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Cryopreservation of Synchytrium solstitiale In Planta

The fungus Synchytrium solstitiale is a candidate for use as a biocontrol agent against Centaurea solstitialis. This obligate parasite can be propagated only in planta, which necessitates development of a method for preserving cultures for longer periods of time for routine biological studies and shipment to other laboratories. Normally, sporangia embedded within the plant tissue release zoospores when submersed in water at temperatures above freezing. To examine what chemicals might inhibit zoospore release, infected tissue was exposed to different suspensions of fungicides. Cycloheximide and benomyl completely inhibited zoospore release or immediately induced encystment from tissue stored in these two chemicals, respectively. A few zoospores were released in suspensions of iprodione and propionic acid but were not motile. However, when tissue stored in iprodione or propionic acid was transferred to fresh distilled water, abundant active zoospores were released. Freezing the infected tissue at different temperatures in different cryoprotectants also affected the release of motile zoospores. Infected C. solstitialis tissue was immersed in water, water plus iprodione, methanol, ethylene glycol, dimethyl sulfoxide, glycerol, skim milk, trehalose, or sucrose and subjected to different temperatures for various periods of time. Some treatments protected the viability of the fungus for a shorter period of time whereas other treatments completely inhibited release. The best results were obtained when infected tissue was stored at -2°C in 0.5 M sucrose where active zoospores were released after 12 weeks of storage. Overall, results obtained from this study demonstrate a technique for long-term storage of S. solstitiale.