Quantification of the proliferation of soil fungi in three dimensions using micro-computer aided tomography

Abstract

Good soil structure is important for sustaining agricultural production and preserving functions of the soil ecosystem. Soil aggregation is a critically important component of soil structure. Stable aggregates enable water infiltration, gas exchange for biological activities of plant roots and microorganisms, living space and surfaces for soil microbes, and contribute to stabilization of organic matter and storage of organic carbon (OC) in soil. Soil aggregation process involves fine roots, organic matter and fungal hyphae. Hyphal proliferation is essential for soil aggregation, and sequestration of OC in soil.

Organic materials and available phosphorus are two of the major factors that influence fungi in soil. Organic materials are a source of energy for saprotrophic microbes and fungal hyphae may increase in the presence of organic matter. Phosphorus is an important element usually found depleted in soil ecosystems. The low availability of phosphorus may limit the biological activity of microbes. Arbuscular mycorrhizal fungi (AM) benefit plants by delivering phosphorus to the root system. However, the density and the length of hyphae of AM fungi do not appear to be increased by available phosphorus.

We do not yet have a mechanism to directly quantify the density of hyphae in soil. A number of indirect methods have been used to visualize distribution of fungi in soil. Reliable analyses of soil are limited because of the use of destructive harvests to gain information. Soils are fragile, and fragility limits opportunity for non-destructive analysis. The soil ecosystem is also complex. Soil particles are dense and the density obscures the visualization of fungal hyphae. Fungal hyphae are relatively fine and information at the small scale (<250 µm) is key to understanding how fungi respond to environmental stimuli. The experiments conducted here developed a new method for the observation of fungi and quantification of hyphae in three dimensions. The methods were first tested using a melanised saprotrophic fungus, 222A. The response of two AM fungi, Glomus mosseae and Glomus pellucidum, growing together to common environmental factors was then quantified. Hyphae were quantified in an artificial soil matrix over 6 week’s incubation using micro-computer aided tomography (microCT). MicroCT provides three dimensional images of mycelia within electron lucent materials and enables the visualization and quantification of hyphae. Starch stimulated proliferation of 222A after 2 weeks. Starch, and starch and K2HPO4, stimulated proliferation of hyphae of AM fungi after 3 and 6 weeks. K2HPO4 stimulated hyphal proliferation only after 3 weeks. The images also indicate that fungal hyphae grew appressed to the surfaces of the particles rather than through the spaces between the particles. The capacity to quantify hyphae in three-dimensional space allows a wide range of questions to now be addressed. Apart from studying mechanisms of carbon turnover, more complex processes may now be considered. Soil is commonly thought of as a black box. That black box potentially is now a shade of grey.