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Page 93 Rutter et al. Extracell Vesicles Circ Nucleic Acids 2023;4:90-106 https://dx.doi.org/10.20517/evcna.2023.04

Table 1. Characteristics of fungal phytopathogen EVs
Transmission electron microscopy/ Dynamic light scattering/
Species [Reference]
Scanning electron microscopy Nano-particle tracking
[60]
Alternaria infectoria Appearance = cup-shaped particles Mean diameters = 50 nm and 100 nm
Range = 20-40 nm
Mean diameter = 28.36 nm
Fusarium oxysporum f. sp. Appearance = cup-shaped and multi-lobed Mean diameter = 155.1 nm
[52]
vasinfectum rosette-shaped particles Mode diameter = 150.0 nm
Fusarium oxysporum f. sp. Appearance = spherical and cup-shaped particles Range = 100-300 nm
[62]
vasinfectum Mean diameter = 120.0 nm
[55]
Fusarium graminearum Appearance = cup-shaped particles Mean diameter = 120 nm
Fusarium graminearum [24] Appearance = cup-shaped particles Mean diameters = 200 nm, 123.8 nm and
232.9 nm
Mode diameter = 93.6 nm, 94.2 nm and
115.2 nm
Zymoseptoria tritici [56] Appearance = cup-shaped particles Range = 100-250 nm
Range = 50-300 nm
Mean diameter = 91.8 nm
Median diameter = 84.0 nm
[57]
Ustilago maydis Appearance = cup-shaped particles N/A
[59]
Colletotrichum higginsianum Appearance = cup-shaped particles Mean diameters = 106 nm and 100-102 nm
Median diameters = 104 nm and 100-102 nm

METHODS OF ISOLATION AND PURIFICATION
Vesicle isolation is the first and most serious challenge in any EV study. Choices made early in the
methodology can have significant impacts on a study’s findings, and variability among laboratories can lead
to conflicting data. International consortiums of EV researchers have made great efforts to standardize the
implementation and reporting of methods [4,63] . Still, when dealing with multiple species, as is the case with
fungal EV research, variability is inescapable.

The majority of fungal EV researchers have adapted their methods from Rodrigues et al., which used
[36]
differential ultracentrifugation (DUC) to isolate EVs from the supernatants of liquid cultures [Figure 1].
These methods remain the gold-standard approaches for fungal EV isolation despite their limitations. DUC,
by its nature, is time-consuming, can require large volumes (hundreds of milliliters to liters) of culture and
often leads to significant losses in the yield of EVs.

At the very beginning, fungi are grown in a liquid culture for several days. There is no standard medium
when growing phytopathogens for EV isolation. Studies have used a range of both natural and synthetic
broths that can be either complete or minimal in composition [Figure 1]. Because the EVs reflect their cell
of origin, differences in the nutritional content of growth media can lead to significant alterations in EV
composition across studies. For example, when grown in two different kinds of media, a defined medium
and a nutrient-rich tissue lysate, Fov produced similar numbers of EVs but with different protein cargos .
[62]
A similar observation was made for the yeast and human pathogen Histoplasma capsulatum, the EVs of
which were altered in both protein and lipid compositions in response to nutrition . It is unknown
[64]
whether these changes merely reflect an altering of EV composition or a shift in the release of different EV
populations.

Another complexity of growing filamentous plant pathogens is the presence of macro-morphologies. Yeast
grows into uniformly dispersed cultures, but filamentous fungi develop large aggregates known as mycelial
clumps and pellets (not to be confused with the kind of pellet produced after centrifugation) . Currently, it
[65]
is unknown whether these dense and sometimes irregular structures affect the diffusion of EVs into the