3.3.75 b>RELATIONSHIP BETWEEN PROSTHEMIUM BETULINUM AND PLEOMASSARIA SIPARIA AND THE DEVELOPMENT OF A SPECIFIC PRIMER RELATIONSHIP BETWEEN PROSTHEMIUM BETULINUM AND PLEOMASSARIA SIPARIA AND THE DEVELOPMENT OF A SPECIFIC PRIMER L PAAVOLAINEN, J HANTULA, T KURKELA and A-M HALLAKSELA Finnish Forest Research Institute, P.O.Box 18, FIN-01 301, Vantaa, Finland Background and objectives Conidiomata and conidia of Prosthemium betulinum Kunze are common in the basal part of naturally pruned fallen birch twigs in southern Finland. This fungus can already be found early autumn in twigs first yellowing, and around the year naturally pruned dead twigs. During winter also asci and ascospores of Pleomassaria siparia (Berk. & Br.) Sacc. are found with P. betulinum in same pustules in detached birch twigs. The aim of this study was to show whether this connection is due to P. siparia and P. betulinum being teleomorph and anamorph of a single species and to develop a specific primer that could detect this fungus straight from the DNA isolated from a birch twig. Materials and methods Monoconidial and monoascospore cultures were isolated on malt extract agar. Morphological characteristics were measured using image analyzer. Total DNA was isolated from fungal cultures and their RAMS markers (with primers DHB(CGA) 5) and ITS as well as 18S rDNA regions were PCR-amplified. The amplified 18S rDNA fragments were digested with restriction enzymes Alu 1 or Msp 1. To develope a specific primer, RAMS markers were cloned and sequenced, and specific primers were designed accordingly. This primer was used to detect P. siparia and P. betulinum from DNA isolated from yellow dead twigs of Betula benduia and Betula pubescens and also the DNA of fungal cultures were PCR-amplified with this primer. Results and conclusions The sizes and structures of conidia of P. betulinum and ascospores of P. siparia were measured and the results were in accordance with those given in the literature. Monoascospore isolates of P. siparia produced typical P. betulinum conidia. Restriction analysis of 18S RDNA did not differentiate between isolates. The analysis of RAMS markers showed a high amount of variation, which, however, was not explained by the origin of cultures. Thus, based on these results we conclude that P. betulinum is the imperfect state of P. siparia. The primer developed on RAMS marker was specific to P. siparia and P. betulinum. The analysis with specific primer distinguished two alleles in agarose gel with very different molecular weights. Two alleles were found also in ITS region. The alleles found with specific primer and in ITS region were linked to each other, suggesting that there are two closely related species with very similar perfect and imperfect stages. Both of these two species were detected in branches taken from both Betula benduia and Betula pubescens.