The mycorrhizal inoculum potential can be measured by different bioassays proposed in the literature. The mycorrhizal inoculum potential indirectly estimates the infective AMF propagules in a given sample and can be used both for field samples and to assess the quality of a monospecific culture.
The MPN (Most Probable Number) measures the presence or absence of mycorrhizal colonization in plants subjected to serial dilutions, with the results being interpreted as a probability of the number of propagules from a statistical table. The assay is indirect as the absolute number of propagules is not measured, however it has the advantage of providing a unique number of propagules for a sample which can be directly compared with that of another sample in the same assay. The downside is that the number has a high 95% confidence interval, even at a low dilutions and with many replicates per dilution. According to Adelman & Morton (1986), all comparisons between samples must be made in just one experiment, that is, under the same conditions and at the same time. The ideal is to carry out at least three tests for each soil sample in order to perform a statistical analysis, something that is rarely done in studies found in the literature. As there are many factors that affect the results, such as environmental factors, type of inoculum and inoculum handling (storage temperature, inoculum drying), comparisons are not possible between different inoculum processed in different ways and tests performed at different times for different people (Wilson & Trinick, 1982).
Two important factors to consider are: a) the dilution factor and b) the number of tubes (replicates) per dilution. The smaller the dilution and the more tubes established per dilution, the smaller the 95% confidence interval. A dilution factor of 10x (10-1, 10-2, 10-3, 10-4...) is generally used with five replicates per dilution.
The amount of time that the test will be conducted depends on the host and the size of the container being used; in all cases it is important to hope that the host plant's root system can grow throughout the substrate containing the inoculum. It is helpful to have some extra conetainers which can be harvested after 4 to 6 weeks to get some idea of the pattern and degree of root growth. At harvest, the roots are washed to remove any material that might stick to them, stained and examined for the presence or absence of mycorrhizal colonization. In dilutions with less inoculum (e.g., 10-4, 10-5), the roots should be carefully examined as the infection units may only be in a few places in the root system. As the examination of colonization is carried out under a dissecting microscope, care must be taken not to confuse mycorrhizal colonization with infection by water molds, especially when field soils are being used. If the person does not have much experience and is unsure whether the colonization is mycorrhizal or not, it is worth checking the colonization under a compound microscope. It should only be counted positive if the fungal structures are actually colonizing the root system; presence of detached root hyphae or spores should not be considered positive.
Below is an example of how to assemble an MPN, modified from the one presented by Bagyaraj & Stürmer (2010):
Material needed:
- Plastic conetainers (150 cm x 2.5 cm) and trays
- Plastic bags (30 x 20 cm)
- Sterile diluent (use a standard diluent to compare multiple samples)
- Sorghum seeds
Procedure:
1) Weigh 30g of field soil (or sample from which you want to measure the MPN) and add it to 270g of the sterilized diluent soil. Shake vigorously to obtain the 10-1 dilution,
2) Remove 30 g of the 10-1 dilution and place it in another plastic bag containing 270 g of the sterilized diluent soil. Shake vigorously to obtain the 10-2 dilution. Establish the following dilutions in the same way until reaching the 10-5 dilution,
3) Distribute the soil from each of the dilutions into the conetainers. Establish five replicates per dilution (total 25 cones if using 5 dilutions).
4) Place sorghum (or another host that is mycotrophic) seeds in each of the conetainers,
5) After seedling emergence, trim to only one plant per conetainer and conduct the test in a greenhouse or growth chamber for 4 to 6 weeks,
6) At harvest, wash the roots gently to remove any soil particles and submit the roots to the Trypan blue staining process (or other procedure that allows you to visualize colonization),
7) Under the dissecting microscope, the presence or absence of mycorrhizal colonization in each of the repetitions is determined. Counting the number of positive conetainers (containing mycorrhiza) at different dilutions is used to calculate MPN values. Use tables from Cochran (1950), Fisher & Yates (1963) or Alexander (1965).
To exemplify the calculations, let's assume that, considering the 5 repetitions for each of the dilutions (10-1, 10-2, 10-3, 10-4, 10-5), the following sequence of numbers of positive conetainers is obtained: 5, 5, 3, 2, 0 This means that all 5 replicates are positive for mycorrhizal colonization at dilutions 10-1 and 10-2, 3 replicates at dilution 10-3, 2 replicates at dilution 10-4, and no replicates at dilution 10-5. For MPN calculation, only three numbers of a given sequence are considered. The first number (N1) specifies the maximum number of positive conetainers at the least concentrated dilution. The other two numbers (N2 and N3) are those corresponding to the next dilutions. In the example above, the combination would be:
N1 = 5, N2 = 3, and N3 = 2
Using the MPN table, the given value for this combination of positive values is 1.4. To obtain the MPN of the infective AMF propagules in the sample, this value has to be multiplied by the medium dilution (in this case 10-3).
Thus, this soil has 1.4 x 10-3 AMF infective propagules per g of soil.
REFERENCES:
Adelman, M. J. and J. B. Morton. 1986. Infectivity of vesicular-arbuscular mycorrhizal fungi: Influence of host-soil diluent combinations on MPN estimates and percentage colonization. Soil Biol. Biochem. 18: 7-13.
Alexander, M. 1965. Methods of Soil Analysis, Part 2, Agronomy Series #9, ASA, Madison, Wisconsin, pp. 1467-1472.
Bagyaraj, J.D. and Stürmer, S.L. 2010. Fungos micorrízicos arbusculares (FMAs). In.: Manual de Biologia dos Solos Tropicais. Eds.: F.M.S. Moreira, E.J. Huising & D.E. Bignell. Editora UFLA, Lavras. Pp.205-225.
Wilson, J. M. and M. J. Trinick. 1982. Factors affecting the estimation of numbers of infective propagules of vesicular arbuscular mycorrhizal fungi by the most probable number method. Aust. J. Soil Res. 21: 73-81.
This method was proposed by Moorman & Reeves (1979) to measure the mycorrhizal inoculum potential in disturbed areas. The authors acknowledge that spore count is one of the most used measures to assess population levels of AMF in soil, however, other propagules such as colonized roots and soil mycelium can be important sources of inoculum (Read et al. 1976).
The bioassay proposed by Moorman & Reever (1979) consists of determining the percentage of colonization (or infection, as stated in the original article) in the root system of maize plants grown for 30 days in disturbed or undisturbed soils. Maize was proposed as a host because of its ability to associate with many AMF species. An important point in this method is that it measures only the viable inoculum whereas in counting spores it is not always possible to distinguish between viable and non-viable spores by visual inspection alone.
In the corn bioassay, the percentage of mycorrhizal colonization or the length of mycorrhizal root is measured. In the original article, Moorman & Reeves (1979) measure % colonization by evaluating the presence of colonization in 100 root segments obtained from the total root system of each replicate. Another method that can be used to measure % colonization is the grid-line method proposed by Giovannetti & Mosse (1980) using a subsample of the root system, which must be weighed first. Mycorrhizal root length should also be measured, especially under conditions where the assay results in different root biomass production between samples (in these cases, % colonization may not reflect the total amount of fungal colonization in the total root system). Regardless of which method you use, the result reflects a measure of primary colonization of AMF (from activated propagules) and secondary colonization (growth of the fungus in the cortex) over a period of time.
This method has the advantage of requiring fewer replicates (and conetainers) to get a result (assuming only one dilution is used), but it is also sensitive to the same parameters that affect the MPN. On the other hand, it requires more work as the % of root colonization has to be carefully measured on each replicate rather than assessing presence or absence.
In the original manuscript, Moorman & Reeves (1979) suggested to establishing the zero, ¼ and 1/40 dilutions, with 5 replicates per dilution and sampling the plants from 5 replicates after 30, 60 and 90 days. This totals 45 conetainers and is more labor intensive compared to MPN. Many modifications are possible from different dilution series to different sampling times. Furthermore, a linear regression equation can be obtained between % colonization and dilution factors instead of % colonization and time.
A harvest date is established for comparative purposes based on host, root growth and environmental conditions. However, it should be considered that the harvest of the bioassay cannot be too early (21 days) as there may be no colonization (in cases where the inoculum potential is very low) nor too late (> 45 days) to avoid secondary colonization (and thus measuring fungus growth within the root and not inoculum potential). Although the variation associated with each dilution is not recorded in the literature, the standard error must be smaller than that of the MPN.
Corn Bioassay as set up at CICG:
1. Maize (Zea mays L.) is our standard host for measuring the MIP of pure cultures as it is mycotrophic, produces many roots in 4 weeks and the fungal structures in the roots are relatively easy to visualize. In some cases, sorghum (Sorghum bicolor) can be used in place of corn.
2. Plants are grown in plastic conetainers (150 cm x 2.5 cm) and 5 replicates are established for each trial. Plants are kept for 30 days in a greenhouse, after which they are harvested to analyze % colonization. In some cases when the inoculum potential is suspected to be high, an additional 2-3 conetainers are established and harvested at 21 days to determine whether the assay should be harvested on that date or maintained for 30 days.
3. The diluent consists of our standard sterilized substrate, a mixture of quartize sand and soil (1:1) with 5% of vermicompost.
4. Only one dilution is established (1/10 or 10% inoculum), even when samples are suspected to be highly infective. In some cases when field soil is being tested, a ½ dilution is made. It is important to assemble the trials all at the same time so that comparisons are appropriate. With just one dilution and 5 replicates, this design maximizes the number of samples that can be tested at the same time to measure the mycorrhizal inoculum potential.
5. Tests are conducted in a greenhouse with controlled light.
6. Roots are harvested, stained and % mycorrhizal colonization measured after 30 days. The average percentage of root colonization of each culture (obtained from the 5 replicates) represents an estimate of the mycorrhizal inoculum potential of each culture and can be compared statistically.
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.
Read, D.J., H.K. Koucheki & T. Hodgson. 1976. Vesicular-arbuscular mycorrhizae in natural vegetation ecosystems. New Phytol. 77: 641-653.
The infection unit method was developed by Franson & Bethlenfalvay (1989) and directly counts the units of infection at the root. In this sense, compared to the MPN and the MIP method, this method can be considered direct as it measures primary mycorrhizal colonization only, that is, that originated by the units of infection resulting from the entry points. One of the advantages of this method is that the relationship between inoculum density and the formation of discrete infection units is linear. The authors state that this method is suitable for determining the amount of inoculum with equivalent potentials for use in short-term experiments.
In the original article, the authors establish several dilutions with the inoculum to be tested, to obtain a linear relationship between the dilutions and the number of infection units. Various dilutions are established based on the mass of the inoculum in relation to the total mass of substrate used. Dilutions in the original protocol are irregular (50%, 12.5%, 3.1% and 0.78%) and no reason why these dilutions were established is given.
The setup and evaluation of the bioassay is very laborious and must necessarily be of short duration (14 days) to ensure that no secondary colonization occurs. This limitation can introduce some variability in the results, depending on the types of propagules present in the inoculum, host species used, and taxonomic composition of the inoculum, all of these factors influencing the probability of contact between the fungal propagules and the root.
Comparisons can be made using the angle of the regression equations obtained and the standard error of the data is relatively low, according to the results of Franson & Bethlenfalvay (1989) based on a study with five isolates (Funneliformis mosseae, Claroideoglomus etunicatum, and Glomus pallidum). Few manuscripts have used this method to measure AMF infectivity and thus its validation remains open.
REFERENCE:
Franson, R. L. and G. J. Bethlenfalvay. 1989. Infection unit method of vesicular-arbuscular mycorrhizal propagule determination. Soil Sci. Soc. Am. J. 53: 754-756.