Changes in fungal community structure in bulk soil and spinach rhizosphere soil after chemical fumigation as revealed by 18S rDNA PCR-DGGE

Abstract

To analyze the impact of chloropicrin and 1,3-dichloropropene on fungal community structure in bulk soil and spinach rhizosphere soil in a field, developed a new nested polymerase chain reaction (PCR) method to facilitate the detection of major fungal taxa and we used the method to monitor 18S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) profiles for 3 years. The cropping system consisted of soil fumigation in September and two subsequent consecutive spinach cultivations each year. The soil was treated with fumigants (chloropicrin at 20 mL m−2 or 1,3-dichloropropene at 32 mL m−2) and covered with polyethylene film for approximately 2 weeks. The nested PCR method with primer pairs AU2/AU4 and GC-FR1/FF390 successfully amplified sequences from all major fungal taxa, but not from plants or oomycetes, which were amplified from rhizosphere soil samples using direct PCR with the primer pair GC-FR1/FF390. The DGGE analysis of the nested PCR products indicated that the chloropicrin treatment had a greater impact on the fungal community than 1,3-dichloropropene, both in terms of the magnitude and duration of the effect. Chloropicrin treatment changed DGGE profiles drastically and reduced the diversity index H′ in both bulk soil and rhizosphere soil 2 months after fumigation. Profiles and diversity indices did not recover completely after 1 year. Bands with high sequence similarity to ascomycetous fungi decreased in intensity and, conversely, bands inferred to represent chytridiomycota became dominant. In contrast, 1,3-dichloropropene did not reduce the diversity index significantly after 2 months. The DGGE profiles of 1,3-dichloropropene plots revealed a smaller change 2 months after fumigation, but became indistinguishable from those of control plots after 6 months. Spinach cultivation also affected the soil fungal community structure because the differences in DGGE profiles between control and chloropicrin plots were smaller in rhizosphere soil than in bulk soil 2 months after fumigation. These results suggest that the rhizosphere effect may contribute to minimizing the effect of chemical fumigation.