FUNGAL DEVELOPMENT

The first step relating to the fungal cultivation is to determine physico-chemical parameters which promote optimum fungal development. Most if not all methods for the production of spores in culture were developed to provide a supply of inoculum for inoculation (Rotem, 1994). Fungal growth is affected by incubation temperature, nutrient media, time period of cultivation, and the type of culture conditions (i.e., stationary or shake culture). Conditions for optimal growth and sporulation were reported to differ within and among fungal species (Gupta et al., 1999). The major work performed about determination of optimal culture conditions was carried out with Alternaria alternata. A.alternata was grown in three different media at 24°C: V8 juice agar (V8), modified V8 juice agar (mV8), and modified Czapeck-Dox agar (mCD) in order to determine suitable media and conditions for optimum yield of spores and mycelial development. The mCD is a synthetic medium widely used in mycological laboratories. Many moulds produce very characteristic colonies on it and may also exude pigmented substances. Aerial growth is often suppressed and sporulation may be enhanced. Some mould, however, grows poorly on this medium and may even fail to sporulate altogether, often because of their inability to synthesize vitamins. The addition of agar to this medium makes it, in reality, a semi synthetic one (Malloch, 1997). The V8 and mV8 are natural media because they are partly or completely composed of natural materials. The V8 juice used in V8 medium probably contains many nutrients that fungi can use, but we have a little idea what they may be. It is a medium that is used routinely in plant pathology and seems to be a good complement to Leonian’s or PDA. Moulds that fail to sporulate on those media often sporulate heavily on V8, or vice versa (Malloch, 1997). Figure 37 shows fungal development after 9 or 14 days of incubation. The growth at 9 days of A.alternata in V8 medium is characterized by a short downy grey mycelium which is dark coloured on its periphery. Culture in mV8 medium presents an abundant white mycelium while mCD medium exhibits a much dispersed mycelium. Sporulation is highest on mCD medium, and lacking on mV8. It is characterized by a dark ring on plate periphery after 14 days on mCD medium (Figure 37d) and appears less dense in V8 medium after 20 days and is more located around central inoculate. Hence, mCD medium promotes sporulation more than vegetative growth. According to Rotem (1994) all Alternaria species sporulate better in media poor in sugars, such as V8 medium prepared without the addition of sugar. Such media suffice for easily sporulating Alternaria. Inhibition of vegetative development by starvation triggered sporulation of A.alternata, A.dauci, and A.solani transferred from an agar medium with nutrients to water agar (Shahin and Shepard, 1979). Spores of A.alternata are formed in long chain of approximately 10 spores or more, either beakless or with very short beaks.

Most species that form spores in chains sporulate easily and usually do not need triggering. Nevertheless, in some cases external induction increases sporulation in such species. Among all triggers for mass production of spores, irradiation is undoubtedly the strongest. Irradiation with UV light is the main element in most formulas for sporulation. For practical purposes it does not matter whether the source of light is a germicidal bulb with short UV wavelengths or a lamp with long UV or near ultraviolet (NUV) wavelength. It also does not matter whether glass or plastic dishes are used for the cultivation of fungi (Rotem, 1994). In species that sporulate in culture without induction, conidiophores* are obviously formed in darkness but become dark and look more typical when exposed to light (Rotem, 1994). Figure 38 shows culture in mCD exposed or not to light after 14 days of incubation at 24±1°C and 90±5% RH. Cultures exposed to light were submitted to horticol neon (blue and red wavelengths are increased). For both sporulation is noticed but higher spore density is observed when cultures are exposed to light. According to Rotem (1994), mechanism of photosporogenesis involves two distinctive phases. The first, or inductive, phase leads to the formation of conidiophores; the second, or terminal, phase leads to the formation of conidia (spore). The temperature and light requirements for these two phases are distinct.