To investigate RNA-dependent RNA polymerase (RdRp) further, mutational analysis of the N-terminal domain of the NS5B protein of Classical swine fever virus was performed. Results show that the N-terminal domain (positions 1-300) of the protein might be divided artificially into four different regions, N1-N4. The N1 region (positions 1-61) contained neither conserved lysine nor conserved arginine residues. NS5B protein with deletion of the N1 region has the capacity for elongative RNA synthesis, but not for de novo RNA synthesis on natural templates. All substitutions of the conserved lysines and arginines in the N2 region (positions 63-216) destroyed RdRp activity completely. Substitutions of the conserved arginines in the N3 region (positions 217-280) seriously reduced RdRp activity. However, all substitutions of the conserved lysines in this region enhanced RNA synthesis and made the mutants synthesize RNA on any template. Substitutions of the conserved arginines in the N4 region (positions 281-300) reduced elongative synthesis and destroyed de novo RNA synthesis. In contrast, substitutions of lysines in this region did not affect RdRp activity significantly. These data indicate that the N3 region might be related to the enzymic specificity for templates, and the conserved lysines and arginines in different regions have different effects on RdRp activity. In combination with the published crystal structure of bovine viral diarrhea virus NS5B, these results define the important role of the N-terminal domain of NS5B for template recognition and de novo RNA synthesis.

Sclerotinia stem rot (SSR) caused by the fungus Sclerotinia sclerotiorum has been an increasing threat to oilseed rape (Brassica napus L.) cultivation. Efficient and environment-friendly treatments are much needed. Here we focus on microbial control. The Pseudomonas fluorescens P13 that was isolated from oilseed rape cultivation soil, proved to be a useful biocontrol strain for application. Morphology, physiological and biochemical tests and 16S rDNA analysis demonstrated that it was P. fluorescens P13 and that it had a broad antagonistic spectrum, significantly lessening the mycelial growth of S. sclerotiorum by 84.4% and suppressing sclerotial formation by 95-100%. Scanning electron microscopy studies attested that P13 deformed S. sclerotiorum mycelia when they were cultured together. P13 did not produce chitinase but did produce hydrogen cyanide (HCN) which was likely one of the antagonistic mechanisms. The density of P13 remained at a high level (≥10(6) CFU/ml) during 5 weeks in the rhizosphere soil and roots. P13 reduced SSR severity at least by 59% in field studies and also promoted seedling growth (p<0.05) at the seedling stage. From these data, our work provided evidence that P13 could be a good alternative biological resource for biocontrol of S. sclerotiorum.

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