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economic and environmental impact of these pathogens and the increased threat of their spread due to
[26]
elevated global temperatures . The purpose of this review is to present the current state of knowledge on
phytopathogenic fungal EVs, how they are isolated, which molecules are associated with them and what role
they play during plant infections.

A BRIEF HISTORY OF FUNGAL EVS
While fungal EV research may seem relatively new, evidence for EVs in fungi has existed for decades. In
fact, the first published images of EV secretion came from a study of the wood-decaying fungus
[27]
Polystictus versicolor . Early on, vesicle-like structures between the plasma membrane and cell wall of
fungal hyphae were referred to as “lomasomes” or “border bodies”. They were thought to be unique to fungi
[28]
and contribute to the formation of the cell wall .

Over the years, multiple studies documented the presence of EVs in fungi using electron microscopy.
[29]
Microvesicle secretion was observed in protoplasts of Aspergillus nidulans , while evidence for exosome
release was observed in freeze-fractured cells of Cryptococcus neoformans . Moreover, in Candida
[30]
[31]
tropicalis, EVs were observed traversing the cell wall , accumulating in the liquid growth medium in the
presence of n-alkanes and adhering to the surface of fungal protoplasts undergoing cell wall
[32]
regeneration .
[33]
Despite these many observations, fungal EVs were not officially isolated until 2007, when Rodrigues et al.
pelleted EVs from liquid cultures of Cryptococcus neoformans, a common environmental yeast and
[36]
opportunistic human pathogen . These vesicles were associated with several virulence-related molecules,
including a major capsular polysaccharide, pigment and proteins [34-36] .

Virulence-related molecules appear to be a common feature of fungal EVs, as subsequent studies examining
different species have shown [37-40] . The presence of such molecules suggests that fungal EVs play a role in
pathogenesis, and a number of findings support this hypothesis. For example, EVs from Cryptococcus
neoformans and Sporothrix brasiliensis enhance the spread of each respective fungus throughout its
host [41,42] , while EVs from Candida albicans can promote the formation of antibiotic-resistant biofilms .
[12]
Through cell-to-cell communication, EVs from virulent strains of Cryptococcus neoformans and
Cryptococcus gattii can also enhance the proliferation and survival of less virulent strains [15,43] .

While fungal EVs can benefit pathogens, they can just as easily work to their detriment. Such vesicles are
often immunogenic, able to induce inflammation and stimulate the antimicrobial activities of host
macrophages [44-46] .

To date, EVs have been isolated from over twenty species of fungi, including both yeasts [37,39-41,43,46-50] and
filamentous fungi [24,51-62] . The majority of these fungi cause disease in humans, a few are valued for their
applications in industry, and a small but growing list are phytopathogens. The latter list of fungi includes:
Alternaria infectoria, the causative agent of wheat black point , Fusarium oxysporum f. sp. vasinfectum
[60]
(Fov), which causes vascular wilt disease in cotton [52,62] , Fusarium graminearum, which causes Fusarium stalk
rot in cereal crops [24,55] , Zymoseptoria tritici, which causes Septoria Tritici Blotch in wheat , Ustilago
[56]
maydis, the causative agent of maize smut , and Colletotrichum higginsianum, which causes anthracnose
[57]
[59]
disease in cruciferous plants [Table 1].