LABOULBENIALES - Agentschap voor Natuur en Bos

Cladosporium (Ascomycota, Dothideomycetes) is one of the largest, mostheterogeneous genera of Hyphomycetes, including endophytic, fungicolous,human pathogenic, phytopathogenic and saprobic species (CROUS et al.,2007). Davidiella is the teleomorph. It seems that incorrectly identifiedsequences are present in the GenBank database, as suggested by BROCK et al.(2009). Blasting the resulting match Dioscorea polystachya (FJ860063) givesDavidiella tassiana (99% identity), Cladosporium cladosporioides (99% identity)and Cladosporium sp. (99% identity) as most common matches, alongDioscorea alata and other Dioscorea polystachya specimens (from the sameresearch group). It is suggested that those sequences of Dioscoreapolystachya with accession numbers FJ860063, FJ860073, FJ860075, FJ860081and FJ860096 and Dioscorea alata with accession numbers FJ860064 andFJ860066 are incorrectly identified.The amplification with ITS5/ITS2 led to useful sequences for only on specimen[flag1151]. The resulting ITS sequence shows, compared with the GenBankdatabase, high similarity with Mycena amabilissima, a member ofbasidiomycotan Agaricomycetes.Amplification using ITS5/ITS2 led to shorter sequences than amplication withITS5/ITS4-A, since with ITS2 as reverse primer only the ITS1 region will beamplified (cfr. Figure XI) (WHITE et al., 1990).Figure XI: Schematic overview of the entire internal transcribed spacer region (ITS), flanking DNA andprimer positions (not drawn to scale).Protocol II was suggested to give consistent results (WEIR & BLACKWELL, 2001a).Yet, it may be stated that the protocol outline was not very clearly described,in both WEIR & BLACKWELL (2001a) and WEIR & BLACKWELL (2001b) on whichprotocol I of this research was based. We tried to communicate with Alex Weirand his students for additional information on the protocols but got no reply.WEIR & HUGHES (2002) suggest that the DNA extraction protocol described inWEIR & BLACKWELL (2001a) is reliable. However, since 2002, no other studies wereperformed using this protocol. The current research suggests that there is a lotof work in finding a real reliable and feasible DNA extraction protocol and PCRamplification procedure for Laboulbeniales.The DNA extraction protocol using Qiagen‟s Puregene Kit A (protocol III) wasunsuccessful, as the protocol using Qiagen‟s DNeasy Plant Mini Kit.Direct addition of thalli to the PCR reaction (protocol V) was unsuccessful,confirming previous reports (WEIR & BLACKWELL, 2001a).P a g e | 42