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Propagules of glomalean fungi must be used immediately or stored for some period of time, once they are extracted or produced. As a future inoculum source or for physiological or immunological comparison, they must be kept alive and infective. To study comparative morphologies (structural or ultrastructural) or to be preserved as vouchers, they must be maintained as close as possible to their natural states.
LIVE PROPAGULES
A. From soilless propagation
Propagules obtained from soilless propagation regimes (e.g. hydroponics, aeroponics, root organ culture) generally are the same as those produced in soil-based media: mycorrhizal roots, external hyphae, spores. In the soilless environment, however, each type of propagule can be harvested separately or in combinations with greater ease. Conditions for successful long-term storage of these propagules remain vaguely defined. If microbial background is low in these systems (which is not as clear-cut as the literature suggests), then the propagation cycle can be extended instead of harvesting the propagules. Once separation has taken place, however, the minimal steps would need to be air-drying and then storage at 4oC. Beyond that, longevity remains an open question.
B. From propagation in solid media
Pot-culture growth media can consist of sand, soil, vermiculite, perlite, or any combinations of these ingredients. Various factors impinge on abundance, viability, and infectivity of propagules, which in turn influence storage potential: (1) carbon partitioning to each propagule type (thus affecting the final proportion of each at harvest), (2) duration of the plant growth period (which can range from 3-12 months), (3) temperature, moisture, and light conditions, and (4) background activity of associated microbiota. The first and last are most important when yield and viability of spores are important factors (e.g. for cultures of Gigaspora or for purposes of spore fractionation of any fungal species). The last also is greatly influenced by (2) and (3), but their influence also is governed by fungal isolate (physiological properties predetermined by its life history and ecology) and the species (morphological properties of spores that might facilitate microbial activity). An example of the former is storage temperatures at 4-10oC for temperate isolates versus 20-25oC for tropical isolates to match propagule requirments to overcome dormancy and germinate. One such comparison is documented by Louis & Lim, 1988 and the references therein). As an example of the latter, Kay Hardie (1984) reports no contamination from surface-sterilized spores from 12-month-old pot cultures of a Rothamsted G. mosseae isolate, whereas spores of a 5-month-old culture had up to 18% contamination. Spores of all G. mosseae isolates synthesize an outer (sloughing) mucilagenous layer that can harbor a wide range of microbiota. This layer likely was completely sloughed in the older culture, thus improving effectiveness of surface-sterilization of underlying spore wall laminae. Spores of many species in Glomus share a similar outer enveloping layer that sloughs with time and age.
Generalizations that drying is disadvantageous for many fungi are erroneous. They seem to be based on a procedure that we find predisposes propagules to higher mortality: harvesting pots moist, chopping roots, mixing contents, and then air-drying to 1-2% moisture content. A safer approach is to dry pots in situ, place pot contents intact (or broken into segments kept as large as possible) in Mason Jars or plastic bags and store at appropriate temperature (see above). Chopping roots and mixing contents should be done only just prior to inoculum usage.
DEAD PROPAGULES
Most of the focus, when it comes to fixed or preserved propagules, is on spores. They contain most of the structural diversity in glomalean fungi and undergo most morphological alterations during storage.
A. Spore vouchers
Conventional preservatives used to date in voucher preparation are able to successfully inhibit growth and reproduction of any associated microorganisms, but they fail to prevent structural changes to phenotypic properties of spores. Available compounds vary greatly in the degree they modify spore characters. From our experience, we rate the worst (where number and magnitude of changes are highest) to best (fewest changes) as follows: phenol, lactophenol, gluteraldehyde, formalin, sodium azide. NONE of these solutions maintain spores in their original condition. We suspect that it is not just chemical action that is leading to these modifications. Mortality alone, concomitant with termination of all maintenance and anabolic processes, probably is the most significant factor leading to many changes: (1) browning of layers in spore wall or spore contents (most commonly occurring in Gigaspora, but also frequent in cream-colored to yellow spores in most other genera), (2) fusion of layers in hyaline flexible inner walls to appear as one (often observed in Acaulospora, Entrophospora, Scutellospora), and (3) loss of layer flexibility (especially in laminae of Gigaspora species or the amorphous layer of the innermost flexible wall of Acaulospora, Entrophospora, and Scutellospora species).
I propose that the only means to prevent unwanted structural changes is to keep spores minimally alive or dormant. Storage of spores in dried whole inoculum retain all of their properties (except color) for 2-3 years if background microbial activity is kept low. Often, spores of Gi. margarita and Gi. rosea turn brown and the contents clear or congeal after 30 days or less storage in 0.5% sodium azide. This occurs much more rarely (both number of cultures and frequency of spores) for spores stored up to six months in dry soil. Mertz et al. (1979) report that spores of Gi. margarita can be stored in water with 250 mg/l Streptomycin sulfate and 100 mg/l Gentamicin for up to one year at 4oC without any loss of viability (as determined by germination). One approach yet to be tried, but which might prove to be highly effective, is to first surface-sterilize spores, treat them for a brief period in antibiotics, and then incorporate them into dry sand or some other inert sterile solid medium. Regardless of purpose, spores extracted in sucrose gradients should be incubated in water at 4oC for a minimum of 24 hours so they have time to equilibrate. It is in this step that the antibiotics could be incorporated.
Physical properties of the solid medium appear to contribute in some way to slower microbial decomposition of stored spores. We have found that those mailed long distances to INVAM this way often are much less subject to fungal contamination or degradation than those transported in water, even when there is no presterilization treatment. One drawback of storage in a solid medium is that spores must be extracted again to view or use them. However, if sand particles greater than 0.5 mm in diameter are used, then only a simple sieving step would be needed.
B. Mycorrhizal vouchers
Most mycorrhizae are preserved by mounting cleared and stained roots in water (sealing the coverslip with nail polish or enamel) or PVLG. The latter has met with considerable success in INVAM, and 12-year-old vouchers have not changed appreciably. Mycorrhizae also can be preserved intact in formalin, gluteraldehyde or sodium azide, although they should be cleared and stained first. Mycorrhizal roots can be stored in sterile distilled water for long periods as long as opportunistic parasites are not present within them. This problem is avoided if the roots are first cleared with 10% potassium hydroxide.
REFERENCES
Louis, I. and G. Lim. 1988. Effect of storage of inoculum on spore germination of a tropical isolate of Glomus clarum. Mycologia 80:157-161.
Hardie, K. 1984. Germination of Glomus mosseae spores isolated from stock pots of different ages. Trans. Br. Mycol. Soc. 83:694-696.
Mertz, S. M., Jr., J. J. Heithaus III, and R. L. Bush. 1979. Mass production of axenic spores of the endomycorrhizal fungus Gigaspora margarita. Trans. Br. Mycol. Soc. 72:167-169