Culture media and incubation

Vigorous growth, sporulation, and pigment production of fusarioid fungi can be achieved on numerous agar formulations. The morphology of fungal structures will vary dramatically depending on the selection of media and growth conditions which may compromise the identification process. In addition, it is also common for fusaria to degenerate and lose viability in culture, particularly when they are grown on nutrient-rich media (Nelson et al. 1983, Nirenberg 1990, Summerell et al. 2003, Leslie & Summerell 2006). Culture conditions and media have been extensively summarised in the literature (Booth 1971, Nirenberg 1990, Nelson et al. 1994, Summerell et al. 2003, Leslie & Summerell 2006). Consequently, we recommend the agar formulations listed in Table 1 to be employed for the isolation and description of fusaria. A summary of the procedures and conditions suitable for work with fusarioid fungi is shown in Fig. 6.

Recommended agar media formulations for the isolation and cultivation of fusaria.

Flow diagram summarising recommended methods for the preservation, identification, and characterisation of fusarioid fungi.

An important condition that must be stressed is that the identification must always be made on the basis of a monosporic culture (a culture produced from a single sporulating conidium, ascospore, or hyphal tip), as multiple species are commonly found to co-occur in the same substrate tissue. A freshly isolated fusarioid strain should be sub-cultured onto at least two different culture media, a relatively rich one suitable for examination of gross morphology, and a nutrient-poor one for micromorphological examination and for further culture propagation. The standard culture setup for initial assessment of growth rates and colony characters i.e., colony pigmentation, diffusible pigments, and colour of sporodochia, is to use potato dextrose agar (PDA) incubated for 1–2 wk. Fusarium and related genera will also grow and sporulate well on malt extract agar (MEA, recipe in Crous et al. 2019a), which can be a suitable alternative for initial isolation and monosporic cultivation. However, MEA should not be used to assess colony or morphological characters. Standard incubation is commonly made in total darkness; however, exposure to light will normally result in a faster and more intense pigmentation. We have observed better colour formation using in-house prepared media rather than commercial formulae. While colony colour cannot be employed as a primary criterion for species identification, it can provide useful means to grossly distinguish related groups and to direct the identification process towards determining genera or species complexes. The high nutrient content of these agar media strongly affects sporulation, commonly resulting in the development of atypical structures. Therefore, we strongly discourage the use of PDA for micromorphological assessment or culture propagation of Fusarium spp. (Nelson et al. 1994, Summerell et al. 2003). Oatmeal agar (OA) is a suitable alternative for strain sub-culturing, allowing for good sporulation with reduced strain degeneration; however, it is not recommended for micromorphological studies.

Carnation leaf agar (CLA), synthetic nutrient-poor agar (SNA), and water agar (WA) are the standard culture media for micromorphological analyses. Also, by reducing culture degeneration, they allow for prolonged storage of actively growing cultures (Nirenberg 1976, Nelson et al. 1983, Leslie & Summerell 2006). Subcultures on CLA will normally produce abundant sporodochia and macroconidia on the surface or around the carnation leaf pieces with consistent morphological features. Incubation at room temperature (20–25 °C) for 1–2 wk under a 12/12 h near-UV light (wavelength 320–400 nm)/dark or near-UV light/cool fluorescent light cycles results in stronger sporulation and good development of sporodochial pigmentation (Nirenberg 1990, Seifert 1996, Summerell et al. 2003, Leslie & Summerell 2006). The use of continuous near-UV light (also commonly termed "blacklight" or UV-A light) is also suitable although it often results in the formation of unusually long macroconidia (Nirenberg 1990), and it can suppress the development of useful morphological characters such as the globose microconidia of Fusarium globosum. Nevertheless, incubation under near-UV light is fundamental since isolates of some species such as Fusarium poae and F. sacchari are known to lack macroconidia or to produce them in only small quantities unless they are stimulated by incubation under a near-UV light source (Leslie et al. 2005, Leslie & Summerell 2006). Fusarium cultures also need adequate aeration to produce conidia reliably and to attain stable growth rates, and hence we discourage the incubation of sealed plates. Carnation leaf agar, SNA, and WA are also suitable for the observation of conidiophore disposition and microconidial arrangements such as the formation of false heads, chains or both. These structures can easily be examined under a dissecting microscope or at low magnification under a compound light microscope (Leslie & Summerell 2006). Examination of micromorphological characters must be carried out using slide preparations mounted in water. Lactic acid, lactophenol and Shear's mounting media can cause considerable shrinking of the structures and can alter the appearance of the cell surface; hence we advise against the use of these mountants for examination of morphological characters in Fusarium and related genera.

Additional culture media, incubation conditions, and protocols are available for induction of sexual characters in Fusarium and related genera (Klittich & Leslie 1988, Leslie & Summerell 2006, Guo et al. 2018, Kim et al. 2019, Santos et al. 2019). Carrot agar (CA) and half-strength CA are the most commonly used media. The crossing procedures are often variations from the protocol of Klittich & Leslie (1988), in which strains of opposite mating types are paired in all possible combinations as male and female parents, together with crosses made against tester strains from known mating populations (Leslie & Summerell 2006). The process can be shortened by reducing the number of combinations to be crossed by first determining the MAT gene alleles carried by each strain by means of specific mating type idiomorph PCR primers (Kerényi et al. 1999, 2004, Steenkamp et al. 2000).