Pythium recalcitrans sp. nov. revealed by multigene phylogenetic analysis

A new species of Pythium collected from grapevine roots (Vitis vinifera) in South Africa and roots of common beet (Beta vulgaris) in Majorca, Spain, is described. The phylogenetic position of the new species was investigated by multigene sequence analyses of the internal transcribed spacers (ITS1 and ITS2) of the rDNA region, as well as three other nuclear and three mitochondrial coding genes. Maximum likelihood phylogenetic analyses based on ITS rDNA and concatenated beta-tubulin and cytrochrome c oxidase II alignment place Pythium recalcitrans together with P. sylvaticum and P. intermedium. Pythium recalcitrans sp. nov. is morphologically almost indistinguishable from other Pythium species that only form hyphal swellings in culture. However its species status is justified by the distinctiveness of the DNA sequences in all the genes examined. In culture P. recalcitrans exhibits fast radial growth, abundant spherical to subglobose hyphal swellings but produces no zoosporangia. Sexual structures are not seen in agar media but form in autoclaved grass blades floated on water. Multiple antheridia (1-7) are encountered with most of them diclinous and crook-necked. Oospores are thin-walled and either aplerotic or plerotic. P. recalcitrans was pathogenic to seedlings of Beta vulgaris and Solanum lycopersicum.     

In vitro and in vivo antagonism of actinomycetes isolated from Moroccan rhizospherical soils against Sclerotium rolfsii: a causal agent of root rot on sugar beet (Beta vulgaris L.)

Aims:  To evaluate the ability of the isolated actinomycetes to inhibit in vitro plant pathogenic fungi and the efficacy of promising antagonistic isolates to reduce in vivo the incidence of root rot induced by Sclerotium rolfsii on sugar beet.
Methods and Results:  Actinomycetes isolated from rhizosphere soil of sugar beet were screened for antagonistic activity against a number of plant pathogens, including S.   rolfsii . Ten actinomycetes out of 195 screened in vitro were strongly inhibitory to S. rolfsii . These isolates were subsequently tested for their ability to inhibit sclerotial germination and hyphal growth of S. roflsii . The most important inhibitions were obtained by the culture filtrate from the isolates J-2 and B-11, including 100% inhibition of sclerotial germination and 80% inhibition of hyphal growth. These two isolates (J-2 and B-11) were then screened for their ability to protect sugar beet against infection of S. rolfsii induced root rot in a pot trial. The treatment of S. rolfsii infested soil with a biomass and culture filtrate mixture of the selected antagonists reduced significantly ( P  ≤ 0·05) the incidence of root rot on sugar beet. Isolate J-2 was most effective and allowed a high fresh weight of sugar beet roots to be obtained. Both antagonists J-2 and B-11 were classified as belonging to the genus Streptomyces species through morphological and chemical characteristics as well as 16S rDNA analysis.
Conclusion:  Streptomyces isolates J-2 and B-11 showed a potential for controlling root rot on sugar beet and could be useful in integrated control against diverse soil borne plant pathogens.
Significance and Impact of the Study:  This investigation showed the role, which actinomycete bacteria can play to control root rot caused by S.   rolfsii , in the objective to reduce treatments with chemical fungicides.     

Phytophthora chrysanthemi sp. nov., a new species causing root rot of chrysanthemum in Japan

A new species of Phytophthora was isolated from stem and root rot of chrysanthemum in the Gifu and Toyama prefectures of Japan. The species differs from other Phytophthora species morphologically, and is characterized by nonpapillate, noncaducous sporangia with internal proliferation, formation of both hyphal swellings and chlamydospores, homothallic nature, distinctive intercalary antheridia, and funnel-shaped oogonia. The new species can grow even at 35°C, with an optimum growth temperature of 30°C in V8 juice agar medium. In phylogenetic analyses based on five nuclear regions (LSU rDNA; genes for translation elongation factor 1α, β-tubulin, 60 S ribosomal protein L10, and heat shock protein 90), the isolates formed a monophyletic clade. Although the rDNA ITS region shows a high resolution and has proven particularly useful for the separation of Phytophthora species, it was difficult to align the sequences for phylogenetic analysis. Therefore, ITS region analysis using related species as defined by the multigene phylogeny was performed, and the topology of the resulting tree also revealed a monophyletic clade formed by the isolates of the species. The morphological characteristics and phylogenetic relationships indicate that the isolates represent a new species, Phytophthora chrysanthemi sp. nov. In pathogenicity tests, chrysanthemum plants inoculated with the isolates developed lesions on stems and roots within 3 days, and the symptoms resembled the ones originally observed. Finally, the pathogen’s identity was confirmed by re-isolation from lesions of infected plants.     

Novel root-fungus symbiosis in Ericaceae: sheathed ericoid mycorrhiza formed by a hitherto undescribed basidiomycete with affinities to Trechisporales

Ericaceae (the heath family) are widely distributed calcifuges inhabiting soils with inherently poor nutrient status. Ericaceae overcome nutrient limitation through symbiosis with ericoid mycorrhizal (ErM) fungi that mobilize nutrients complexed in recalcitrant organic matter. At present, recognized ErM fungi include a narrow taxonomic range within the Ascomycota, and the Sebacinales, basal Hymenomycetes with unclamped hyphae and imperforate parenthesomes. Here we describe a novel type of basidiomycetous ErM symbiosis, termed 'sheathed ericoid mycorrhiza', discovered in two habitats in mid-Norway as a co-dominant mycorrhizal symbiosis in Vaccinium spp. The basidiomycete forming sheathed ErM possesses clamped hyphae with perforate parenthesomes, produces 1- to 3-layer sheaths around terminal parts of hair roots and colonizes their rhizodermis intracellularly forming hyphal coils typical for ErM symbiosis. Two basidiomycetous isolates were obtained from sheathed ErM and molecular and phylogenetic tools were used to determine their identity; they were also examined for the ability to form sheathed ErM and lignocellulolytic potential. Surprisingly, ITS rDNA of both conspecific isolates failed to amplify with the most commonly used primer pairs, including ITS1 and ITS1F + ITS4. Phylogenetic analysis of nuclear LSU, SSU and 5.8S rDNA indicates that the basidiomycete occupies a long branch residing in the proximity of Trechisporales and Hymenochaetales, but lacks a clear sequence relationship (>90% similarity) to fungi currently placed in these orders. The basidiomycete formed the characteristic sheathed ErM symbiosis and enhanced growth of Vaccinium spp. in vitro, and degraded a recalcitrant aromatic substrate that was left unaltered by common ErM ascomycetes. Our findings provide coherent evidence that this hitherto undescribed basidiomycete forms a morphologically distinct ErM symbiosis that may occur at significant levels under natural conditions, yet remain undetected when subject to amplification by 'universal' primers. The lignocellulolytic assay suggests the basidiomycete may confer host adaptations distinct from those provisioned by the so far investigated ascomycetous ErM fungi.     

Rhizoctonia solani AG 11 isolated for the first time from sugar beet in Poland

Two isolates of Rhizoctonia solani AG11 were isolated from sugar beet seedlings from South-west Poland. Both isolates gave C2 reactions in anastomose pairings with the tester isolates of AG11. The membership of both isolates to AG11 was confirmed by analysis of pectic isozyme profiles, and by verification that the internal transcribed spacer sequences of both isolates matched the references in the GenBank database. Both AG11 isolates formed white-beige to creamy-colored mycelium with wide concentric zonation. One of them formed light-colored sclerotia. The average daily rate of hyphal growth at 21 °C was 22.8 mm and 22.6 mm on PDA. They were mildly pathogenic to sugar beet seedlings due to the mycelial and secondary metabolites’ activity. The sensitivity to fungicides typically used in sugar beet protection was different for each isolate; one of them (isolate ID11) was less sensitive to thiram than the other (isolate ID3). This article discusses the worldwide occurrence of R. solani AG11, expands the currently known host range, shows its broad world distribution in regions of moderate climate, and confirms the isolates’ low frequency.     

天山北坡甜菜内生菌分离鉴定及其动态变化

对新疆昌吉和石河子两地种植的甜菜内生菌进行了分离、鉴定和分析,结果表明甜菜内生菌多属于细菌,其中假单胞菌 (Pseudomonas sp. )和芽孢菌类(Bacillus sp.)的分离频率分别在33.2%～59.2%和12.7%～28.1%,是甜菜植株中的优势内生菌群.16S rDNA 和 ITS 序列同源性比较和系统发育分析表明内生菌具有丰富的多样性.根中内生菌的多样性高于茎、叶,昌吉地区种植的甜菜中分离出的内生菌种类较多.从感病品种及生长不良甜菜植株中分离出的内生菌种类比较丰富.通过回接分离及利用扫描电镜观察内生菌在植物体内分布发现,内生菌能够定殖于甜菜块根.     

Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton

Analyses of small subunit ribosomal RNA genes (SSU rDNAs) have significantly influenced our understanding of the composition of aquatic microbial assemblages. Unfortunately, SSU rDNA sequences often do not have sufficient resolving power to differentiate closely related species. To address this general problem for uncultivated bacterioplankton taxa, we analysed and compared sequences of polymerase chain reaction (PCR)-generated and bacterial artificial chromosome (BAC)-derived clones that contained most of the SSU rDNAs, the internal transcribed spacer (ITS) and the large subunit ribosomal RNA gene (LSU rDNA). The phylogenetic representation in the rRNA operon PCR library was similar to that reported previously in coastal bacterioplankton SSU rDNA libraries. We observed good concordance between the phylogenetic relationships among coastal bacterioplankton inferred from SSU or LSU rDNA sequences. ITS sequences confirmed the close intragroup relationships among members of the SAR11, SAR116 and SAR86 clades that were predicted by SSU and LSU rDNA sequence analyses. We also found strong support for homologous recombination between the ITS regions of operons from the SAR11 clade.     

Ribosomal DNA sequence divergence and group I introns within the Leucostoma species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., ascomycetes that cause Cytospora canker of fruit trees

Leucostoma species that are the causal agents of Cytospora canker of stone and pome fruit trees were studied in detail. DNA sequence of the internal transcribed spacer regions and the 5.8S of the nuclear ribosomal DNA operon (ITS rDNA) supplied sufficient characters to assess the phylogenetic relationships among species of Leucostoma, Valsa, Valsella, and related anamorphs in Cytospora. Parsimony analysis of the aligned sequence divided Cytospora isolates from fruit trees into clades that generally agreed with the morphological species concepts, and with some of the phenetic groupings (PG 1-6) identified previously by isozyme analysis and cultural characteristics. Phylogenetic analysis inferred that isolates of L. persoonii formed two well-resolved clades distinct from isolates of L. cinctum. Phylogenetic analysis of the ITS rDNA, isozyme analysis, and cultural characteristics supported the inference that L. persoonii groups PG 2 and PG 3 were populations of a new species apparently more genetically different from L. persoonii PG 1 than from isolates representative of L. massariana, L. niveum, L. translucens, and Valsella melastoma. The new species, L. parapersoonii, was described. A diverse collection of isolates of L. cinctum, L. persoonii, and L. parapersoonii were examined for genetic variation using restriction fragment length polymorphism (RFLP) analysis of the ITS rDNA and the five prime end of the large subunit of the rDNA (LSU rDNA). HinfI and HpaII endonucleases were each useful in dividing the Leucostoma isolates into RFLP profiles corresponding to the isozyme phenetic groups, PG 1-6. RFLP analysis was more effective than isozyme analysis in uncovering variation among isolates of L. persoonii PG 1, but less effective within L. cinctum populations. Isolates representative of seven of the L. persoonii formae speciales proposed by G. Défago in 1935 were found to be genetically diverse isolates of PG 1. Two large insertions, 415 and 309 nucleotides long, in the small subunit (SSU) of the nuclear rDNA of L. cinctum were identified as Group 1 introns; intron 1 at position 943 and intron 2 at position 1199. The two introns were found to be consistently present in isolates of L. cinctum PG 4 and PG 5 and absent from L. cinctum PG 6 isolates, despite the similarity of the ITS sequence and teleomorph morphology. Intron 1 was of subgroup 1C1 whereas intron 2 was of an unknown subgroup. RFLP patterns and presence/absence of introns were useful characters for expediting the identification of cultures of Leucostoma isolated from stone and pome fruit cankers. RFLP patterns from 13 endonucleases provided an effective method for selecting an array of diverse PG 1 isolates useful in screening plant germplasm for disease-resistance.     

Pythium abappressorium--a new species from eastern Washington

A new species of Pythium isolated from wheat and apple roots in eastern Washington is described. Pythium abappressorium sp. nov. is characterized by abundant appressoria. Plerotic oospores and sporangia are formed from the appressoria and remnants of the appressoria remain attached to the base of sporangia at maturity. Smaller appressorial swellings, reminiscent of hyphal swellings, are also formed within the appressoria. Pythium abappressorium is pathogenic to wheat, causing damping-off and stunting, but is not pathogenic to apples. The fungus can grow in the temperature range 5 to 30 C, with an optimum of 20 C. The sequence of the ITS1 region of the rDNA did not match the sequences from a worldwide collection of over 1200 isolates, including types and neotypes, suggesting that this species has not been previously described.     

Expansion of the sooty blotch and flyspeck complex on apples based on analysis of ribosomal DNA gene sequences and morphology

Sooty blotch and flyspeck (SBFS) is a late-season disease of apple and pear fruit that cosmetically damages the cuticle, resulting in produce that is unacceptable to consumers. Previous studies reported that four species of fungi comprise the SBFS complex. We examined fungal morphology and the internal transcriber spacer (ITS) and large subunit (LSU) regions of rDNA of 422 fungal isolates within the SBFS complex from nine orchards in four Midwestern states (USA) and compared them to previously identified species. We used LSU sequences to phylogenetically place the isolates at the order or genus level and then used ITS sequences to identify lineages that could be species. We used mycelial and conidial morphology on apple and in culture to delimit putative species. Thirty putative species found among the Midwest samples were shown to cause SBFS lesions on apple fruit in inoculation field trials. Among them Peltaster fructicola and Zygophiala jamaicensis have been associated previously with SBFS in North Carolina. The LSU analyses inferred that all 30 SBFS fungi from Midwestern orchards were Dothideomycetes; one putative species was within the Pleosporales, 27 were within Dothideales, and two putative species could not be placed at the ordinal level. The LSU sequences of 17 Dothideales species clustered with LSU sequences of known species of Mycosphaerella.     

Molecular phylogeny and paclitaxel screening of fungal endophytes from Taxus globosa

We studied the endophytic mycoflora associated with Taxus globosa, the Mexican yew. The study localities; Las Avispas (LA), San Gaspar (SG), and La Mina (LM) were three segments of cloud forest within the range of Sierra Gorda Biosphere Reserve, México. Overall, 245 endophytes were isolated and 105 representative Ascomycota (morphotaxons) were chosen for phylogenetic and genotypic characterization. Maximum likelihood analyses of large subunit of ribosomal RNA (LSU) rDNA showed well-supported clades of Dothideomycetes, Eurotiomycetes, Leotiomycetes, Pezizomycetes, and Sordariomycetes. Analyses of ITS rDNA groups showed 57 genotypes (95% sequence similarity), in general consistent with the phylogenetically delimitated taxa based on LSU rDNA sequences. The endophyte diversity measured by Fisher's α, Shanonn, and Simpson indices was ca. three-fold and ca. two-fold greater in LM than in LA and SG respectively. A screening for paclitaxel using a competitive inhibition enzyme immunoassay showed 16 positive isolates producing between 65 and 250 ng l(-1). The isolates included Acremonium, Botryosphaeria, Fusarium, Gyromitra, Nigrospora, Penicillium, three novel Pleosporales, and Xylaria.     

PYTHIUM SYLVATICUM鉴定及其专一性PCR引物

从杭州采集的水稻、棉花和大豆猝倒苗中分离到国内新记录腐霉种Pythium sylvaticum。该腐霉为异宗配合种,菌丝膨大体球形或柠檬形,雄器异丝生,藏卵器光滑,每个藏卵器上1~3个雄器,雄器常在接近藏卵器处形成二叉状分枝,卵孢子不满器。测定了该种4 个菌株的核糖体内转录间隔区(ITS)的序列,根据与59种腐霉ITS序列的比较,设计了P. sylvaticum种专一性引物PSF1和PSR2。实际结果表明:该引物能从11种共14株腐霉DNA中特异性地扩增P. sylvaticum,从而与其它10种腐霉区分。     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

Comparative study of Gymnodinium mikimotoi and Gymnodinium aureolum, comb. nov. (=Gyrodinium aureolum) based on morphology, pigment composition, and molecular data

Light and electron microscopy, nuclear-encoded LSU rDNA sequences, and pigment analyses were performed on five geographically separate isolates of Gymnodinium mikimotoi. The morphological variation between the isolates equals that found within the isolates. The nuclear-encoded LSU rDNA sequences were nearly identical in all isolates, and molecular analyses using maximum likelihood, parsimony, and neighbor joining showed the geographical isolates as an unresolved clade. Based on the available data it is concluded that the European isolates, formerly identified as Gyrodinium aureolum , Gyrodinium cf. aureolum , or Gymnodinium cf. nagasakiense , are conspecific with the Japanese Gymnodinium mikimotoi. An isolate from the Pettaquamscutt River, USA, is suggested to represent what Hulburt (1957) described as Gyrodinium aureolum. The LSU rDNA sequence data and ultrastructural characters in this isolate closely resemble those of Gymnodinium fuscum , the type species of Gymnodinium , and Gyrodinium aureolum Hulburt is therefore renamed Gymnodinium aureolum (Hulburt) G. Hansen, comb. nov.     

Taxon-specific oligonucleotide primers for detection of Glomus etunicatum

The 5.8S subunit and flanking internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (rDNA) from spores of Glomus etunicatum MD107, MD127, TN101, and FL329 were amplified by polymerase chain reaction (PCR) using ITS1Kpn and ITS4Pst as primers. The amplification products (597, 599, 598, and 613 bp, respectively) were cloned and sequenced. The similarity among ITS region sequences from MD107, MD127, and TN101 was 99%, whereas the sequence similarity between the ITS regions of these three DNAs and that from FL329 was 91%. The 5.8S rDNA sequences of all four G. etunicatum isolates were identical. In contrast, major dissimilarities in the corresponding rDNA sequence regions of other glomalean taxa were observed. Oligonucleotide sequences unique to G. etunicatum were tested for their specificity in PCR amplification of genomic DNA from spores of 55 isolates comprising 29 glomalean fungi: 18 isolates of G. etunicatum, five G. intraradices, three G. claroideum, 16 other Glomus isolates, and 11 other glomalean taxa from each of four other genera. The G. etunicatum isolates were from a broad range of geographic regions and soils. The oligonucleotide pair GETU1:GETU2 primed specific amplification of an oligonucleotide sequence (approximately 400 bp) present in all G. etunicatum. This primer pair did not prime PCR when template consisted of DNA from any of the other glomalean fungi or any of the non-mycorrhizal controls, including roots of corn (Zea mays). In addition, the pair successfully detected G. etunicatum in nested PCR using a primary PCR product amplified from highly diluted extracts of colonized corn roots using modified ITS1:ITS4 primers. In the phylogenetic analysis of Glomus 5.8S and ITS2 rDNA region sequences, which included 500 bootstrap data sets, confidence in the G. etunicatum branch was very strong (90%) and clearly independent of G. claroideum and G. intraradices, to which it is very closely related. Accepted: 15 October 2000     

Calosphaeria canker of almond caused by Calosphaeria pulchella in Iran

Trunk diseases are a potential threat for the almond industry in Northern Iran. In a survey on fungal agents associated with almond die back and decline in East Azerbaijan province, located in Northwest of Iran, acremonium-like hyphomycete isolates with hyaline and simple conidiophores and hyaline cylindrical phialides with a finely pigmented apical region and deep, flaring collarettes were repeatedly recovered from symptomatic tissues. Fungal isolates were identified as Calosphaeria pulchella based on cultural and morphological characteristics. The identity of the species was further confirmed based on the sequence data from LSU and ITS rDNA regions. A phylogeny inferred using ITS sequence data, clustered the Calosphaeria isolates together with Calosphaeria pulchella isolates in GenBank known from Prunus persica and Prunus avium. The results of pathogenicity assay using an excised shoot method showed that Calosphaeria pulchella is highly pathogenic on almond. The distribution and host range of this new pathogen on almond and the impact of the disease on almond industry in this region remain to be studied.     

Cryptic speciation in Protoceratium reticulatum (Dinophyceae): Evidence from morphological,molecular and ecophysiological data

The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20–25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.     

Study on DNA diversity of Iranian populations of Erysiphe betae causal agent of sugar beet powdery mildew

107 samples of E. betae were collected on infected leaves from all over Iranian beet cultivation areas. Their choosing were based on geographical and host origin(sugar beet, red beet, fodder beet and wild beet). 30 isolates were single colonized and grown on sugar beet susceptible genotype 7233. 107 specimens were analyzed by restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers (ITS) and 5.8s DNA which previously amplified by the polymerase chain reaction (PCR) with 2 universal primers, ITS1 and ITS4. PCR product was affected by 9 different restriction enzymes. PCR product was a 645 bp band for all of the isolates. 3 restriction enzymes; CfoI, MspI and HaeIII could cut this fragment into smaller bands, but electrophoretic patterns were identical for all of the isolates. 30 single colonized isolates were used in RAPD experiments. In RAPD-PCR experiment genetic diversity was investigated with 30 isolates from different parts of the country. 59 random primers were used and then 21 primers that displayed good consistency and reproducibility were selected. Most of the primers revealed identical patterns between 3 to 14 bands. 5 primers that showed more polymorphism were selected to analyze 30 isolates. For these 5 primers 61 distinct bands were obtained which 62% of these bands were polymorphic. Results indicated that there is no relationship between cluster grouping and geographical origin and the isolates showed a high similarity.     

Aspergillus ochraceus micromycetes—producers of extracellular proteinases—protein C activators of blood plasma

Natural isolates of Aspergillus ochraceus micromycetes from soil and plant remains from various regions have been screened. The isolated strains were characterized by similar cultural and morphological features and an identical nucleotide sequence in the ITS1-5,8S-ITS2 region of rDNA. The ability of the extracellular proteinases of A. ochraceus micromycetes to activate protein C of blood plasma has been established. Differences are revealed in the accumulation of proteinases activating protein C and proteinases with thrombinand plasmin-like activities in the growth dynamics of producers.