Effects of freeze–thaw stress during soil storage on microbial communities and methidathion degradation

Prior to registration and commercialization, crop protection products (CPP) need to be tested for their environmental impact and fate. For fate studies, laboratory microcosm experiments are performed with defined natural soils. Quality control of these soils represents a challenge, since it is often difficult to obtain fresh soil and soil storage may affect soil characteristics. According to guidelines of the Organization for Economic Cooperation and Development (OECD) frozen storage is recommended for periods exceeding three months. The present study was performed to investigate the influence of soil frozen storage on microbiological soil characteristics (MSCs) and CPP mineralization rates. For this purpose an agricultural soil was frozen for four days, thawed, and subsequently equilibrated and monitored during 42 days under controlled conditions. Substrate-induced respiration (SIR) rates increased shortly after thawing and returned to reference levels within ten days. Degradation of the CPP methidathion appeared unaffected in the soils that had been frozen. Conversely, we found that soil DNA contents and direct cell counts decreased by 24 and 22%, respectively, in the frozen stored soil. Impacts on soil microbial population structures were analyzed by use of domain-specific PCR of small subunit ribosomal RNA genes for Eucarya, Bacteria, and Archaea in conjunction with restriction fragment length polymorphism (RFLP) analysis. Since the Archaea community appeared most sensitive to the freezing stress, cloning and sequencing was performed to identify marker genes for freezing stress sensitive and resistant groups. Novel specific PCR detections were developed for a freezing stress resistant and a freezing stress sensitive group. These new tools allowed monitoring and comparison of archaeal population shifts without using RFLP analysis. Over all, the molecular biological approach revealed that the initial MSCs were not restored after 42 days equilibration of the frozen soil, even though SIR rates and mineralization of one specific CPP appeared unaffected. Our results demonstrate that specific molecular tools can identify sensitive indicators of altered MSCs and may be applied for the description of stress effects that can occur during soil storage.