Graduate Thesis Or Dissertation
 

An improved selective medium for the isolation of Verticillium dahliae Kleb

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  • Sodium polypectate (SPP) was added to an ethanol-streptomycin agar (ESA) medium to determine if it would provide a selective substrate for the growth of Verticillium dahliae and for isolation of the fungus from soil and plant tissues in the presence of saprophytic fungi. Sodium polypectate improved greatly the formation and pigmentation of microscierotia in four different isolates of V. dahliae. Adding SPP to ESA medium also improved the recovery rate of the fungus from soil and from infected plants. At high inoculum densities, SPP did not increase colony numbers, and ESA without SPP apparently allowed the recovery of all viable fungus propagules. However, adding SPP to the medium, even at levels as low as 0.25 g/l, increased the size, microsclerotial formation, and pigmentation of the colonies in every case. All the colonies on ESA-SPP medium were very distinct and they could be observed and counted easily and rapidly with the unaided eye. The time required for formation and pigmentation of microsclerotia was shortened by five to seven days when SPP was added to the medium. The results suggest that V. dahliae utilizes SPP primarily to form pigmented resting structures rather than vegetative mycelium. In soil dilutions, addition of SPP up to 1.0 g/l of ESA increased the recovery of V. dahliae. At higher concentrations of SPP, vigorous growth of saprophytic soil fungi inhibited the growth and masked the presence of V. dahliae. Microsclerotial production by V. dahliae was obtained in shake cultures by adding SPP to a 0.1 percent water agar or a 0.1 percent Czapek Dox solution, both containing 0.76 percent ethanol and 101 ppm streptomycin. The intensity of production was always related to the level of SPP in the media. Water agar containing SPP yielded many more microsclerotia than Czapek Dox containing SPP, at all tested levels. For best microsclerotial production the combined effect of SPP and ethanol was necessary in all experiments. Improvement of ESA medium by addition of appropriate amounts of SPP will allow more refined studies in the biology of V. dahliae and the knowledge gained from such experiments should enhance our understanding of this important plant pathogen and eventually aid in its control.
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