Properties of infective propagules at the suborder level (Glomineae versus Gigasporineae)
(from INVAM Newsletter Vol. 3, No. 2; September, 1993)

Putative infective propagules of glomalean fungi include: (1) spores (formed by all taxa), (2) hyphae from mycorrhizal root fragments (formed by all taxa), (3) vesicles (formed by members of Glomineae only) and (4) auxiliary cells (formed by members of Gigasporineae only). The literature is fairly confusing as to the importance of each propagule type in fungal life cycles. As a consequence, their taxonomic and ecological significance has remained fairly ambiguous. This situation does not bode well, because such knowledge is crucial to any biological or ecological interpretation of mycorrhizal processes. It also is essential in culture management, which is the impetus for writing this article.

Almost all data on propagule infectivity has been collected using organisms in Glomus and Acaulospora species as experimental units (Biermann and Linderman, 1983; Hall, 1976; Jasper et al., 1989; Tommerup and Abbott, 1981). Scutellospora calospora was incorporated into comparative analyses in one study (Jasper et al., 1989) and organisms of two Gigaspora species examined in another (Biermann and Linderman, 1983). Unfortunately, results from study of fungi in a family (Glomaceae) of one suborder (Glomineae) have been generalized to include those in a family (Gigasporaceae) in a separate suborder (Gigasporineae), despite evidence that form and function may be different in each.

In the development and maintenance of germ plasm of fungi in all taxonomic groups, we have amassed anecdotal evidence of significant differences in infectivity linked to geneology of fungal organisms. The most prominent and consistent differences are between members of Gigaspora and Glomus (representing distinct differences at the suborder level), so they are the primary focus of this discussion.

At this juncture, we caution the reader to keep in mind that ecological (selection) pressures drives local life cycle adaptations, so that many differences are likely to exist between geographic isolates of a species involving traits such as infectivity, primary colonization rates, secondary colonization rates, foraging ability of extraradical hyphae, survivability, tolerance to microhabitat changes, etc.

Spores appear to be the only infective propagules of species in this genus. Biermann and Linderman (1983) showed that neither root pieces or auxiliary cells produced mycorrhizae in geranium plants. We have noted the following observations that support their results:

1. Pot culture inocula consisting of only mycorrhizal roots and auxiliary cells fail to produce new mycorrhizae in subculture attempts. This has been observed most often with whole inoculum of Gi. rosea, Gi. margarita, and Gi. decipiens isolates stored at 4^oC for one year or longer and all spores had completely degenerated.
2. Attempts to use hyphae with auxiliary cells as inoculum consistently fail to produce new mycorrhizae. As first noted by J. Kough (pers. comm. in Biermann and Linderman, 1983) and observed also in our pot cultures, auxiliary cells appear to peak prior to sporulation and then decline in numbers. Thus, these structures have a different role than as propagules, such as temporary storage of carbon compounds.
3. Spores from pot cultures are so infective that 80-100% of new cultures started from single spores are successful in our regime (Mycotaxon 48:491).
4. Spores of Gigaspora collected directly from the field often are as infective as those collected from trap cultures (except when there is obvious hyperparasitism or senescence of spores).
5. When spores of different fungi in Gigaspora and Glomus are present in the same trap culture and inoculated separately onto sorghum seedlings, Gigaspora (and Scutellospora) never cause contamination problems in the Glomus (or Acaulospora) cultures. This contamination, if it were to occur, would have to originate from stray hyphae not detected among (or within) the spore preparation.

If spores are the only infective units in Gigaspora, then counts in research plots or in cultures provide some measure of inoculum potential and are amenable to ecological interpretations. Some evidence exists that abundance of germinable spores correlates with the Mean Infection Percentage (MIP) infectivity assay (Gemma and Koske, 1988).

Biogeographic distribution of species may also be explained more completely by the role of spores as vehicles of dissemination and as the ONLY propagule forming new individuals. Gigaspora species appear to have a somewhat limited distribution in Europe (Walker, 1992), a pattern that may hold true on other continents as well. If so, then constraints on species distribution may have less to do with ancestor-descendant relationships among organisms of a species than with limits on range of spore dissemination and establishment of new mycorrhizae.

All of these conclusions and interpretations are based on propagules disturbed by extraction and inoculation procedures. Thus, it has yet to be determined if hyphae from mycorrhizal roots or auxiliary cells can initiate new infections when the root-mycelium network is not disturbed. We hypothesize that results of such experiments also will be negative because the behavior or fungal propagules in Gigaspora is so consistent.

We suspect that many properties of Gigaspora are shared by Scutellospora, but not enough evidence has been documented to make any strong conclusion. Clearly, however, auxiliary cells of Scutellospora are not infective.

Almost all parts of the soma of fungal organisms in Glomus appear to function as infectious propagules. We have observed the following patterns:

1. Pot cultures can be started from (i) detached hyphae, (ii) mycorrhizal roots, and (iii) spores of many species, particulary those which form intraradical spores (e.g. G. diaphanum, G. intraradices).
2. Inocula from stored pot cultures retain some level of infectivity even when spores are no longer present.
3. Spores of many species are not as infective as hyphae or mycorrhizal roots. For example, the success rate of cultures started from single spores usually ranges from 10-50% in our regime.
4. Spores of Glomus species collected directly from the field often are not infective, whereas whole inocula (containing roots and hyphal fragments) almost always are infective.
5. When spores of different fungi in Gigaspora and Glomus are present in the same trap culture and inoculated separately onto sorghum seedlings, Glomus (or Acaulospora) sometimes contaminate Gigaspora (or Scutellospora) cultures.

The wide distribution of Glomus in almost any habitat may relate, in part, to flexibility of diverse propagules to start new individuals. This diversity of propagules also suggests that spore count data have little value in interpreting life cycle processes or community dynamics (except as a measure of species composition).

The literature supports the tentative conclusion that Acaulospora and Entrophospora have similar flexibility in kinds of infective propagules. In the former genus, at least, dormancy factors, differential infectivity with disturbance or drying, etc. complicate the picture (Jasper et al., 1989; Tommerup and Abbott, 1981). However, we hypothesize this variation to be ecologically-derived rather than inherited from parental organisms (genealogy/taxonomy).

Geneological and ecological causation in life-cycle differences among geographic isolates (or ramets sensu Rayner) have yet to be distinguished in experimental comparisons. Differences in propagule infectivity are treated as a species-level, or genealogical-based, property. Comparisons must depend on careful choice of germ plasm based on differences in: (1) taxonomic position, (2) geographic origin, and (3) climatic conditions at site of origin (particularly in relation to phenology of the plant host).

With respect to culture development, we have become very concerned about carry-over of infective hyphal fragments of Glomus or Acaulospora species when separating spores of the same morphotype for inoculation onto sorghum. Situations have arisen wherein a "pure" culture of one organism started with multi-spore inoculum suddenly becomes "contaminated" with another organism in the third or fourth propagative cycle. The contaminant often is traceable to the trap culture from which both originated. To minimize this problem, we now take very careful steps to exclude all visible hyphae from spore preparations.

Spores of the same morphotype are collected with a glass pipet having an extruded tip (< 0.5 mm opening) and transferred to a watch glass. Adding more water and swirling, all visible debris and hyphae are removed by pipetting. Spores are transferred to another watch glass and the same operation repeated. The spores then are carefully examined for presence of any hyphae or debris before being placed on seedling roots.

REFERENCES

Biermann, B. and R. G. Linderman. 1983. Use of vesicular-arbuscular mycorrhizal roots, intraradical vesicles and extraradical vesicles as inoculum. New Phytol. 95: 97-105.

Hall, I. 1976. Response of Coprosma robusta to different forms of endomycorrhizal inoculum. Trans. Br. Mycol. Soc. 67: 409-411.

Jasper, D. A., A. D. Robson, and L. K. Abbott. 1989. Soil disturbance reduces the infectivity of external hyphae of vesicular-arbuscular mycorrhizal fungi. New Phytol. 112: 93-99.

Tommerup, I. and L. K. Abbott, 1981. Prolonged survival and viability of vesicular-arbuscular mycorrhizae hyphae after root death. Soil Biol. Biochem. 13: 431-433

Gemma, J. N. and R. E. Koske. 1988. Seasonal variation in spore abundance and dormancy of Gigaspora gigantea and in mycorrhizal inoculum potential of a dune soil. Mycologia 80: 211-216.

Walker, C. 1992. Systematics and taxonomy of the arbuscular endomycorrhizal fungi (Glomales) -- a possible way forward. Agronomie 12: 887-897