3). This study suggested that spore dispersal in aerosol is an important route for the epidemiology of plant fungal disease, and these data will contribute to the development of new strategies for the effective alleviation and control of plant diseases. [ABSTRACT FROM AUTHOR]