Isolating and Culturing Fungi

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Despite the benefits of mycorrhizal associations, application has not been widely used on a commercial scale. For commercial development, large quantities of inoculum must be produced. Since AM are obligate biotrophs, they must be grown and maintained on living plant roots. This is often problematic because of the high risk of introducing plant pathogens and other contaminants into the culture system (Jarstfer and Sylvia 1997). In contrast, EM fungi may grow on agar media in the absence of a host plant root or from the vegetative mycelium of fruiting bodies collected directly from the field (Molina and Palmer 1982).

The first step in culturing mycorrhizal fungi is producing stock of the individual fungal isolate on host plant roots. For AM fungi, spores or colonized root fragments from the stock are used to produce larger quantities of inoculum for growth on soil-based or soil-free substrates (Schenck and Perez 1990). Although large amounts of EM fungal spores can be easily collected in the field, spores are rarely used to isolate EM fungi. Instead, many inoculation programs use the EM vegetative mycelium for its effective growth and storage on agar (Molina and Palmer 1982).

There are several benefits to using a soil-based culture system. Soil-based inocula are easy to produce, highly infective, and can be stored for several months or years. Jarstfer and Sylvia (1997) outline the most basic procedure for isolating and culturing spores on plants in sterile soil. Host plants propagated from seed are preferred over cuttings because they are easily disinfected. Disinfecting fungal propagules prior to inoculation is also critical because other microorganisms may be propagated with or instead of the AM fungi (Jarstfer and Sylvia 1997). To avoid contamination, cultures should be isolated from nonsterile environments. Cultures are typically grown for 4-6 months, ensuring sporulation of all genera (Sieverding 1991), and are then stored as air-dried soil at room temperature (Dodd and Thomson 1994). Fungal inoculum can be stored for long periods (>5 years) as air-dried soil; however, the viability of individual isolates during storage remains uncertain (Jarstfer and Sylvia 1997). The most convenient use of soil-based systems has been for inoculating nursery grown plants that are later transplanted in the field. This type of culture system may be otherwise too cumbersome for extensive use on a landscape-scale (Jarstfer and Sylvia 1997).

Soil-free systems, like hydroponics and aeroponics, were developed to overcome the limitations and drawbacks associated with soil-based systems. Culturing fungi in soilless media provides greater control over the physical and chemical characteristics of the growth medium and minimizes the detrimental impacts of contamination with other organisms (Jarstfer and Sylvia 1995). As such, the ideal conditions conducive to AM development are capable of being achieved. Better control of nutrients in soil-less systems can result in greater root proliferation and higher numbers of spores per centimeter of colonized root length (Sharma et al. 2000). Colonized roots and spores free of substrate allow for more efficient production and distribution of inocula (Jarstfer and Sylvia 1997). Consequently, soil-free systems produce greater propagule densities than soil-based pot cultures of the same age (Jarstfer and Sylvia 1995). Aeroponic culture in particular allows for easy extraction of AM fungal propagules, and mycorrhizal roots can be sheared to produce high-density inoculum that is both efficient and easy to handle (Jarstfer and Sylvia 1995). Commercial nurseries currently use aeroponic culture systems for revegetation programs where on-site production of inocula allows for the use of fresh mycorrhizal propagules at optimal times (Jarstfer and Sylvia 1995).

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