Trap Cultures

Trap cultures are established using field soil in order to produce healthy AMF spores in large quantities. The technique was proposed by Gilmore (1968) and more recently disseminated by the work of Stutz & Morton (1996). There are many implications (usually not considered in published work) associated with AMF spores recovered directly from the field. First, in most soils, AMF communities are composed of several species, of which three or four are dominant in terms of sporulation while the majority occur in low amounts (Stürmer & Siqueira, 2006). Second, AMF spores can remain in the soil for years without being totally degraded, with only the spore wall remaining without viable content. Furthermore, the spore phenotype may be altered due to chemical characteristics of the soil, microbial activity, humidity, among others. These last two points directly affect the accurate identification of species in from field soils. Finally, spores represent only those fungi that have reached sufficient biomass within the root to allow sporulation (Gazey et al., 1992). The establishment of trap cultures is an effective way to circumvent some of these situations because: a) spores obtained from these cultures are of good quality and can be used to establish pure cultures (=monospecific), b) assessment of AMF diversity in survey-type of studies can be complemented, and c) trap culture can be used as inoculum source composed of an assemblage of fungi for experimental purposes.

However, results of a trap culture are quite variable and depend on several conditions such as the initial mycorrhizal inoculum potential of the soil, the host used, and the growth conditions of the trap culture (temperature, light). In some cases, it can act as an ecological filter not allowing sporulation by some species and at the same time allowing cryptic species, not detected in the field, to sporulate in trap cultures (Stürmer, 1998; Moreira-Souza et al., 2003 ). For example, in two cycles of trap cultures established with native soil from Araucaria forests, A. foveata, A. spinosa and Claroideoglomus etunicatum were recovered only in the first cycle, while Ambispora gerdemannii, Gigaspora margarita and Cetraspora pellucida were recovered only after two cycles of culture (Moreira-Souza et al., 2003). Our experience in soils from mesic regions has shown that the dominant species, in terms of sporulation in the field, are those that preferentially sporulate in trap crops (Leal et al., 2009). In arid regions, changing moisture conditions in trap crops appears to be a factor that encourages sporulation by cryptic species (Stutz & Morton, 1996).

For those interested in establishing single cultures of AMF, the establishment of trap cultures is undoubtedly an important first step. Even though not all fungal species will sporulate in trap cultures, species that sporulate produce good amounts of healthy spores, with all the morphological characteristics to allow an accurate identification and serve as a propagule for the beginning of a pure culture. 

The choice of the host plant is particularly important in the establishment of trap cultures, since the host plant roots represent the “Petri dish” where the fungi will colonize, exchange nutrients and acquire enough biomass to initiate the sporulation process. Thus, one of the first criteria for choosing a host plant is how well it develops under the greenhouse conditions where trap cultures are grown. In addition to the plant adaptation to the local condition, the amount of root that the plant produces must be taken into account, bearing in mind that a greater root biomass means more space for the fungus to grow and sporulate. Thus, C4 grasses are generally considered good AMF multipliers and commonly used for the establishment of trap cultures and pure cultures. Grasses are known to have a fasciculated root system which results in a large root area that can be occupied by fungi. Associated with this morphological characteristic, the metabolism of a C4 plant allows for greater photosynthetic efficiency, thus ensuring a large amount of photoassimilates translocated to the root system that can be used by the fungus. Thus, plants such as sorghum (Sorghum bicolor, S. sudanense), Pensacola (Paspalum notatum) and Brachiaria (Brachiaria decumbens, B. brizantha) have been widely used as host plants for AMF multiplication.

METHOD

The method used in CICG is adapted from that described by Stutz & Morton (1996) and consists of mixing field soil with sterilized sand. Bever et al. (1996) used a different procedure to establish trap cultures called “transplanted trap cultures” where intact plants collected from the field were transplanted into a pot with an AMF-free substrate and sporulation assessed after 3-4 months.

Soil and roots are collected from the area of ​​interest. It is important that during sampling the roots of host plants are included as they serve as propagules to initiate new mycorrhizal colonization. The roots must then be cut into small pieces (1-2 cm long) and homogenized with the soil.

This soil + roots is mixed with sterilized sand in a 1:1 ratio (50% field soil + 50% sterile sand). The homogenization can be done inside a plastic bag or even in a clean plastic tray.

 This mixture is placed in 1.5 kg plastic pots (or 0.8 Kg Deep pots) with openings at the base. Trap cultures can be set up with only one host or multiple hosts. With only one hosts, seeds of Brachiaria decumbens or Sorghum sudanense are sown and covered with a substrate of soil:sterilized quartz sand (1:1) (or some other sterilized substrate). It's important to add a good amount of seeds and we put in 40-60 seeds per pot. Remember two things: 1) roots are the “Petri dishes” of AMF - if there are few plants in the pot, sporulation can take longer and plants can grow very tall without necessarily investing in root, and 2 ) choose a C4 plant that is mycorrhizal dependent and grows well under your greenhouse conditions. Alternatively, trap cultures can be set up using multiple hosts and we use at the CICG a combination of Kalanchoe daigremontiana, Bidens pilosa, Belamcanda chinensis, and Schinus terebinthifolia.

The cultures are then grown in a greenhouse for at least 4 months, at which time two soil cylinders (50 ml each) are extracted to check sporulation. During this time, fertilization is done only if necessary and with a low phosphorus solution (we use Hogland’s solution with 10% P). 

After this period, watering is suspended and the pots are allowed to dry in situ in an environment with low natural light so that the drying process is slow (1-3 weeks). If there is interest in extracting spores to obtain pure cultures, the trap cultures are dried only to the point of eliminating most of the moisture, leaving the substrate a little moist before storing.

Trap cultures are stored in zip-lock plastic bags for a minimum period of 1 month before recovering the AMF spores to be used to establish pure cultures. This period seems to be important when there are species of some genera to break the dormancy of the spores.

If sporulation is not adequate after a period of 4 months, the crops can be stored dry for a period and then reactivated or simply remove the aerial part and sown again. 

Establishment of trap culture: