Molecular characterization and identification ofSaprolegnia by restriction analysis of genes coding for ribosomal RNA

Abstract

Restriction fragment length polymorphisms (RFLPs) in two regions of the ribosomal DNA (rDNA) repeat unit were examined in 33 strains representing 18 species of Saprolegnia. The Polymerase Chain Reaction (PCR) was used to separately amplify the 18S rDNA and the region spanning the two internal transcribed spacers (ITS) and the 5.8S ribosomal RNA gene. Amplified products were subjected to a battery of restriction endonucleases to generate various fingerprints. The internal transcribed spacer region exhibited more variability than the 18S rDNA and yielded distinctive profiles for most of the species examined. Most of the species showing 100% similarity for the 18S rDNA could be distinguished by 5.8S + ITS restriction polymorphisms except for S. hypogyna, S. delica, S. lapponica, and S. mixta. The rDNA data indicate that S. lapponica and S. lapponica and S. mixta are conspecific with S. ferax, whereas there is no support for the proposed synonymies of S. diclina with S. delica and of S. mixta with S. monoica. Results from cluster analysis of the two data sets were very consistent and tree topologies were the same, regardless of the clustering method used. A further examination of multiple strains in the S. diclina-S. parasitica complex showed that restriction profiles are conserved across different strains of S. parasitica originating from the U.K. and Japan. HhaI and BsaI restriction polymorphisms were observed in isolates from the U.S. and India. The endonuclease BstUI was diagnostic for S. parasitica, generating identical fingerprints for all stains regardless of host and geographic origin. Except for the atypical strain ATCC 36144, restriction patterns were also largely conserved in S. diclina. Correlation of the rDNA data with morphological and ultrastructural features showed that S. diclina and S. parasitica are not conspecific. Restriction polymorphisms in PCR-amplified rDNA provide a molecular basis for the classification of Saprolegnia and will be useful for the identification of strains that fail to produce antheridia and oogonia.

Saprolegniaceae identified on amphibian eggs throughout the Pacific Northwest, USA, by internal transcribed spacer sequences and phylogenetic analysis

“…Our S. diclina group was the most geographically homogeneous of our groups with larger sample sizes: 13 of 14 samples came from the Bitterroot Mountain region of Idaho-Montana. Also consistent with Molina et al (1995) was our finding of a close relationship between S. ferax and S. hypogyna.…”

“…Fourteen isolates grouped strongly with S. diclina ATCC 90215. The strong support for our S. diclina group (bootstrap 100) and our S. parasitica group (bootstrap 93; n 5 2 egg isolates) concurs with the conclusion by Molina et al (1995) that S. diclina and S. parasitica are closely related but separate species. Our S. diclina group was the most geographically homogeneous of our groups with larger sample sizes: 13 of 14 samples came from the Bitterroot Mountain region of Idaho-Montana.…”

“…Species identification has been more challenging because it has required presence of the sexual structures, the oogonia and antheridia. More recently molecular identification has been accomplished with selected Saprolegniaceae using the internal transcribed spacer (ITS) and 5.8S regions of ribosomal DNA (rDNA) (Molina et al 1995, Leclerc et al 2000. The most complete molecular phylogeny of this family to date identified 10 genera and 40 species through analyses of ITS and the large ribosomal subunit (LSU) (Leclerc et al 2000).…”

Preliminary Investigations of Disease-Causing Organisms in the White-Clawed Crayfish Austropotamobius Pallipes Complex From Streams of Northern Italy

“…Recently, molecular studies were also carried out based on these features (MOLINA et al, 1995;DIÉGUEZ-URIBEONDO et al, 1996). In the strain of Saprolegnia we isolated, only asexual reproduction had occurred and so, as we not observed the characteristic of secondary cysts and not performed molecular studies, we were unable to determine the species.…”

Saprolegnia brachydanis, A New Oomycete Isolated from Zebra Fish

“…Traditionally, the generic definition was mainly based on the asexual characters, especially the mode of zoospores discharge, whereas species differentiation was mainly on the features of sexual reproductive organs including oogonia, antheridia, antheridial origin, and oospore [4,5,12,15]. However, many of these characters may be variable in one species or similar in different species [8] and some strains either lose the ability to produce sexual reproduction or cannot be induced to form them under laboratory conditions [13,18]. So, it is difficult to make definitive identification only using traditional morphological criteria in species level.…”