Cryopreservation of Radopholus similis, a tropical plant-parasitic nematode

Abstract

For obligate plant-parasitic nematodes, cryopreservation has advantages over the usual preservation methods on whole plants or axenic culture systems, because the latter two are laborious and time and space consuming. In addition, cross contamination among different isolates can occur easily. Moreover, specific genetic studies require maintenance of the original population. The nematode under investigation, Radopholus similis, is a plant-parasitic nematode from the humid tropics. Therefore, any treatment at low temperatures is likely to add extra stress to the nematode, making the development of a cryopreservation protocol extremely difficult. In this paper, we describe experiments to achieve a successful cryopreservation protocol for the tropical nematode R. similis using vitrification solutionbased methods based on a well defined mixture of cryoprotectants in combination with ultrarapid cooling and thawing rates. A two-step treatment was used consisting of an incubation in glycerol followed by the application of a vitrifying mixture of methanol, glycerol and glucose. After cryopreservation, the pathogenicity of the nematodes was not altered, since they could infect and reproduce on carrot discs after recovery in the Ringer solution. The cryopreservation method described can be used for routine cryopreservation of R. similis lines from different origins.

Emerging molecular knowledge on Radopholus similis, an important nematode pest of banana

“…In females that have not mated with a male 50–60 days after the fourth moult, self‐fertilization takes place with rod‐shaped spermatids produced in the ovatestis and matured in the spermatheca (Kaplan and Opperman, 2000). The study of this nematode is relatively easy, as it multiplies quite quickly on carrot discs (Speijer and De Waele, 1997), and nematodes can even be conserved by cryopreservation (Elsen et al. , 2007).…”