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Trap Cultures

Trapping is necessary to obtain many healthy spores of colonizing fungi for identification and as inoculum to establish monospecific cultures. Spores collected directly from a field soil suffer from many problems: (a) they appear healthy but are not viable (some persisting as dead husks for years or possibly decades), (b) they lose or change appearance of their structural characters in response to root pigments, soil chemistry, temperature, moisture, and microbial activity, and© they represent only those colonizing arbuscular fungi with enough activity and biomass to trigger sporulation. Trap cultures yield very different results, depending on all of the biotic and abiotic factors that go into growth, reproduction, and the partitioning of colonization and sporulation. Our experience indicates when species richness is high and colonization by all members of the assemblage is relatively low (e.g. arid environments), most if not all indigenous fungi will sporulate after 2-3 successive propagation cycles. When species richness and colonization are both moderate to high, often only the most aggressive species will sporulate and 2-3 successive propagation cycles could have the opposite effect of yielding just 1-2 sporulating species. We consider trap cultures to be important in the following situations:

Example: In the early days (1982-83), many cultures were started from field-collected spores, with 150-200 the normal range of spores applied to a single plant root system. Few of these were successful (< 10%), and those that were often yielded pure cultures of “weedy” fungal species such as Paraglomus occultum, Archaeospora trappei, or Acaulospora morrowiae. When these soils were set up as trap cultures and spores from those used to inoculate seedlings, success rate sometimes was 100% (usually > 75%).


METHOD

Rhizosphere soil (attached to roots and dug up with a shovel or some other tool) is collected. A root ball is ideal. Shoots are removed at the crown and roots are chopped into small fragments and mixed with thoroughly the associated soil using an ax or some other cutting utensil.

Roots dug with shovel  Roots, pot and pastic bag


Hatchet and soil sampleThis chopped blend is mixed 1:1 (v/v) with autoclaved coarse sand. We use gallon zip-loc bags for the mixing process, because it is easy to “massage” the bag to break up small clumps of soil or roots and improve chances for a homogeneous product. This mix then is transferred to a 15-cm plastic pot. Smaller pot sizes should be avoided if possible (especially cone-tainers), because roots fill the volume too quickly, quit growing, and increase competition among species for limited root substrate. Larger pots also work well, but optimal sporulation takes longer (our hypothesis of the basis for this).


Seeds planted in a potSeed pots with a highly dependent C4 grass (we use sudangrass; Norm Schenck used bahia grass in Florida because it tolerates hotter greenhouse temperatures; Frank Pfleger used big bluestem in Minnesota). We overseed (80-100 seeds/pot) for a variety of reasons ( explanation). Other hosts can be used, but care must be taken to avoid coarse or sparsely rooted species (which may be wonderful for single species) because they have more constrained niche space that is likely to exclude slow-growing fungal species.


Three different size seedlingsCultures are grown in a greenhouse or growth room for at least four months. Some sporulation occurs during the first three months, but it is during the fourth month when plant shoots (and roots) have ceased growth and carbon seems to be repartitioned to sporulation rather than mycorrhizal development. A longer time often is undesirable because other fungi often colonize the cultures which can lead to hyperparasitism and poorer conditions for dry storage. Fertilization is kept to a minimum, being applied only when plants show signs of phosphorus deficiencies (purpling of leaf sheaths), or nitrogen deficiency (chlorosis of young leaves).


Seeds started in small potsPots are left to dry undisturbed in a shaded room with a fairly stable temperature so that the drying period is not too rapid (1-2 weeks). We use an abandoned dish-washing room with one shade-drawn window.

Spores should be extracted before pot contents are dry to obtain the brightest and cleanest specimens. If there is any chance that spores are to be collected to set up monospecific cultures within 3 months of harvest, we generally only dry long enough to eliminate free moisture when contents are bagged (to be sure again of retrieving highly infective propagules).


Trap cultures in zip-locs stored on shelfAll trap cultures are stored in gallon zip-loc plastic bags for at least 30 days before spores are extracted for inoculation onto seedlings. Spores of many species (particularly those in Acaulospora and Entrophospora) appear to require a dormancy period before they are infective, but this can vary with habitat. The 30-day rest period appears to reduce or eliminate this variable.

For trap cultures that are to be propagated for 1-2 additional cycles, the bagged material is used as inoculum (undiluted) and set up within 30 days of harvest. If sporulation is extremely low, then the pots aren’t harvested, but shoots removed and the pot contents reseeded.