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MIP Assays as a Screening Tool

With so many accessions in the culture collection, and a paucity of information of ecotypic variation, etc., we sometimes need to test isolates for their ability to colonize in an experimental environment designed by a user (at their request usually).

A soil environment example

A biology professor at WVU mentored a student who wanted to conduct experiments examining fungal uptake of copper and zinc at plant-toxic concentrations. We did not have records of any fungi in our collection with tolerance to these conditions, so we selected several strains that were aggressive colonizers in acidic soils. The researcher wanted to grow the assay host in sand culture so that the nutrient/ionic environment in the pots was as controlled as possible. An MIP assay was set up to screen five different fungal strains using the experimental host and five different copper and zinc levels. The results indicated that one strain of Rhizophagus clarus was highly aggressive at all but the highest Cu and Zn levels, whereas other strains were infective only at the lowest cation concentrations. We then were able to confidently recommend this strain for the experiment.

A plant host example

Anecdotal data collected from culturing numerous fungi on different hosts suggested that discrimination/compatibility differed between woody perennial versus herbaceous annual species. For example, Claroideoglomus etunicatum in a mixed inoculum preferentially colonized and persisted in roots of corn, sorghum, red clover, and fescue and rarely was found in roots of apple and grape. Conversely, Rhizophagus intraradices frequently dominated or became the sole colonizer in woody perennial species. To test compatibility of fungi for different hosts to be used in a comparative experiment would be to set up MIP assays using the same growth medium and the same fungal strains, but varying host species between assays. The extent of mycorrhizal colonization in 30 days (or whatever time frame is best for the hosts being examined) is interpreted as directly proportional to plant compatibility. Assay results in this type of screen are tentative indicators because they do not even address compatibility interactions (leading to persistence/loss) occurring over the long term where fungal communities in roots can shift as a result of environmental fluctuations and possibly even host phenological changes.

A geographic example

Disturbed sites often are targets for introduction of fungal inoculum. But questions if whether such a move is necessary or whether inoculum can be more efficiently placed can be important, and they can be answered to some extent by “mapping” infectivity of various points on a site with MIP assays. In this case, we have selected the dominant (or most mycotrophic) plant species in the community as assay host, created a mix of various soil samples from the site to create an “averaged” growth medium, and then run all site samples (containing native fungi) simultaneously. When results of the assays are obtained, points are ordered from those with the highest to those with the lowest colonization (highest to lowest infectivity). For example, in Mingo County, West Virginia, crushed rock from a coal-mining operation was layered over one acre and planted with corn. Infectivity was nil at the beginning of the growing season. However, at the end, colonization as high as 15% was obtained from some points in the field. A pattern emerged wherein colonization was higher in one half of the field compared to the other, and this correlated with two different sources of rock which varied in calcium content (and thus pH).