An evaluation of 18S rDNA approaches for the study of fungal diversity in grassland soils.

Fungal community structure and diversity in two types of agricultural grassland soil were investigated by amplified 18S ribosomal DNA restriction analysis (ARDRA) and 18S ribosomal DNA sequence analysis. These two grassland sites represent a species-rich old hay meadow and an agriculturally improved site with low floristic diversity. Two primer sets were used in combination to amplify approximately 550 bp of rDNA from three major fungal groups, the zygomycetes, basidiomycetes, and ascomycetes, and clone libraries were created for each site. 18S ARDRA was used to analyze 170 rDNA clones, and three diversity indices were calculated. A small-scale culturing analysis was also carried out and the most common isolates analyzed using ARDRA and sequence analysis. The soil fungal community revealed by the rDNA approaches was significantly different from that produced by this limited culture-based analysis. Twenty-eight soil-derived clones were sequenced, and many represented fungal taxa rarely reported in culture-based studies. The PCR-based techniques detected differences in diversity between the two fungal communities and changes in patterns of dominance that paralleled higher plant diversity. The results suggest that 18S rDNA-based approaches are a useful tool for initial screening of fungal communities, and that they represent a more comprehensive picture of the community than plate culturing.

An analysis of the instability kinetics of plasmid pHSG415 during continuous culture of Escherichia coli.

The effect of dilution rate on the instability kinetics of Escherichia coli RV308(pHSG415) during glucose-limited continuous culture is examined. Two nonlinear models are fitted to the data, both of which characterize the plasmid-host system in terms of the rate parameters R (for the plasmid segregation rate) and d mu (for the specific growth rate difference between plasmid-free and plasmid-bearing single cells). In the first model, both R and d mu have constant values with respect to time. In the second, either R or d mu is represented as a time-dependent function. Although both models fit the data equally well, it is demonstrated that the constant rate parameter model gives results which appear to be misleading. A comparison is also made among some of the many plasmid instability models (both mass-balance and segregated) which have appeared in the literature. It is found that all of these give identical trajectories and differ only in the definitions of the rate parameters used.

A nonlinear technique for the analysis of plasmid instability in micro-organisms.

A nonlinear technique to calculate parameters for segregational instability and differences in cellular growth-rate for plasmid-bearing micro-organisms growing in batch or continuous culture is presented. This method is compared with an approximate technique based upon linear regression. The accuracy and sensitivity of the results are evaluated by use of simulated data and biological data taken from experiments with Pseudomonas aeruginosa(pGSS15) and Escherichia coli(pHSG415). It is demonstrated that the nonlinear analysis gives results which are significantly more accurate and which show much better agreement with the data. Consequently, the new analysis leads to quite different conclusions with regard to the nature of the instability of the plasmid-bearing strain. This method offers an opportunity to study the genetic and physiological aspects of plasmid instability and so aid the design and optimization of cloning vectors.