Quantification of Active Vesicular-Arbuscular Mycorrhizal Infection Using Image Analysis and Other Techniques

Methods for the objective measurement of vesicular-arbuscular mycorrhizal infection in the roots of four species of host plant are described. The methods are based on vital staining of transverse sections of fresh roots, followed by evaluation of occurrence and/or numbers of living arbuscules and hyphae per section. The most useful method involved freeze-sectioning roots embedded in gelatin containing glycerol and DMSO, followed by staining the sections with nitroblue tetrazolium overnight to reveal succinate dehydrogenese activity. Two systems of computer-aided image analysis were evaluated for measurement of the number, perimeter and cross-sectional areas of intercellular hyphae and arbuscules. This approach, which is clearly objective, is an advance on previous methods which were based on ranking sections or segments of root with respect to the intensity of development of arbuscules. The data were used to calculate the area of interface between plant and fungus in infected roots. Results are presented showing that the contribution of arbuscules to the total infection and area of interface declines markedly as plants and infections age. Differences in infection in different host/fungus combinations are also described.