Soil samples were obtained from surface of upland field (U; Gleysol) at Itoshima, Fukuoka, Japan, where vegetable had been cultivated under conventional field management, and from the surface horizon of forest soil (F; brown forest soil) at Wajiro-Hgashi, Fukuoka, Japan. Commercial manure (M) made from rice straw and cattle feces was used in this study. To test vials (5 replicates) including Biolog Universal Growth Medium (BUGM; BIOLOG Hayworth, CA, USA) broth [22] , serial 10-fold dilutions (10⁻⁴ to 10⁻¹⁰) prepared from samples (1 g fresh wt.) were inoculated. After 3 days incubation at 30˚C, bacterial DNA in each vial was extracted described previously [20] [21] and purified by the conventional methods.

Using the V2 forward primer (41f), and the V6 reverse primer (1066r) [23] , 16S rDNA was amplified according to the former study [18] [19] . After restriction digestion of the PCR product (10 μl) by each of 10 units of the restriction enzyme, Hae III or Hha I or Rsa I or Scr F1 (Takara Bio Co. Ltd., Shiga, Japan) in Low salt buffer solution (10xLow salt buffer, Takara Bio Co. Ltd.) and 5 folds dilution by de-ionized water (for Low salt buffer), restriction fragment lengths were measured by microchip electrophoresis system (MCE-202 MultiNA; Shimadzu Co., Ltd., Kyoto, Japan).

The newly constructed database was used for this research, which was edited using the method of Watanabe and Okuda [17] described previously [19] . For 41f/1066r primers, 30,844 post-amplification sequence files, which were consisted from 1379 bacterial genera, including uncultured and unidentified bacteria, were mainly re- edited using small subunit rRNA files in RDP II release 9_61 [24] under 5-bases mismatches in the both in primer annealing sites.

As the reference MERFL database was edited from the homogeneous 16S rDNA sequences, the measured MERFL digested from the homogeneous 16S rDNA had to be used for phylogenetic estimation.

The major RFs, which had the highest relative mole concentration (ratio of fluorescent intensity to fragment size) and represented as H in Table 1, were selected among the mixed heterogeneous FRs as described previously. The 2nd major RFs, represented as M in Table 1, were similarly selected among the remained mixed heterogeneous FRs after subtraction of the major RFs. The 3rd major gene, represented as L in Table 1, were similarly selected using the remained mixed heterogeneous FRs after subtraction of the 2nd major RFs.

The pairwise distance (D_AB) between the measured RFLP (A) and the theoretical RFLP (B) was calculated according to Nei and Li [25] . For similarity search, the theoretical MERFLP (B) having the smallest pairwise distance (D_ABME), which was an average of all the D_ABs for used restriction enzymes, to the measured MERFLP (A) were searched in the reference database as described previously (Watanabe et al., 2008). Similarity (%) in Table 1 was calculated as the following equation; (1-D_ABME) × 100.

In phylogenetic estimation, identical theoretical MERFL (100%) was searched preferentially by using all the 4 measured MERFL data at first. When the completely identical theoretical MERFL was not found, combinations of 3 restriction enzymes were used for the next searches (Table 1). When the completely identical theoretical MERFL (100%) was not found, combinations of 2 restriction enzymes were used for the next searches (Ta- ble 1). When the completely identical theoretical MERFL (100%) was not found using 2 restriction enzymes, the theoretical MERFL having the highest similarity to the measured MERFL was indicated in Table 1 [17] [19] .

After differentiation of the measured MERFLs into 8 groups (A~J) based on the phylogenetic estimation. Numbers of each group were estimated by MPN for five-tube, three-decimal-dilution experiment (Table 2). Confidence limits shown in Table 2 were obtained using FDA’s Bacterial Analytical Manual [26] .

Affiliations of fifty MERFLs were summarized in Table 1. The MERFLs in this study was found to have a higher bacterial diversity than those in the former studies as the followings; all of the 50 MERFLs were divided into 47 OTUs, then ratio of total number of the OTUs to that of MERFLs was 94%, which was higher than that of upland field using selective medium (62.2%) [20] , that of manures during composting (60.4%) [21] and that of commercial food products (34.6%; unpublished results). The higher diversity of MERFLs was caused from higher bacterial diversity of samples and non- selectivity of the used incubation medium. They were divided into 8 groups for the MPN calculation as the followings; Actinobacteria (Group A, 10 MERFLPs), Bacillus spp. (Group B, 5 MERFLs), the other Firmicutes (Group C, 11 MERFLs), α-Proteobacteria (Group D, 4 MERFLs), β-Proteobacteria (Group E, 8 MERFLs), γ-Proteobacteria (Group F, 7 MERFLs), δ-Proteobacteria (Group G, 3 MERFLs), and the other gram negative bacterial group (Group H, 2 MERFLs) (Table 1).

^aGrouping was based on affiliation by MERFL; Actinobacteria (Group A), Bacillus spp. (Group B), the other Firmicutes (Group C), α-Proteobacteria (Group D), β-Proteobacteria (Group E), γ-Proteobacteria (Group F), δ-Proteobacteria (Group G), and the other gram negative bacterial group (Group H); ^bThe 1st letter in vial indicates samples; “F” stands for the sample from forest soil, “M” stands for the sample from commercial manure, and “U” stands for the sample from upland field soil . Exponential of vial number represents the decimal dilution of the vial. The 2nd number of vial number (1 - 5) represents number in 5 replicates for the each decimal dilution. H of last letter represents MERFL originating from the major 16S rDNA, M represents from the 2nd major 16S rDNA, and L represents from the 3rd major 16S rDNA; ^cRestriction enzymes used for similarity search; “Ha”, “R”, “Sc”, and “Hh” stand for Hae III, Rsa I, Scr F1, and Hha I. For the measured MERFLP which had no completely identical theoretical MERFLP, the theoretical MERFLP having the highest similarity using all the RFLPs was presented with the similarity as described in the materials and method; ^dSpecies name (accession number) of the theoretical MERFL having the highest similarity with the measured MERFL; ^eAdditional name (accession number) of the theoretical MERFL using the different restriction enzymes; ^fDifferent accession number of the theoretical MERFL in the same group using the different restriction enzymes.

^aGroups: A: B. cereus, B: Bacillus spp., C: Clostridium, D: The other Fumicutes, E: Actinobacteria, F: Proteobacteria, G: Prevotella, H: Cytophagales, I: Gram negative bacteria; ^bTotal number of bacteria.

The precision of the affiliations of each MERFLs was lower than that of the former studies. With respect to the major MERFL, ratio of the MERFLs with 100% similarity to the corresponding theoretical MERFLs (43.3%) was lower than that of field soils using selective medium (90.5%) [20] , that of the manures during composting (62.9%) [21] [23] , and that of the commercial food products (59.6%). The lower precision of the affiliations was caused from higher bacterial diversity of sample. As the diversity of sample became higher, the each MPN vial included many kinds of bacteria, which made it difficult to select MERFLs originated from homogenous 16S rDNA.

There was a large difference in microbial properties among the three samples as the followings. In the forest soil (F), Group E, which included Burkholderia sp., Ralstonia sp., and Alcaligenes sp., was numerically dominant bacterial group (3.64 × 10⁶ MPN g⁻¹ dry soil), followed by Group F (1.32 × 10⁶ MPN g⁻¹), Group G (0.006 × 10⁶ MPN g⁻¹), and Group H (0.006 × 10⁶ MPN g⁻¹) (Table 2, Figure 1). The some bacterial group detected here, e.g., Burkholderia spp., was reported to be detected using clone library sequencing [27] except for phyla Acidobacteria which could not be detected by the culture based method [28] .

In the commercial manure (M), Group A, which included Streptoverticillium salmonis, Mycrococcus sp., Streptomyces bikiniensis, and Microbacterium ulmi, was numerically dominant bacterial group (30.8 × 10⁶ MPN g⁻¹), followed by Group D (26.0 × 10⁶ MPN g⁻¹), which included Agrobacterim sp., Sphingomonas sp., Erythrobacter citreus and Erlichia sp., Group E (17.1 × 10⁶ MPN g⁻¹), which included Alcaligenes sp., and Ralstonia sp., Group G (11.2 × 10⁶ MPN g⁻¹), which included various sulfate reducing bacteria and Chondromyces robustus, Group C (1.71 × 10⁶ MPN g⁻¹), which included Paenibacillus gluconolyticus, Eubacterium cylindoides, and Staphylococcus sp., Group F (0.5 × 10⁶ MPN g⁻¹), and the Group H (0.05 × 10⁶ MPN g⁻¹) (Table 2, Figure 1).

The microbial property of M was different from those of the manures during composting in the former paper as the followings [21] : Total bacterial number (3.64 × 10⁸ MPN g⁻¹) was lower than that of the manure after thermophilic phase (7.89 × 10¹⁰ MPN g⁻¹), and that after maturing phase (14.83 × 10¹⁰ MPN g⁻¹). The reason of the lower number was attributed to an absence of Bacillus spp., which was the dominant bacteria in thermophilic phase, and the decrease of the other Firmicutes, sulfate reducing bacteria and the other gram negative bacterial group, which were dominant bacteria in maturing phase [21] . While α and β-Proteobacteria, which once disappeared after maturing phase, recovered in considerable number, and Actinobacteria, which increased during maturing phase, was remained [21] .

In the upland field (U), Group C, which included Paenibacillus sp., and Weissella paramesenteroides, was numerically dominant bacterial group (4.41 × 10⁶ MPN g⁻¹), followed by Group A (2.14 × 10⁶ MPN g⁻¹), which

included Mycobacterium sp., Corynebacterium genitalium, and Arthrobacter citeus, Group B (2.14 × 10⁶ MPN g⁻¹), which included B.cereus, B.fusiformis/B.spaericus and B.firmus/B.smithii/B.azotoformans, and Group F (0.35 × 10⁶ MPN g⁻¹), which included Pseudomonas sp. (Table 2, Figure 1). Number of Bacillus spp. was similar to that of the upland Andosol field in the former study using selective medium for Bacillus spp. (1.58 × 10⁶ MPN g⁻¹), and those estimated by dilution plate method (2.30 × 10⁶ CFU g⁻¹) [20] . The most bacterial groups detected here were reported to be detected using culture-based method [19] , or DGGE [9] , or clone library sequencing [29] except for phyla Acidobacteria [28] .

There was a difference between the total bacterial number estimated by MPN using all the amplified vials and those of the sum of the each bacterial MPN (Table 2). In the forest soil (F), the sum of the each MPN (4.7 x10⁶ MPN g⁻¹) was higher than that of the total bacterial MPN (2.9 × 10⁶ MPN g⁻¹). The over estimation was caused from the reason that some positive vial was repeatedly counted not only as the major MERFLs, but also as the 2^nd major, and the 3^rd major MERFLs. In the forest soil, two 2^nd major MERFLs and one 3^rd major MERFL were additionally counted as the positive results to the 6 major MERFL. In the manure (M), the sum of the each MPN (74.9 × 10⁶ MPN g⁻¹) was lower than the total bacterial MPN (312 × 10⁶ MPN g⁻¹). Differentiation of the whole MERFLs into the 6 sub-groups was the major factor of the underestimation (Table 2). Because not all bacteria in each vial were detected by the method due to the PCR bias, the MPN scores of each group were lower than the true MPN scores. This under estimation could be decreased by conjugating the small sub-groups into the larger group with higher MPN score. In upland field soil (U), the sum of the each MPN (9.0 × 10⁶ MPN g⁻¹) was much lower than that of the total bacterial MPN (4088 × 10⁶ MPN g⁻¹). The under estimation was also caused from low PCR amplification rate of the numerically dominant bacteria, for which bands detected in the highest dilutions (10⁻⁸ and 10⁻⁹) were too weak to afford visible fragments after restriction digestion. The other our research indicated that amplification rates of some Antinobacteria and Firmicutes were low, and the other Actinobacteria was not amplified by the used PCR condition (unpublished results). The analysis by a new PCR condition including newly designed PCR primer for these bacteria will be presented in the following manuscripts.