Manual book for microbial and nematode community analysis using soil DNA
To elucidate how soil microbes and nematodes are involved in the phenomena in agricultural fields, such as crop growth, pest infestation and suppressiveness, and green house gas emission, we need to collect as much as possible the information of soil biota, and compare it across soil samples. For this purpose, an optimized and standardized analytical method is required for each group of soil organisms. Although PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis), a method to analyze microbial communities using DNA fragments, is prevailing since 1990, collection of biological information has not yet been made sufficiently, because experimental conditions are different between analyses. Therefore, to standardize the analytical conditions, we have optimized PCR-DGGE procedure for each of bacterial, fungal and nematode communities, and published a manual book (Fig. 1).
Optimization of analytical conditions
For the primer set selection, the most critical point of the conditions, we tried three to four sets for bacteria and fungi, to amplify soil-extracted DNA, and conducted DGGE to compare the band numbers and the diversities of the band patterns between the primer sets (Fig. 2, fungal test as an example).
For nematodes, we selected a single primers set in advance based on the clarity of the band patterns, and confirmed its detection ability, by comparing community structures (frequencies of nematode taxa) as revealed by the clone type analysis with the primer set, and those by morphological identification under a microscope (Fig. 3).
After these examinations we determined the most suitable primer set for each of soil bacterial, fungal and nematode analysis. For these primer sets, we also determined the optimized conditions in PCR amplification, electrophoresis and other experimental steps (Table 1).
In addition, we developed "DGGE marker" for the three organism groups, sets of DNA fragments of known species, to compare DGGE band patterns across gels. The "marker" has been already available commercially. With the standardized and optimized DGGE procedures we developed, clear and sharp band patterns can be produced (Fig. 4).
Soil DNA data base
We expect to elucidate the mechanisms in which soil biota affect agricultural production, by analyzing the biological information as revealed by our DGGE procedure. Actually, NIAES is now collecting soil samples, or the DNA extracted from soils, under various types of agricultural management at different localities from the northern to the southern Japan, and is conducting DGGE to construct a soil DNA database. Our final goal is to determine the relationships between the biological properties evaluated by DGGE band patterns, and the soil physicochemical properties as affected by cultivation practice.