| | ||||||
| | | | | | | |
Many different methods have been proposed to culture glomalean endomycorrhizal fungi, with a bewildering array of claims and counterclaims. All involve a plant host, either intact or as root explants. Methodological differences focus mainly on differences in the cultural environment, the most dramatic being the interface between fungus, plant root, and external matrix. The traditional (and most widely used) approach has been to grow a plant host in a solid growth medium consisting of one or a combination of the following solid growth media: soil, sand, peat, vermiculite, perlite, clay, or various types of composted barks. Alternative efforts have centered on growing bare-rooted mycorrhizal plants in hydroponic films (Elmes et al., 1984) or in aeroponic mist chambers (Hung and Sylvia, 1988). Excised Ri T-DNA transformed roots also were developed to grow fungi axenically on agar media (Diop et al., 1992). An underlying motivation for the break from pot culturing regimes has been to work directly with roots or fungal spores and hyphae in an environment free of organic or inorganic particulate matter. Aeroponic chambers establish conditions for production of a patented "sheared root" inoculum, although similar propagules may be equally infective from roots grown in solid media. Root organ cultures provide the only empirical means at the present time to monitor closely behavioral and physiological interactions at the root-fungus interface.
When
the focus is strictly on inoculum production, pot culture methods using soil
in combination with other inert ingredients have clear superiority in the range
of germ plasm cultured (see figure), either singly or in highly flexible combinations.
Incremental increases in constraints on host-fungus compatability appear to
exist when the culturing system moves from soil to agar media (see figure).
The dramatic reduction in germ plasm diversity that can be accommodated may
be a function of a decreasing capacity of some fungal organisms to accommodate
an increasingly "foreign" external environment for ingress, establishment,
growth, and sporulation. Clearly, the physical and chemical environments change
radically, which in turn affect physiological processes in both symbionts. To
some extent, this constraint may be deceptive because it also is a function
of the number of fungi tested in these systems. Another advantage of pot culture
methods in inoculum production is that the substrate in which mycorrhizae develop
provide a natural medium for prolonged storage after a drying step.
Many arguments have been invoked against use of pot cultures: contamination by saprobes, pathogens, and other mycorrhizal fungi, low propagule yields, and long duration between setup and harvest. Unfortunately, the same problems can appear in ANY culture system. I have seen, or heard about, contamination by saprobes and algae in hydroponic, aeroponic, and root organ cultures. Yield of infectious propagules in root organ culture is a fraction of that obtained in pot or aeroponic systems with the same culture period. A majority of problems in any of these systems are solved using common sense solutions: careful planning and management, and a vigilant quality control program.
Not all solid substrate growth media support the wide range of germ plasm indicated in the figure above. Moving into the realm of soilless media is a tricky business, and few generalizations can be made. Soilless environments often are not any less variable or hazardous to mycorrhizal development than soils. The choice of soilless medium for inoculum varies with particular goals. If inoculum is to be bulked and applied to a separate target crop, then the most important properties of the culture medium-host combination is optimization of fungal growth (maximum mycorrhizal roots and extraradical hyphae) and sporulation (maximum extraradical survival structures), together with properties that do not modify the application environment (a problem that sometimes plagued the clay-based carrier of Native Plants, Inc.). Productivity of the culture host is not as much of an issue, so higher fertility or other factors that facilitate plant growth, but inhibit mycorrhizal development, can be avoided. Application of inoculum in transplantable mycorrhizal seedlings is a more difficult task, because optimization of seedling vigor and growth with fertilizer treatments of the media must be balanced against the inhibitory effects these amendments may have on mycorrhiza formation. Some media (e.g. some barks, peats) may have inherently high levels of phosphorus and other nutrients.
Sand-based media
Sand alone is not a good substrate if plant growth responses are needed. There is no buffering capacity to the medium, so soluble nutrients must be continuously added to maintain host growth. Solution levels to optimize host growth may be too high (in the absence of fixation) for optimal fungal growth. Millner and Kitt (1992) solved this problem in a sand-based semi-hydroponic system by using an automated watering and fertilization regime. Their explicit purpose was to rapidly produce maximum numbers of fungal propagules in the shortest possible time. In some cases, sporulation in this system exceeded that of sand-soil mixes. The greatest advantage of this system is the absence of soil or other particulates that clutter spore extracts following sucrose density-gradient centrifugation and a higher probability (but not a certainty) of lower background microbial activity on root, hyphae, and spore surfaces. Thus, spores and hyphae are much easier to harvest and prepare for a variety of assays.
A combination of sand and vermiculate mixed in a ratio of 3:1 (v/v) was developed by Liyange (1989) to culture fungi in INVAM when it was situated at the University of Florida. A Long Ashton fertilizer solution was applied every two days, with bahia grass as host. My experience with this mixture suggests that it produces similar propagule yields to a sand medium, with less danger of dessication. In particular, Acaulospora species sporulated more abundantly. This result is significant, because organisms in Acaulospora have yet to be cultured successfully in aeroponic or root organ cultures. Spores extracted from a culture of this mix are clean if algal growth is kept to a minimum. Hyphae often have fine particulates coating the surface, but they can be cleaned with a short sonication treatment.
Soil in combination with sand provides additional buffering capacity, so that fertilizations can be reduced. The tradeoff, in this mix, is the introduction of mineral elements that may have toxic effects on plants or fungal symbionts following sterilization or during plant growth. The soil environment can never be precisely defined, no matter how thorough the chemical and physical analysis. Despite these pitfalls, sand in combination with a low phosphorus loamy mineral soil (proportion of 2:1 v/v) is the standardized mix used in INVAM because of its low maintenance and high success in culturing fungi from a wide range of habitats globally (Morton et al., 1993). An empirical benefit is the capability to determine growth responses concomitant with inoculum production. A disadvantage of this environment is a background level of dead spores and other soil debris that can confound recognition and harvest of spores for those who are inexperienced. When adherent organic particles to spores and hyphae are not wanted, a sonication treatment usually suffices.
Peat-based media
Peat moss, alone or in mixtures with perlite, vermiculite, or bark, rarely are used alone for inoculum production. There are some good reasons for this. Peat-based media usually are high in organic matter and do not adsorb P, two conditions which have strong potential to inhibit mycorrhizal colonization and sporulation (with even moderate fertility amendments). In our experience, inoculum yields in peat-based media are highest when they contain at least 25% soil. Buffering capacity appears to be the major constraint on propagule yield. Graham and Timmer (1984) found that rock phosphate provided a steady supply of P in peat-based media, without any ill effects to mycorrhization or plant growth responses. Conversely, readily solubilized P amendments (e.g. superphosphate) suppressed mycorrhizal development.
Vermiculite and perlite, when added with peat, shows no evidence of creating any ill effects to either the host, the fungus, or the mycorrhizal association. Perlite becomes a hinderance if the need ever arises to extract and separate fungal spores and hyphae. Unlike vermiculite, perlite does not break down. Perlite, however, also traps considerable quantities of hyphae (and spores when they are less than 100 µm in diameter) in pores, and these propagules can be collected cleanly only with considerable manual effort (and dexterity).
Calcined clay-based media
Plenchette et al. (1982) report success at inoculum production in calcinated montmorillonite clay, or "Turface". Our one attempt to use Turface was an abysmal failure (no colonization detected). Others report similar success or failure. Causes for these extremes in results are not clear, but they appear to be linked to vendor origin and associated differences in substrate preparation and levels of toxic impurities in the medium. It is possible that thorough washing of the clay might reduce or eliminate this variation, but I am not sure this has been tested.
Bark-based media
Composted barks (soft or hard woods) have not been used extensively as a pot culture substrate. Tests by a horticulture student in our lab revealed that composted and noncomposted hardwood bark mixes inhibited mycorrhizae formation by an inoculum "cocktail" of five fungal isolates in three genera. Specific barks may be more conducive to mycorrhization. Biermann and Linderman (1983) were successful at growing G. intraradices (reported as G. fasciculatum) in Douglas Fir bark. These conflicting results suggest that the use of bark alone or in combination with other solid components requires rigorous pretesting, preferably against a soil-based potting medium as a positive control.
In summary, each culture system and the various solid media that can be used alone or in various combinations in pot environments have their own unique advantages and disadvantages, depending the goals and applications of individual users. The choice is yours!
REFERENCES
Biermann, B. and R. G. Linderman. 1983. Effect of container plant growth medium and fertilizer phosphorus on establishment and host growth response to vesicular-arbuscular mycorrhizae. J. Amer. Soc. Hort. Sci. 108:962-971.
Diop, T. A., G. Becard, and Y. Piche. 1992. Long-term in vitro culture of an endomycorrhizal fungus, Gigaspora margarita, on Ri T-DNA transformed roots of carrot. Symbiosis 12: 249-259.
Elmes, R. P., C. M. Hepper, D. S. Hayman, and J. O'Shea. 1984. The use of vesicular-arbuscular mycorrhizal roots by the nutrient film technique as inoculum for field sites. Ann. Appl. Biol. 104: 437-441.
Graham, J. H. and L. W. Timmer. 1984. Vesicular-arbuscular mycorrhizae development and growth response of rough lemon in soil and soilless media: Effect of phosphorus sources. J. Amer. Soc. Hort. Sci. 109:118-121.
Hung, L.-L. and D. M. Sylvia. 1988. Production of vesicular-arbuscular mycorrhizal fungus inoculum in aeroponic culture. Appl. Environ. Microbiol. 54: 353-357.
Liyange, H. D. 1989. M.S. Thesis, University of Florida, Gainesville.
Millner, P. D. and D. G. Kitt. 1992. The Beltsville method for soilless production of vesicular- arbuscular mycorrhizal fungi. Mycorrhiza 2: 9-15.
Morton, J. B., S. P. Bentivenga, and W. W. Wheeler. 1993. Germ plasm in the International Collection of Arbuscular and Vesicular-arbuscular Mycorrhizal Fungi (INVAM) and procedures for culture development, documentation and storage. Mycotaxon 48:491-528.
Plenchette, C., V. Furlan, and J. A. Fortin. 1982. Effects of different endomycorrhizal fungi on five host plants grown on calcined montmorillonite clay. J. Amer. Soc. Hort. Sci. 107:535-538.