Quantifying Vesicular-Arbuscular Mycorrhizae Using a Morphometric Technique

Abstract

In an attempt to quantify the amount of VA mycorrhizal colonization, many authors have obtained data from views of whole roots. This approach yields qualitative but not quantitative data. Quantitative methods have been developed which employ biochemical assays for chitin and the use of thin sections. However, these techniques are often laborious. A technique employing the basic principle of morphometric cytology on squashed roots is offered as a compromise. By employing a symmetrical grid of dots over the image of a root squash, counts of invaded cortical cells can be made. This technique is faster to apply than sectioning and yields quantitative information concerning the percentage of the cortical cells containing arbuscules. In all studies involving vesicular-arbuscular mycorrhizae the occurrence of colonization must be determined at least on a qualitative basis. However, many studies, especially those dealing with the efficiency of the association in phosphorous uptake or growth increase experiments, require more quantitative approaches. In the past, a variety of methods have been employed to quantify the percentage of host tissue colonized by the mycorrhizal fungus. Observations of per cent colonization at the light microscope level range from verbal descriptions (very slight to abundant) (12) and numbers of plants colonized (14) to estimations of colonized cortical tissue (13). More analytical approaches have been used to estimate the per cent colonization using lengths of whole roots (2), or the amount of fungus in root segments (1, 3, 4, 7, 8, 17, 19). Recently, several methods of estimating per cent colonization have been reviewed (3, 7). Four of the more common methods for determining per cent colonization have been compared and several conclusions drawn (7). Most early measurements based on presence or absence of mycorrhizal fungus in 1 cm root segments overestimated colonization by a factor of two and the most accurate method involves the use of 1 cm root segments in Petri dishes with an underlying grid pattern for analysis. Using this method Giovannetti and Mosse (7) obtained values for the per cent of 1 cm segments containing mycorrhizal fungus in maize roots which ranged from 23 to 48%. Biermann and Linderman (3) applied the technique of estimating the length of 1 cm root segments colonized in three separate species. Values ranged from 32 to 73% root length colonized compared with 65 to 83% for root segments colonized. In these reviews (3, 7) as well as in many other studies (1, 4, 8, 17) authors gave values for per cent colonization ranging from 20 to 80%. Of what significance are these figures? These authors (1, 3, 4, 7, 8, 17) are not saying that 20 to 80% of the root volume is occupied by the fungus. These authors do not indicate (in most cases) whether the fungus which is present is in the form of intercellular hyphae, vesicles, degenerated or mature arbuscules, nor do they indicate a basic quantity such as the per cent cortical cells invaded. All of these methods of analysis suffer from the fact that the data are derived from two-dimensional projections of three-dimensional space. Invaded cells lying over uninvaded cells are counted the same as invaded cells lying