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 client researcher.

A soil environment example. A biology professor at WVU had a student who wanted to conduct experiments examining uptake of a glomalean fungal isolate by 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 fungi 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 as described using a standardized host (the one to be used in the experiment), with treatments being five different copper and zinc levels applied to assay cone-tainers for each fungus tested. The results indicated that one isolate of Glomus clarum was highly aggressive at all but the highest Cu and Zn levels whereas other isolates lost infectivity at lower levels. We then were able to recommend this fungus with some degree of confidence that it would grow and function in the experiment.

In the MIP above, the two far left assays are controls (no amendments), and then each assay to the right is the same fungal inoculum mixed with increasing amounts of amendments used in a commercial mix (humics, yucca powder). In the two far right assays, shoots and roots grew poorly and no colonization was detected.

A plant host example.

Anecdotal data collected from culturing numerous fungi on different hosts suggest that discrimination/compatibility of accessions on woody perennial versus herbaceous annual can be quite marked. For example, Glomus etunicatum in a mixed inoculum tends to preferentially colonize and persist roots of corn, sorghum, red clover, and fescue and rarely is found in roots of apple and grape. Conversely, Glomus intraradices frequently dominated in the latter woody perennial species (to the extent it was the only fungus present in roots of some plants). One way to test compatibility of fungi for different hosts to be used in an experiment is to set up MIP assays using a standardized growth medium (the same used in the experiment) and the same fungi, but varying host species. The amount of 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 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).