RAPID AND SENSITIVE DETECTION OF ROSELLINIA NECATRIX IN ROOTS AND SOILS BY REAL TIME SCORPIONPCR
L. Schena, A. Ippolito
Two primer pairs specific for Rosellinia necatrix, designed from the internal transcribed space (ITS) regions, were utilised to develop a molecular detection method suitable for large-scale analyses of soils and plant materials. One primer (R15) was modified to obtain a Scorpion primer for detecting a specific 71 bp amplicon by fluorescence emitted from a fluorophore through a self-probing PCR assay. Primer R15 Scorpion in combination with a reverse conventional primer (R18) produced a more intense fluorescent signal compared to a previously reported pair of Scorpion primers. Primer specificity was assessed both by means of BLAST (Basic Local Alignment Search Tool) analyses to exclude the presence of similar sequences in other microrganisms among available DNA databases (Gen- Bank) and by using genomic DNA from a large number of R. necatrix isolates and other fungi from several hosts and different geographic areas. Simple, rapid, and effective procedures for direct DNA extraction from soil and plant materials were developed to yield DNA of purity and quality suitable for PCR assays. Combining these protocols and a single amplification with primer R15 Scorpion-R18 it was possible to detect the pathogen in naturally and artificially infected host tissues. To detect the pathogen from infested soils, and to improve the sensitivity on host tissue, a nested Scorpion-PCR with conventional (R3-R8) and Scorpion (R15 Scorpion-R18) primers was utilised. The reliability of the entire procedure was assessed using both artificially and naturally infested soils and plant materials from 26 different woody species. Compared to traditional detection methods based on baiting and fungal isolation on agar media, the molecular approach proved to be more rapid, sensitive, reliable, and enabled large scale analyses.