A novel mechanism behind aerial dispersal of pycnidiospores

Pycnidia are asexual fruiting bodies formed by diverse Ascomycete fungi, comprising many destructive horticultural pathogens within Diaporthales, Pleosporales, and Botryosphaeriales. These fungi disperse over long distances via airborne sexual ascospores, while their asexual pycnidiospores are thought to spread locally, mainly by water splash. However, in Botryosphaeriaceae, including Lasiodiplodia theobromae, the sexual stage is rarely observed, never reported in Israel, yet disease exhibits frequent long-distance aerial dispersal of pycnidiospores. This research aimed to elucidate the mechanisms underlying pycnidial burst and aerial dispersal of pycnidiospores, using L. theobromae as a model. Field observations correlated new infections with seasonal relative humidity (RH). In vitro findings and scanning electron micrograph confirmed that high RH triggers pycnidial bursts, while aerial pycnidiospore dispersal follows subsequent RH decline. Wind tunnel experiments revealed that pycnidiospores were released at RH >70%, and their travel distances increased with wind speed (2.0 m s^-1), with spores detected up to the tunnel's end. The pycnidial analysis revealed high osmolyte concentrations (~0.17 M; sugars and glycerol), generating internal pressure of 4.18 atmospheres. These findings support a new mechanism for pycnidiospore release: under high RH, moisture permeates the closed pycnidia, dissolves osmolytes, and increases osmotic pressure to trigger pycnidial bursts. As RH drops, the mucilage surrounding the pycnidiospores dries, enabling the pycnidiospores to disperse by the wind over long distances. This mechanism was observed across Botryosphaeriaceae species infecting different trees. These results provide an overlooked mechanism for long-distance dispersal of pycnidiospores mediated by humidity-driven pycnidial burst and wind-assisted transport, with implications for disease epidemiology and management in agricultural systems.IMPORTANCEAirborne dispersal of fungal plant pathogens is generally attributed to sexual spores, whereas asexual pycnidiospores are widely assumed to spread locally via water splash. This assumption is difficult to reconcile with frequent long-distance disease outbreaks caused by fungi, that is, Botryosphaeriaceae, in which the sexual stage is rare or absent, including Lasiodiplodia theobromae. Here, we demonstrate that pycnidiospores can disperse aerially over long distances under rainless conditions through a previously unrecognized mechanism. We show that high relative humidity induces pressurized pycnidial bursts driven by osmolytes, including sugars and glycerol, generating sufficient internal pressure to rupture pycnidia. As humidity subsequently declines, released pycnidiospores dry and become wind-dispersible. This mechanism explains how asexual pycnidiospores of various species can contribute to long-distance disease spread and challenges long-standing assumptions about fungal dispersal ecology. Our findings have broad implications for understanding fungal epidemiology and improving disease forecasting and management in agricultural systems.