Browsing by Author "Bäcklund, Kirsi"

Boreal forest soil contains large nitrogen resources. Most of the nitrogen is bound to humified material and proteins. However, plants can directly utilize only simple nitrogen compounds as ammonium, nitrate and simple amino acids. The amount of these simple nitrogen compounds is very small in forest soil. Trees form ectomycorrhizal symbiosis with a large number of fungi. Ectomycorrhizal fungi receive carbon compounds from host trees and trees receive nutrients from fungi. Some ectomycorrhizal fungi produce proteases which are enzymes that hydrolyze proteins. This symbiosis might be important in utilizing the large nitrogen resources in forest soils and supplying nitrogen to host plants. The main aim of this study was to measure if ectomycorrhizas produce proteases in the field and to find out if the mycorrhizal protease activities change during the year. The aim was also to find out if proteolytic activity is found in the soil fluid and to see if the activities in soil fluid are related to mycorrhizal activities. Mycorrhizal fungi were isolated to form pure cultures and their protease activities were measured and species were identified. As background information mycorrhizal, nonmycorrhizal and dead root tips were calculated to see how their amounts change during the year. Also different kinds of protease producing mycorrhizal morphotypes were calculated. Samples were taken in Hyytiälä Forestry Field Station in Central Finland monthly from March to October. Fifteen soil cores were collected each month. If possible, eight ectomycorrhizas were randomly picked from the pine roots in the humus layer of each soil core. Protease activities were measured from the ectomycorrhizas and from the soil solution obtained from the homogenized humus layer using Protease Fluorescent Detection Kit (PF0100-1KT, SIGMA). Detection limit of the kit was enzyme activity that is equivalent to 5 ng of trypsin control activity. Results were read from the trypsin standard curve so the protease activities of the samples are equivalent to fluorescence of certain amount of trypsin control. Pure cultures were isolated to Hagem’s agar plates and species were identified by doing PCR from the ITS gene region and sequencing. Root tip and morphotype calculations were done by using a microscope. 12 % of all ectomycorrhizal root tips produced proteases. The smallest activities couldn’t be detected because of the detection limit of the kit. Ectomycorrhizal protease production varied between 0–12 ?g/m2 of soil. Statistically non-significant protease production peaks were observed in spring and autumn. The protease activity of the soil fluid varied mainly between 0–200 ?g/m2 of soil. The highest activity was detected in the frozen soil in March, when the variation was large and an average of 800 ?g of protease was measured per m2 of soil. The protease activities in the soil fluids were not related to the protease activities of the ectomycorrhizas. 17 pure cultures were achieved. Some of them couldn’t be identified to species level. Part of the pure cultures produced proteases. Number of mycorrhizal root tips was large in spring, decreasing in early summer and increasing again towards autumn. About half of the mycorrhizal morphotypes produced proteases. The most important conclusions are that ectomycorrhizal fungi produce proteases in the field and a lot of protease activity is also found in the soil fluid. The used method is sensitive and suitable for measuring protease activities directly from mycorrhizal root tips and soil fluid. Seasonal variation in the protease activities may occur both in mycorrhizas and in soil fluid.