Effects of pH and temperature on growth ofSaprolegnia diclina andS. parasitica isolated from various sources

Saprolegnia diclina andS. parasitica isolated from three sources could germinate in strong acidic conditions. Growth ability correlated with the species of fungi rather than with the sources from which they were isolated.S.diclina isolates appeared to germinate at a pH condition as low as 3.5, whereasS. parasitica isolates could not germinate at below pH 3.8.S. parasitica isolates from visceral mycoses still showed good growth at 30°C, whereas other isolates did not. Also,S. parasitica isolates from visceral mycoses produced more abundant motile zoospores, and continued to do so for a longer period of time (28 d), thanS. parasitica isolates from external saprolegniasis andS. diclina isolates.     

Some biochemical characteristics of fungi isolated from salmonid eggs

Fungal isolates from salmonid eggs displayed apparently unique patterns of biochemical characteristics at both the generic and specific levels. of the five genera examinedAchlya andPythium were able to assimilate 13–16 out of 19 carbohydrates.Aphanomyces was able to assimilate only glucose and starch, which was assimilated by all isolates. Members ofSaprolegnia displayed identical patterns of carbohydrate assimilation, except forS. hypogyna, which was also able to assimilate melibiose, in common withAchlya, Pythium, andLeptolegnia. Pythium was the only genus capable of assimilating salicin. OnlyAchlya andP. monospermum were able to assimilate rhamnose. In terms of amino acid assimilation isolates ofSaprolegnia ferax andS. diclina displayed an identical patterns, as did isolates ofS. parasitica andS. hypogyna. OnlyAphanomyces frigidophilus isolate was capable of assimilating cysteine. All genera exceptPythium assimilated glutamine, a fundamental amino acid. All isolates exhibited lipase and fatty acid esterase activities but no cellulase acitivity. The biochemical characteristics discovered in this study offer possibilities for identification and classification of these fungi, which are discussed herein.     

Molecular genetic characterization of bacterial isolates causing brown blotch on cultivated mushrooms in Japan

The polymerase chain-reaction (PCR) was used to amplify 16S ribosomal DNA (16S rDNA) from bacteria, identified asPseudomonas tolaasii orP. fluorescens, causing brown blotch on cultivated mushrooms in Japan. PCR-amplified 16S rDNA was analyzed on the basis of nucleotide sequence and restriction fragment length polymorphisms (RFLP) to determine the specific identity of isolates. Banding patterns obtained through PCR using primers corresponding to repetitive extragenic palindromic sequences of enteric bacteria (REP-PCR) were used to determine the relatedness of conspecific isolates. AllP. tolaasii isolates and a mushroom pathogen identified asP. fluorescens had identical RFLP patterns and partial 16S sequences, and are considered conspecific. An isolate ofP. fluorescens from creamery wastes (IFO 3507) differed slightly from isolates ofP. tolaasii in both 16S sequence (0.8%) and RFLP patterns (d=0.08), and had almost entirely different REP-PCR bands (d=0.88–1.0). Phylogenetic analyses based on 16S sequences indicated thatP. tolaasii andP. fluorescens are close members ofPseudomonas sensu stricto. REP-PCR shows promise in characterizing isolates pathogenic on different mushroom crops. Two isolates ofP. tolaasii pathogenic onPleurotus ostreatus had identical banding patterns, but three isolates fromLentinula edodes showed the greatest diversity. Contribution No. 312 of the Tottori Mycological Institute, Totori, Japan.     

Restriction polymorphisms in the internal transcribed spacers and 5.8S rDNA ofSaccharomyces

Polymorphism in enzymatically amplified ribosomal DNA (rDNA) were examined in 18 strains ofSaccharomyces. Restriction patterns generated from the region spanning the internal transcribed spacers (ITS) and the 5.8S rDNA produced two clusters corresponding toS. bayanus andS. cerevisiae. The type culture ofS. carlsbergensis (ATCC 76529), which could not be separated from theS. cerevisiae group by small subunit (SSU) rDNA patterns, showed aScrfI profile that was distinct from all the other strains. The type culture ofs. intermedius var.turicensis (ATCC 76519) is assigned toS. bayanus on the basis of the combined results of SSU and ITS restriction analyses.S. kluyveri occurred at a separate branch of the distance tree and is unrelated to any of the strains. Results were in general agreement with reported DNA homologies and are discussed in relation to other molecular and genetic data.     

Authentication of ATCC strains in theSaccharomyces cerevisiae complex by PCR fingerprinting

Restriction fragment length polymorphisms in the rDNA internal transcribed spacer region (ITS) of 18 yeast strains currently assigned toSaccharomyces cerevisiae, S. pastorianus, andS. bayanus were examined. Primers complementary to the ITS region were used to amplify the ITS rDNA by the polymerase chain reaction (PCR). The products were digested with 10 endonucleases and cluster analysis was used to generate a phenogram from the restriction fragment data. Three strains ofS. cerevisiae (ATCC 10609, 26250, and 66162) exhibited restriction patterns that were different from the type strain but identical to those of theS. bayanus-S. pastorianus cluster. In contrast,S. pastorianus (ATCC 76671) showed restriction profiles that were different from its type strain but were identical to the type strain ofS. cerevisiae (ATCC 18824). These results suggest that the three strains ofS. cerevisiae should be reassigned to eitherS. pastorianus orS. bayanus, and the strain ofS. pastorianus (ATCC 76671) should be reclassified asS. cerevisiae.     

Identification ofArmillaria species from hokkaido by analysis of the intergenic spacer (IGS) region of ribosomal DNA using PCR-RFLP

The intergenic spacer (IGS) region, which is located between the 3′ end of 26S ribosomal DNA (rDNA) and the 5′ end of 5S rDNA, of sixArmillaria species from Hokkaido was investigated using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Restriction with onlyAlu I could distinguishA. mellea subsp.nipponica from the other species. WithAlu I andDde I,A. ostoyae andA. gallica could be distinguished from the other species. Digestion withAlu I resulted in two patterns (types A and B) ofA. singula and three patterns (types A, B, and C) ofA. jezoensis. One pattern (type B) of the former species and two patterns (types B and C) of the latter species were each different from those of the other species.Armillaria sinapina gave only oneAlu I digestion pattern, which was identical to that ofA. jezoensis (type A) andA. singula (type A). However, by digestion withDde I,A. singula (type A) could be distinguished fromA. jezoensis (type A) andA. sinapina.     

RFLP analysis of the PCR-amplified 28S rDNA inRhizoctonia solani

RFLP analyses of a portion of the 28S rDNA gene region were conducted by using four restriction endonucleases for 57 isolates of 13 intraspecific groups (ISGs) representing 7 anastomosis groups (AGs) ofRhizoctonia solani. Variations in the PCR-amplified rDNA products and the polymorphisms on digestion with restriction enzymes (BamHI,HaeIII,HhaI andHpaII) were observed among three AGs, AG 1, 2 and 4. These differences were also conserved among some ISGs of AG 1 and AG 2. Among ISGs of AG 1, the pattern of rDNA fragments of AG 1-IA obtained by digestion withHpaII was significantly different from those of AG 1-IB and IC. Such difference in the fragment pattern was also observed among AG 2-1, 2-2 IIIB and 2-2 IV by the digestion withHhaI andHpaII. A dendrogram derived from the restriction enzyme data showed that ISGs from AG 1 and AG 2 can each be subdivided into distinct groups, those are distantly related to the majority isolates of the other AGs.     

Detection of ‘long-haired’ Saprolegnia (S. parasitica) isolates using monoclonal antibodies

The ability of five monoclonal antibodies (Mabs) raised against a pathogenic Saprolegnia parasitica isolate from brown trout to detect and differentiate between isolates with bundles of long hairs (S. parasitica) and other Saprolegnia species was determined by means of an indirect immunofluorescence assay. Four of the Mabs used recognized some of the long-haired S. parasitica isolates but also cross-reacted with other Saprolegnia species without bundles of hairs and with Achlya sp. The other Mab (named 18A6) was able to differentiate between the asexual and most of the sexual isolates in the group of long-haired S. parasitica isolates, but did not recognize Achlya sp. or the Saprolegnia species without bundles of hairs, with the exception of S. hypogyna. These results indicate that isolates with bundles of long hairs are closely related with other members of genus Saprolegnia and share several antigens. However, Mab 18A6 seems to recognize an epitope that is expressed mainly in the asexual isolates in the long-haired S. parasitica isolates.     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.     

Rapid detection ofRhizoctonia species, causal agents of rice sheath diseases, by PCR-RFLP analysis using an alkaline DNA extraction method

Restriction fragment length polymorphism (RFLP) analysis for DNA products amplified by the polymerase chain reaction (PCR) was used for the direct detection ofRhizoctonia solani AG 1 IA and AG 2-2 IIIB,R. oryzae, R. oryzae-sativae andR. fumigata from the diseased rice sheaths. A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract parasite DNA from diseased rice sheaths. 28S ribosomal DNA (rDNA) derived from fungal genomic DNA extracted by the alkaline method was specifically PCR-amplified. The results of PCR-RFLP analysis for DNA samples from artificially inoculated rice sheath tissues with eachRhizoctonia spp. and the corresponding culture on the medium using two restriction enzymes.HhaI andMspI, showed identical polymorphisms. PCR-RFLP analysis using DNA samples from naturally infected rice sheath tissues also revealed the possibility of direct diagnosis ofR. solani AG 1 IA,R. oryzae andR. oryzae-sativae.     

Restriction enzyme analysis of the nuclear 45s ribosomal DNA of six cultivated Alliums (Alliaceae)

Estimates of the phylogenetic relationships among cultivated and wildAllium species would benefit from identification of objective molecular characters. Restriction fragment length polymorphisms in the nuclear 45s ribosomal DNA (rDNA) were identified among two of five accessions of each of six cultivated Alliums. Restriction enzyme sites forBamHI,DraI,EcoRI,EcoRV,SacI, andXbaI were mapped. Different lengths of the rDNA repeat unit among the cultivated Alliums were due to sizes of the intergenic spacer. Nineteen polymorphic restriction enzyme sites were discovered and used to estimate phylogenetic relationships. Cladistic analyses based on Wagner parsimony were completed without an outgroup and resulted in two equally most parsimonious trees of 22 steps. A combined analysis of differences at RE sites in the ribosomal (19 characters) and chloroplast (15 characters) DNA generated a single most parsimonious tree of 39 steps. Single trichotomies were observed at 40 and 41 steps. Strict consensus of the three trees of 41 or fewer steps consisted of a lineage forA. tuberosum, a second forA. ampeloprasum andA. sativum, and a third forA. cepa, A. fistulosum, andA. schoenoprasum. Estimates of phylogenetic relationships based on variability at restriction enzyme sites in the rDNA and chloroplast DNA agree with the classification scheme ofTraub. Because of the predominance of autapomorphies, restriction enzyme analysis of the nuclear 45s rDNA is of limited use in estimating phylogenies amongAllium sections. However it is useful in the establishment of interspecific hybridity.     

Taxonomic and population differentiation of mitochondrial diversity inPinus banksiana andPinus conforta

We have studied two mitochondrial DNA polymorphisms in 741 individuals from 16 allopatric populations ofPinus banksiana Lamb. andPinus contorta Dougl. Restriction fragments of both polymorphisms distinguished the two species qualitatively, except in aP. Banksiana population whose ancestors were involved in hybridization withP. contorta.COXI-associated restriction fragments were monomorphic within species, whileCOXII-associated restriction fragments were highly variable inP. contorta (H_es=0.68). Population differentiation was substantial inP. contorta (F_st=0.31 among subspecies; mean F_st=0.66 within subspecies) and consistent with predictions for maternally inherited markers. Plant mitochondrial markers appear to be useful for the investigation of seed migration routes, hybridization and introgression, breeding zone designation, and the development of germ plasm conservation sampling strategies.     

On the conspecificity ofAnopheles fluviatilis species S withAnopheles minimus species C

Anopheles fluviatilis andAn. minimus complexes, each comprising of at least three sibling species, are closely related and important malaria vectors in Oriental Region. RecentlyAn. fluviatilis species S, which is a highly efficient malaria vector in India, has been made conspecific withAn. minimus species C (senior synonym) on the basis of homology in 335 base pair nucleotide sequence of D3 domain of 28S ribosomal DNA(rDNA). We examined the conspecificity of these two nominal species by obtaining and analysing the DNA sequences of nuclear ribosomal loci internal transcribed spacer 2 (ITS2) and D2-D3 domain of 28S rDNA (28S-D2/D3) from those ofAn. fluviatilis S andAn. minimus C. We found that the sequences ofAn. fluviatilis S are appreciably different from those ofAn. minimus C with pair-wise distance (Kimura-2-parametre model) of 3.6 and 0.7% for loci ITS2 and 28S-D2/D3, respectively. Pair-wise distance and phylogenetic analyses using ITS2 sequences of members of Minimus and Fluviatilis Complexes revealedthat An. fluviatilis S is distantly related toAn. minimus C as compared to any other members of the Fluviatilis Complex. These findings suggest that the two nominal species,An. fluviatilis S andAn. minimus C, do not merit synonymy. The study also confirms that the reported speciesAn. fluviatilis X is synonym with species S.     

Actinophages and restriction enzymes fromMicromonospora species (Actinomycetales)

Summary To develop a screening procedure for the detection of restriction endonucleases in micromonosporae and catellatosporae based on efficiency of plating, eight different actinophages were isolated from soils enriched withMicromonospora species and one fromCatellatospora-enriched soil. The lytic actinophages all contained double-stranded DNA and the majority appeared, when examined by electron microscopy, to belong to Ackermann's type B1 since they had isometric heads and noncontractile tails. One actinophage was classified as type C1 because of its isometric head and very short noncontractile tail. The host ranges of the actinophages were determined on strains ofMicromonospora and selected species from other actinomycete genera of cell wall chemotype II. Type II restriction enzymes were isolated fromM. echinospora ssp.echinospora (ATCC 15837),M. purpurea (ATCC 15835) andM. zionensis (LL-100-125) and were designatedMecI,MpuI andMziI, respectively. Restriction enzymesMecI andMpuI are isoschizomers ofXhoI, whileMziI is an isoschizomer ofPvuII.     

Morphological aspects ofSaprolegnia diclina Type 1 isolated from pejerrey,Odonthetes bonariensis

Saprolegnia diclina Type 1 (syn.S. parasitica) discovered in pejerrey,Odonthetes bonariensis, is described and illustrated herein.     

Mitochondrial genome size variation and restriction fragment length polymorphisms in threePhaseolus species

Summary Restriction patterns of mitochondrial DNA (mtDNA) from threePhaseolus species were examined to estimate their relative genome sizes and to determine the level of interspecific variability and relatedness. Three restriction endonucleases that produced relatively simple profiles were identified and used to determine the genome size of the three species. Taking into account fragment stoichiometries, the average estimates across enzymes were 456, 324, and 400 kb, respectively, forP. vulgaris, P. coccineus, andP. acutifolius. Restriction fragment length polymorphisms (RFLPs) differentiated the species when the mtDNAs were digested with seven endonucleases and hybridized with five cosmid clones covering ca. 200 kb of mtDNA sequences. Proportions of shared restriction fragments between every two species were computed as F-values and demonstrated thatP. vulgaris andP. coccineus are more related to each other than either is toP. acutifolius, and that the latter has a similar degree of relationship to the other two species.     

Morphological variation among members of theStellaria longipes complex:S. longipes,S. longifolia,andS. porsildii (Caryophyllaceae)

Morphological relationships were investigated among diploidStellaria porsildii, polyploidS. longipes, and diploidS. longifolia. Canonical discriminant analysis, based on a priori assumptions to maximize differences among groups, showed thatS. longipes clusters equally distant between the two diploid species along an axis connecting the diploids' centroids, but it differs along an axis perpendicular to this axis. The intermediacy along the former axis is evidence thatS. longipes is an amphiploid derived fromS. longifolia andS. porsildii. The divergence along the latter axis may be attributable to adaptively valuable heterotic traits which were retained following amphidiploidization. The only morphological discontinuity occurred between the two diploids, whereas the morphological range ofS. longipes overlapped the range of both diploids forming a continuum. The lack of discrete clusters is likely due to hybridization and introgression withS. longifolia on one hand, and convergence of traits betweenS. longipes var.monantha andS. porsildii on the other. High a posteriori assignments in classificatory discriminant analysis supports the separation ofS. longipes var.monantha from otherS. longipes specimens. AlthoughS. longipes var.monantha grouped close toS. porsildii, the two groups separate based on leaf shape traits. Overall results support, firstly, the hypothesis thatS. porsildii is a diploid parent species which by hybridizing withS. longifolia gave rise to polyploidS. longipes. Secondly, results suggest thatS. longipes var.monantha converged morphologically towardsS. porsildii relatively recently due to ecological specialization, and merits distinction at least as a variety ofS. longipes.     

Restriction enzyme mapping of ribosomal DNA ofSchistosoma spindale andS. leiperi (Digenea) and its application to interspecific differentiation

Restriction maps were determined for the ribosomal RNA gene complex ofSchistosoma spindale andS. leiperi. The restriction map of theS. spindale rRNA gene complex was found to differ from those of other schistosomes previously described by the presence of an additionalEcoRI site in the non transcribed spacer region. In common withS. mattheei andS. margrebowiei, bothS. leiperi andS. spindale appear to have insertions in the transcribed spacer region, relative toS. mansoni.EcoRI digests of genomic DNA, probed with pSM 889, enabled differentiation ofS. spindale andS. leiperi from four other species of schistosome.     

Relationships between species ofLeymus,Psathyrostachys, andHordeum (Poaceae,Triticeae) inferred from Southern hybridization of genomic and cloned DNA probes

We have used total genomic DNA as a probe to size-fractionated restriction enzyme digests of genomic DNA from a range ofTriticeae species from the generaLeymus Hochst.,Psathyrostachys Nevski, andHordeum L., and hybrids betweenHordeum andLeymus to investigate their taxonomic relationships. Genomic Southern hybridization was found to be an effective and simple way to assess the distribution and diversity of essentially species-specific and common, repetitive DNA sequences, and is hence especially useful in evolutionary studies. The DNA sequences ofH. vulgare seem to diverge substantially from those ofH. brachyantherum, H. lechleri, H. procerum, andH. depressum. The genome ofThinopyron bessarabicum shows little homology to those of theLeymus species investigated, confirming thatT. bessarabicum is not an ancestral genome inLeymus. Although the genomes ofLeymus andPsathyrostachys share substantial proportions of DNA sequences, they include divergent repeated sequences as well. Hybridization with a ribosomal DNA probe (pTa 71) showed that the coding regions containing structural genes encoding the 18 S, 5.8 S, and 26 S ribosomal RNA were conserved among the species investigated, whereas the intergenic spacer region was more variable, presenting different sizes of restriction fragments and enabling a classification of the species. The rye heterochromatin probe pSc 119.2 hybridized to DNA fromH. lechleri andT. bessarabicum, but not to DNA from the other species investigated.     

Experimental transmission and pathogenicity of some zoosporic fungi toTilapia fish

Seventeen fungal species belong to six genera were recovered from the four organs ofTilapia fish and the most common wereSaprolegnia ferax, S. diclina, Achlya dubia, A. americana, A. racemosa andA. flagellata, Dictyuchus sterile,Pythium undulatum andAphanomyces sp. Severe infection followed by death of all fish was incited byS. parasitica andS. ferax through experiment I. 30–70% ofT. nilotica andT. galileae were killed through experiment II byS. parasitica andS. ferax. T. galileae was more susceptible to fungal infection thanT. nilotica.