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MEAN INFECTION PERCENTAGE (MIP) METHOD
This
assay measures the percentage mycorrhizal colonization in an assay host over
time, after the host has been grown in a series of inoculum dilutions. The goal
with the different dilutions is to obtain data which can fit into a linear regression
equation. Then, direct statistical comparisons can be made. This is a comparative
assay in which a set of assays are measured relative to a reference assay (usually
some sort of positive control. There is not a 1:1 correspondence between number
of infectious propagules and the assay result. For example, if one wishes to
compare commercial inoculants (all of which differ in important variables such
as fungal species composition, properties of isolates in this species mix, and
substrate composition), then a reference assay would be an inoculum of known
properties from which an informed comparison can be made.
In each assay, the statistic being measured can be either percentage colonization or mycorrhizal root length. The method of measuring % colonization by counting number of mycorrhizal segments out of 100 taken from a subsample of total roots per growth tube is not very accurate, especially at lower dilutions. The preferred method, and the most widely used one, is the grid-line intersect method (Giovannetti and Mosse, 1980) using a weighed subsample of roots. Mycorrhizal root length should be measured more often, especially if conditions in each assay result in different root biomass accumulation (in which case % colonization measurements may not reflect the actual amount of fungal colonization in the total root system). Regardless, the amount of mycorrhizal colonization includes a measure of both primary ingress (from propagules) and secondary spread (new infection units from those already established) within a finite time period. This assay has the advantage of requiring fewer tubes to obtain a result, but it also is sensitive to environment and the other parameters mentioned above for the MPN assay. The trade-off is the more labor-intensive task of carefully estimating percentage of mycorrhizal roots in each tube rather than scoring for presence/absence.
The method calls for five replications of each dilution. In the seminal paper on this method (Moorman and Reeves, 1979), dilutions ranged from zero to 1/40, with sampling times at 30, 60 and 90 days. This approach is quite laborious and often not needed. Many modifications are possible, from a standardized dilution series (2-fold, 5-fold, 10-fold) to different sampling times. In addition, regressions of percentage colonization against dilution factor could be plotted instead of colonization against time. A set harvest date is established based on host, root growth, and environmental conditions surrounding the assay.
Key considerations are: (i) a date early enough (e.g. 21-30 days) to keep secondary colonization low and (ii) a date late enough to insure measurable levels of colonization in all the different samples being tested. Published results do not specify the amount of variation in results of each dilution, but the standard error undoubtedly is much lower than the confidence limits of MPN assays.
Our MIP assays are conducted as follows:
1. Corn is our standardized assay host because it is highly mycotrophic, produces abundant roots in three weeks, and fungal structures in the roots are easy to stain and visualize.
2. Plants are grown in plastic cone-tainers (4 x 18 cm), four replicates per assay. Usually 2-3 additional replicates are set up to examine % colonization at 21 days to determine if the assay should be harvested at that time or maintained for another 9 days (30 days is a maximum because of complications from secondary colonization, especially if different fungi are present amongst assays).
3. Diluent consists of sterile sand (if changes in medium chemistry are important) or a mix of sterile sand and low-P, low organic matter soil (2:1 v/v) adjusted to pH 6.2.
4. When samples are assumed to by highly infective, two dilutions generally can be used: 1/10 and 1/20. In our experience with testing field soils, pot culture inocula, and commercial inoculants, a 1/10 dilution is adequate. It is important to set up the assays so that ALL relevant sample comparisons are conducted at the same time. With only one dilution, this creates a better design to maximize number of treatments.
5. Assays are carried out in a growth room with controlled light, temperature, and humidity conditions.
6. Roots are harvested, stained, and mycorrhizal colonization measured at either 21 days or 30 days. On occasion (mostly commercial inoculants obtained from users rather than a company representative), infectivity is so low that we extend the assay for 45 days to see if any propagules in the medium are active.
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| In this MIP assay, six different inoculant formulations are being tested (with a nonamended control on the far left, so the stimulatory effects of amendments is quite obvious). The two formulations on the far right are having a severe inhibitory effect on plant shoot and root growth, whereas those in the middle are greatly stimulating plant biomass. If the fungi present in all of these treatments grew at a similar rate (if the formulation ingredients didn't have a similar effect on the fungi, which were the same in all samples except the control), then percentage colonization would be lower in cones with high root biomass and higher in cones with fewer roots. To get a good measure of the proportion of roots colonized, fungal colonization must be compared against total root length. |
REFERENCES
Moorman, T. and F. B. Reeves. 1979. The role of endomycorrhizae in revegetation practices in the semi-arid West. II. A bioassay to determine the effect of land disturbance on endomycorrhizal populations. Amer. J. Bot. 66: 14-18.
Giovannetti, M. and B. Mosse. 1980. An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol. 84: 489-500.