From ISTA 7-032 method:
After 5-15 days incubation, look for infected seeds on NP-10 agar, or on blotter paper.
Verticillium dahliae: Examine plates for miscrosclerotia, conidiophores and conidia typical of V. dahliae.
Microsclerotia: Black survival structures that are masses of melanised fungal cells ranging from 10–230 µm in diameter (Qin et al., 2008; Sorensen et al., 1991).
Conidiophores and conidia: Verticillate, tree-like structures with phialides borne in whorls on the conidiophores, and clumps of hyaline, single-celled conidia borne at the end of each phialide (du Toit et al., 2005; du Toit and Hernandez-Perez, 2005).
To determine the potential effect of seed treatments on V. dahliae, it is important to distinguish between viable and dead microsclerotia on the seeds by observing development of typical verticillate conidiophores on the same seed as those on which microsclerotia are observed. The presence of microsclerotia on a seed without conidiophores, or without the presence of newly developed microsclerotia in the blotter paper is an indication that the Verticillium sp. on that seed is not viable (du Toit et al., 2009; du Toit et al., 2010; du Toit and Hernandez-Perez, 2005).
Verticillium spp. can be misidentified with Acremonium spp. which form conidiophores that resemble those of V. dahliae. However, the mycelium of Acremonium spp. tends to develop into ‘rope-like strands’ by the 9 and 14 d readings, from which individual conidiophores branch off at right angles, and which do not normally form phialides in distinct verticillate whorls (i.e., the phialides are not verticillate). Also, Acremonium spp. do not form microsclerotia.
Pther species of Verticillium have been found associated with spinach seed, e.g. V. nigrescens, which has been re-named Gibellulopsis nigrescens (Iglesias-Garcia et al., 2013; Villarroel-Zeballos et al., 2012). This fungus forms conidiophores that resemble those of V. dahliae, but does not form microsclerotia. Also, G. nigrescens forms tiny, black chlamydospores in NP-10 agar medium that are much smaller than microsclerotia and uniformly round, but does not form chlamydospores on blotter paper. Verticillium tricorpus also has been observed on spinach seed, and tends to form a yellow pigment in NP-10 agar (unlike V. dahliae), larger microsclerotia than those of V. dahliae, and the microsclerotia tend to be scattered in a random pattern in and on the NP-10 agar medium whereas those of V. dahliae tend to form in concentric rings.
