Isolation and morphologic characterization of microsclerotia of Verticillium dahliae isolate from soil

Abstract

The isolation of Verticillium dahliae from soil is necessary for studies of ecology and virulence and for designing preventative control strategies for Verticillium wilt diseases. In this study a methodology was described for improving the estimation of Verticillium inoculum density in soil and for extracting pure cultures of V. dahliae from such platings for studies of culture characteristics. The efficacy for recovery of V. dahliae colonies from soil platings was compared on a Modified Sodium Polipectate Agar Medium (MSPA) to that obtained on plates of MSPA covered by a 600 μm thick sterilized permeable cellophane sheet (MSPA-C). Soil samples from 15 olive orchards were processed by wet sieving. Then 1 mL of the suspensions containing the soil particles ranging from 35 to 150 μm in size in 100 mL of water were plated onto the two media, using 10 replications. Plates were incubated 14 days at 22°C. Then, the number of V. dahliae colonies were counted using a stereoscope after the soil residues were removed under running water for MSPA plates, or without removing the soil for MSPA+C plates. Colonies forming microsclerotia on the cellophane were recovered by cutting little squares of the sheet. The microsclerotia were removed from the cellophane by shaking in water and by treatment in an ultrasonic bath. The suspensions were then sieved through a filter connected to a pressure vacuum pump and the resulting residue was plated onto MSPA, PDA or acidified PDA media. Colonies which were reasonably free of contaminants were transferred again to obtain pure cultures. The morphology of the microsclerotia produced by these cultures were characterized by measuring length and width and values compared to those of two reference isolates, V117 (cotton defoliating) and V4 (cotton non-defoliating). Results showed a high variability in the morphological features of the microsclerotia produced by the isolated cultures. The MSPA-C medium was effective for estimating the inoculum density of the pathogen in soil and also for recovery of pure culture of V. dahliae. This technique provides an useful tool for studies of ecology, virulence of populations of the pathogen in soil and risk assessment. © 2005 Asian Network for Scientific Information.