Evaluation of the selective respiratory inhibition method for measuring the ratio of fungal:bacteria activity in acid agricultural soils

A procedure for the measurement of the fungal and bacterial contribution to substrate-induced respiration was tested in three arable soils. Glucose and different amounts of cycloheximide (eukaryote inhibitor) and streptomycin sulfate (prokaryote inhibitor) were added to soil suspensions, and respiration (CO2 evolution) was measured. Streptomycin sulfate concentrations from 10 to 120 mg ml-1 soil solution caused a stable inhibition of respiration. Amounts of cycloheximide ranging from 5 to 35 mg ml-1 showed an increasing inhibition. In a test with separate and combined addition of the antibiotics at maximum inhibitory concentrations, inhibition by streptomycin was completely overlapped by cycloheximide. This indicated non-target inhibition which may lead to overestimation of fungal respiration. Experiments with sterilized soils inoculated with either fungi or bacteria confirmed that streptomycin selectively inhibited bacteria. Cycloheximide, however, did not only inhibit fungal respiration already at 2 mg ml-1, but also increasingly inhibited bacterial respiration at increasing concentrations. Only at < 5 mg cycloheximide ml-1 was the condition of selective fungal inhibition fulfilled. When 2 mg cycloheximide and 10 mg streptomycin sulfate ml-1 were applied, the sum of the separate inhibitions almost equalled the combined inhibition by the mix of both inhibitors in field samples. This method yielded fungal:bacterial respiration ratios of 0.50 to 0.60, and confirmed the dominance of bacteria in Dutch arable soils. The ratios obtained by the selective inhibitors were not correlated with, and were higher than, ratios of fungal:bacterial biovolume (0.19 to 0.46) as determined by microscopy and image analysis. Similar measurements in a forest soil (A-horizon) raised doubts on the reliability of the fungal inhibition by cycloheximide in this soil. It is concluded that the separate:combined inhibition ratio should always be checked, and comparison with other approaches is recommended.