Molecular Identification of Fungi Isolated from Infected Redclaw Crayfish, Cherax quadricarinatus

Abstract

In Malaysia, the redclaw crayfish, Cherax quadricarinatus is widely known as freshwater lobster due to its lobster-like appearance and habitat. Even though the exact year in which the redclaw species was introduced into the country is unknown, commercial scale culturing activity of this species has been in record since 2003 in the southern part of Peninsular Malaysia. Crayfish plague is a water mould caused by an oomycete Aphanomyces astaci which contributes for high mortality rate in indigenous crayfish species throughout Europe. However, the effects of the plague can vary among species, regions and local populations. Traditionally, the crayfish plague agent was seen to have only the freshwater crayfish as hosts. The identity of the pathogen is only assumption and not confirmed by research because of difficulties in cultivation and ambiguous morphological characteristics. In this study, we demonstrate the use of ITS86F and ITS4 primer pair to amplify the ITS2 region in the isolated fungi from infected C. quadricarinatus. The primer aligned with sequences from a range of fungal species within the Ascomycota and Basidiomycota clades. A total of 14 fungi were isolated from the infected C. quadricarinatus. With the constructed neighbour-joining phylogenetic tree, 7 of the ITS2 sequences were identified to most closely related to 9 fungal species within the Ascomycota clade, 5 sequences in Basidiomycota clade and 2 sequences were closely related to endophyte culture collection. Results show fungi other than A. astaci, could cause crayfish plague. These isolated fungi might be linked to the A. astaci. Genetic analysis provides easy identification of the large majority of fungi at the species level, regardless when little DNA is available. Moreover, there are very low quantities of the pathogen DNA in the sample that are detectable by some of the molecular methods.