Mapping species differences for adventitious rooting in a <Emphasis Type="Italic">Corymbia torelliana</Emphasis> × <Emphasis Type="Italic">Corymbia citriodora</Emphasis> subspecies <Emphasis Type="Italic">variegata</Emphasis> hybrid

Quantitative trait loci (QTL) detection was carried out for adventitious rooting and associated propagation traits in a second-generation outbred Corymbia torelliana × Corymbia citriodora subspecies variegata hybrid family (n = 186). The parental species of this cross are divergent in their capacity to develop roots adventitiously on stem cuttings and their propensity to form lignotubers. For the ten traits studied, there was one or two QTL detected, with some QTL explaining large amounts of phenotypic variation (e.g. 66% for one QTL for percentage rooting), suggesting that major effects influence rooting in this cross. Collocation of QTL for many strongly genetically correlated rooting traits to a single region on linkage group 12 suggested pleiotropy. A three locus model was most parsimonious for linkage group 12, however, as differences in QTL position and lower genetic correlations suggested separate loci for each of the traits of shoot production and root initiation. Species differences were thought to be the major source of phenotypic variation for some rooting rate and root quality traits because of the major QTL effects and up to 59-fold larger homospecific deviations (attributed to species differences) relative to heterospecific deviations (attributed to standing variation within species) evident at some QTL for these traits. A large homospecific/heterospecific ratio at major QTL suggested that the gene action evident in one cross may be indicative of gene action more broadly in hybrids between these species for some traits.     

Cytological studies of <Emphasis Type="Italic">Agaricus brasiliensis</Emphasis>

Agaricus brasiliensis is a medicinal mushroom native to Brazil. It was first identified as Agaricus blazei and its scientific name continues to be debated. We examined the cytology of different Brazilian commercial strains of A. brasiliensis and the nuclear behavior of strain CS1 during basidiospore development using fluorescent microscopy. All strains have multinucleate hyphae and no significant differences in nuclei numbers were observed between them. Basidia from A. brasiliensis strain CS1 are typically tetrasporics and produce binucleate basidiospores, demonstrating that a postmeiotic mitosis occurs during basidiospore development. This result suggests that A. brasiliensis is primarily a heterothallic species.     

Chromosomes are eliminated in the symmetric fusion between <Emphasis Type="Italic">Arabidopsis </Emphasis><Emphasis Type="Italic">thaliana</Emphasis> L. and <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> Willd.

In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed. Wang Minqin and Zhao Junsheng contributed equally to the work.     

Genetic variability and molecular phylogeny of <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> species-complex isolates from Iran,and notes on the systematics of Asiatic populations

The Pleurotus eryngii species-complex includes taxa of the northern hemisphere growing in association with plants of the family Apiaceae (umbellifers). In this study, 45 Pleurotus strains were isolated from five different host-plants: Ferula ovina, F. assa-foetida, Smyrniopsis aucheri, Kellusia odoratissima, and Cachrys ferulacea; all plant species, with the exception of C. ferulacea, are reported for the first time as hosts for this fungal group. Random amplified polymorphic DNA-PCR (RAPD) analysis and nucleotide sequence data from the internal transcribed spacer of the nuclear rRNA genes (ITS1, 5.8S and ITS2) were used for assessing genetic diversity and for determining phylogenetic relationships among the populations studied. Results permitted the grouping of the strains studied into three major clusters corresponding mainly to the nature of the host-plant: the first included isolates collected from Ferula spp. only, the second included isolates originating from C. ferulacea only but from various sampling locations, and the third included all K. odoratissima and S. aucheri associated strains plus a few isolates collected from F. ovina and C. ferulacea. The grouping of the Iranian material, in conjunction with the position in the resulting phylograms of other previously obtained P. eryngii complex sequences, revealed that the first cluster is related to the asiatic ‘P. nebrodensis’ (or to the asiatic Ferula spp. associated Pleuroti), the second forms a rather distinct lineage which is linked with reference strains originally classified as P. fossulatus, whereas the third cluster falls within the main part (or the “core”) of this complex, i.e., P. eryngii. Pleurotus populations growing on umbellifers in Iran seem either to have recently diverged through a sympatric speciation process based mainly on ecological factors (e.g., P. fossulatus), or they form part of a rather wide agglomerate associated with various host-plants where exchange of genetic material is still in progress (i.e., P. eryngii).     

<Emphasis Type="Italic">Ochroconis calidifluminalis</Emphasis>, a Sibling of the Neurotropic Pathogen <Emphasis Type="Italic">O. gallopava</Emphasis>, Isolated from Hot Spring

Two strains resembling the neurotropic fungus Ochroconis gallopava were isolated from hot spring river water (IFM 54738 and IFM 54739). The isolates showed optimal growth at 42°C, while the maximum growth temperature was 49°C, thus having temperature relationships similar to those of O. gallopava. Colonies were light olive green, with a color change to dark reddish brown after several passages, which was also observed in O. gallopava. Conidia were indistinguishable from those of O. gallopava. The antifungal susceptibility profile of the isolates was also similar to that of O. gallopava, except for a lower susceptibility to micafungin. The two isolates had 100% homologous rRNA genes including the internal transcribed spacer (ITS) region and the D1/D2 region of the large subunit. The gene fragments, as O. gallopava, could be amplified with species-specific rDNA primers, and loop-mediated isothermal amplification designed for O. gallopava yielded positive results in the two isolates. However, homologies with O. gallopava in ITS and D1/D2 regions were 79.2 and 95.9%, respectively, widely exceeding generally accepted species boundaries. These differences were corroborated in virulence tested in experimental infection. The two isolates did not kill a mouse even until 28 days. However, mortalities of four O. gallopava strains ranged from 40 to 100%. The new isolates mainly affected the kidneys; whereas O. gallopava had a strong preference for the brain. We therefore propose a new species, Ochroconis calidifluminalis, for the two isolates.     

The constitution of incompatibility factor and mating characteristics of spore isolates in a bipolar mushroom, <Emphasis Type="Italic">Pholiota nameko</Emphasis>

Pholiota nameko is a wood-rotting edible mushroom that carries a bipolar A incompatibility factor gene. The linkage analysis of the multiple allelomorphic A factor gene demonstrated that sexual reproduction produced a monospore isolate carrying a new A factor gene in addition to two parental mating types of isolates. However, 10%–30% of the modified monospore isolates could not produce a dikaryon with both of the parental monokaryons by crossing. It is concluded that the bipolar A incompatibility factor gene of P. nameko is constituted of two functional subunits, Aα and Aβ, which might be successively located beside each other with an apparent genetic distance of 0.3 centi-Morgan between them on the same chromosome. Further, some monospore isolates that did not conjugate with both parental monokaryons could produce dikaryons with different monokaryotic stocks with either one of the parental mating types. This result suggests that the crossing capability of these isolates were essentially those for one of the mating types of the parental monokaryons, but that their function for mating activity was made partially by unequal crossing-over in the process of sexual recombination. Received: May 1, 2001 / Accepted: December 5, 2001     

Isolation and characterization of melanin pigment from <Emphasis Type="Italic">Pleurotus cystidiosus</Emphasis> (telomorph of <Emphasis Type="Italic">Antromycopsis</Emphasis><Emphasis Type="Italic">macrocarpa</Emphasis>)

Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of bacteria and fungi. For more than 40 years, fungi have been known to produce pigments called melanins. Melanin pigment production by mushrooms was not intensively studied. The present study was carried out on isolation and characterization of melanin from an edible mushroom Pleurotus cystidiosus var. formosensis. The mushroom produced dark mucous mass of hyaline arthrospores on mycelium. The coremia exclusively produced dikaryotic arthrospores with the remnant of a clamp connection. Continuous cell extension and division in the coremium stipe supplied cells for arthroconidiation at the coremium apex, which is surrounded by a liquid droplet (coremioliquid). The black coloured coremea (conidia) were produced by Antromycopsis macrocarpa (anamorph of P. cystidiosus) when cultured on potato dextrose agar medium. The agar plate was incubated at continuous light illumination for high amount of pigment (coremea) production. The slimy layer of the coremea was extracted and partially purified by alkaline and acid treatment. The black pigment was confirmed as melanin based on UV, IR and EPR spectra apart from chemical analysis. This is the first report on characterization of melanin obtained from Pleurotus cystidiosus var. formosensis.     

Comparative analysis of <Emphasis Type="Italic">Pasteurella pneumotropica</Emphasis> isolates from laboratory mice and rats

Selected biochemical and genetic characteristics of the wild-type strains of Pasteurella pneumotropica isolated from mice and rats were investigated and compared in order to determine the significant differences among the isolates. The isolates were divided into six groups on the basis of the patterns of carbon source utilization in the host rodents. The genome sizes were determined by electrophoretic analysis, and the mean genome size of the isolates from mice was larger than that of the isolates from rats (P < 0.05). Cluster analysis of the rpoB sequences discriminated five clusters; the differences might have correlated with the host associations. Principal component analysis (PCA) based on both the biochemical and genetic characteristics revealed total 44 strains discriminated into three groups comprising the host-dependent and host-independent groups. Although the P. pneumotropica isolates were mainly classified on the basis of the host rodents by the examinations, the existence of isolates that could not be discriminated on the basis of the host rodents alone was confirmed by the PCA. These results indicated that the P. pneumotropica isolates could be further classified by taxonomic analysis and also suggested the existence of a host-independent group in addition to the host-dependent groups.     

Discovery of <Emphasis Type="Italic">Ophiostoma tsotsi</Emphasis> on <Emphasis Type="Italic">Eucalyptus</Emphasis> wood chips in China

Ophiostoma species such as O. quercus are the most frequent causal agents of sapstain of freshly felled hardwood timber and pulpwood. Many species are regarded as economically important agents of wood degradation. The aim of this study was to identify a collection of Ophiostoma isolates, resembling O. quercus, found on stained Eucalyptus pulpwood chips in China. DNA sequences of the internal transcribed spacer regions, including the 5.8S region, of the ribosomal DNA, and parts of the β-tubulin and elongation factor-1α genes, revealed that the isolates were not O. quercus. Surprisingly, they represented O. tsotsi, a wound-infesting fungus recently described from hardwoods in Africa. In addition, sequence data from an isolate from agarwood in Vietnam, identified in a previous study as belonging to an unknown Pesotum species, were also shown to represent O. tsotsi. A high level of genetic variability was observed among isolates of both O. quercus and O. tsotsi. This was unexpected and suggests that both species have been present in Asia for a significant amount of time.     

The identity of <Emphasis Type="Italic">Penicillium</Emphasis> sp. 1, a major contaminant of the stone chambers in the Takamatsuzuka and Kitora Tumuli in Japan,is <Emphasis Type="Italic">Penicillium paneum</Emphasis>

Penicillium appeared as the major dweller in the Takamatsuzuka Tumulus (TT) and Kitora Tumulus (KT) stone chambers, both located in the village of Asuka, Nara Prefecture, in relation to the biodeterioration of the 1,300-year-old mural paintings, plaster walls and ceilings. Of 662 Penicillium isolates from 373 samples of the TT (sampling period, May 2004–2007) and the KT (sampling period, June 2004–Sep 2007), 181 were phenotypically assigned as Penicillium sp. 1 which shared similar phenotypic characteristics of sect. Roqueforti in Penicillium subg. Penicillium. Fifteen representative isolates of Penicillium sp. 1, 13 from TT and 2 from KT, were selected for molecular phylogenetic analysis. The 28S rDNA D1/D2, ITS, β-tubulin, and lys2 gene sequence-based phylogenies clearly demonstrated that the three known species P. roqueforti, P. carneum and P. paneum in sect. Roqueforti, and all TT and KT isolates grouped together. In addition to this, TT and KT isolates formed a monophyletic group with the ex-holotype strain CBS 101032 of P. paneum Frisvad with very strong bootstrap supports. So far, P. paneum has been isolated only from mouldy rye breads, other foods, and baled grass silage. Therefore, this is the first report of P. paneum isolation from samples relating to the biodeteriorated cultural properties such as mural paintings on plaster walls.     

Study of CTX-M Type of Extended Spectrum β-Lactamase among Nosocomial Isolates of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in South India

Data on CTX-M type extended-spectrum β-lactamase (ESBL) produced by Gram-negative bacteria by molecular methods are limited from India. This study was conducted to investigate the prevalence of CTX-M type ESBL producing Escherichia coli and Klebsiella pneumoniae from nosocomial isolates in a tertiary care hospital in southern India. A total of 179 clinical isolates of K. pneumoniae (n = 72) and E. coli (n = 107) were obtained in a period of 3 months and assessed for ESBL production phenotypically. Associated resistance to a panel of antibiotics and Minimum Inhibitory Concentration for 3rd generation cephalosporins was determined. Phenotypically ESBL positive isolates were subjected to PCR for bla _CTX-M gene using two sets of primers for the simultaneous detection of all the five major groups of CTX-M types. All the positive isolates were then subjected to a group specific PCR to detect the prevalent group. Out of 179 isolates, 156 (87.1%) were positive for ESBL phenotypically, which includes 39.2% of K. pneumoniae and 60.8% of E. coli. All of them were examined by PCR using two primers for the presence of bla _CTX-M genes. Among the 156 phenotypic positive isolates, 124 (79.4%) were positive for bla _CTX-M genes, of which 45 (36.2%) were K. pneumoniae, 79 (63.7%) were E. coli. When the 124 positive clinical isolates were further tested with CTX-M group-specific primers, all were positive for the CTX-M-1 group. Our findings document evidence of the high prevalence of multidrug resistant CTX-M group 1 type ESBL among nosocomial isolates in this region. High co-resistance to other non-β-lactam antibiotics is a major challenge for management of ESBL infections. This is alarming and calls for the judicious use of carbapenems, especially in developing countries. This has significant implications for patient management, and indicates the need for increased surveillance and for further molecular characterization of these isolates.     

Comparative analysis of <Emphasis Type="Italic">iol</Emphasis> clusters in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> strains

The present study was designed to expand genetic knowledge of myo -inositol (MI) metabolism in Lactobacillus casei. Twenty-four L. casei isolates of dairy origin were tested for the presence of iol cluster. PCR screening revealed eight strains encoded functions involved in MI utilization, of which one strain was able to use MI as carbon source. To gain a deeper understanding of the function of iol genes, four of the eight observed iol clusters were subjected to the full sequencing procedure. The results showed that the iol cluster was not a common feature among dairy L. casei strains. In addition, the four iol clusters were highly similar to one another in terms of sequence similarity and operon architecture. However, abundant polymorphisms that comprised a majority of synonymous mutations were detected throughout the full sequences. Three of them distributed among iolB, iolC, and iolT genes were found in linkage to MI-negative phenotype. Compared with other bacterial iol clusters, the iol cluster of L. casei showed a high similarity with that of Bacillus subtilis.     

Immunological and biological relationship among flagellin of <Emphasis Type="Italic">Pseudomonas aeruginosa,Burkholderia cepacia</Emphasis>, and <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>

The flagellar protein (flagellin) was isolated and purified from strains of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. A significant difference was observed in the molecular weight of different flagellin preparations obtained from these bacterial isolates. Antiserum prepared against S. maltophilia flagellin did not react with flagellin of P. aeruginosa or/and B. cepacia on Immunoblot or in indirect ELISA. In addition the anti-flagellin did not agglutinate P. aeruginosa and B. cepacia. No inhibition of motility of P. aeruginosa and B. cepacia was observed in presence of antiserum; though the latter inhibited the motility of S. maltophilia. The results of the present study prove that no specific relationship existed among all the studied flagellar proteins obtained from closely related bacteria.     

Characterization of the nematocidal toxocyst in <Emphasis Type="Italic">Pleurotus</Emphasis> subgen. <Emphasis Type="Italic">Coremiopleurotus</Emphasis>

Toxocysts of the genus Pleurotus are blastoconidia-like ovoid structures surrounded by a liquid droplet containing a toxin that paralyzes nematodes. This study investigated toxocyst development using a strain S396 of Pleurotus cystidiosus subsp. abalonus (subgen. Coremiopleurotus). The surface of the liquid droplet was found to be an elastic envelope. When a nematode touches the toxocyst, the envelope adheres to the worm and bursts. Toxocysts are induced simultaneously with coremia formation in the absence of nematodes and developed only from aerial hyphae in which nuclear division had ceased. In the early stage of toxocyst development, liquid springs repeatedly from the tip of the sterigma-like stipe before ovoid (blastoconidium-like structure) formation. A certain substance in the liquid might polymerize to form the envelope while the ovoid simultaneously budded in the droplet. The nucleus tends to locate near the toxocyst, especially in early stage of toxocyst development. Each dikaryotic cell predominantly formed one or two toxocyst(s) while each monokaryotic cell predominantly formed one. In rare cases, a nucleus existed in the toxocyst, suggesting the possibility that the toxocyst is a vestigial blastoconidium.     

<Emphasis Type="Italic">Lactobacillus</Emphasis> spp. associated with early childhood caries

A group of 69 lactobacilli was isolated from caries lesions and root canals of early childhood caries (ECC) affected children treated in the Department of Pedodontics (Children’s Teaching Hospital, Brno, Czech Republic). Biochemical and physiological properties of all strains were characterized by API 50 CH kit and conventional tube tests. The rep-PCR fingerprinting with the (GTG)₅ primer was used for genotypic grouping of the isolates. The (GTG)₅-PCR fingerprinting grouped all analyzed strains into a few clusters in nearly full agreement with phenotype identification results and clarified the taxonomic position of 13 biochemically unidentified strains. In total, 20 strains of Lactobacillus fermentum, 17 L. rhamnosus, 14 L. casei/paracasei, 7 L. gasseri, 7 L. salivarius and 4 L. plantarum were identified. Mixtures of two or even three Lactobacillus spp. were isolated from a few root canal content samples. Results obtained by biotyping and (GTG)₅-PCR were generally comparable except for L. gasseri strains that were not biochemically identified. The (GTG)₅-PCR fingerprinting was shown to be quicker, easier to perform and more reliable than biotyping. Our results imply this molecular method as a good tool for screening and identification of Lactobacillus spp. inhabiting dental plaque.     

Occurrence of the parasitic sea lamprey<Emphasis Type="Italic">Petromyzon marinus</Emphasis> on western North Atlantic right whales <Emphasis Type="Italic">Eubalaena glacialis</Emphasis>

Few data exist on the marine distribution and host organisms of the parasitic sea lamprey, Petromyzon marinus. Some observers have speculated that cetaceans serve as hosts for these fish based on scars, but few lamprey – cetacean interactions have been described in detail in the literature. Here we discuss 35 previously unreported records of sea lampreys that were observed while attached to western North Atlantic right whales, Eubalaena glacialis, during the period 1984 – 2002. Of these observations, 11 were photographically documented with images of sufficient quality to identify the lamprey as P. marinus based on morphological characteristics. The majority of the attachments were recorded in the Bay of Fundy during the summer months when P. marinus are preparing to spawn. It is unknown how lampreys might benefit from this association or what cost may be incurred by their right whale hosts. Feeding and transport are two possible reasons for the attachments.     

Antagonistic activity of bacteria isolated from crops cultivated in a rotation system and a monoculture against <Emphasis Type="Italic">Pythium debaryanum</Emphasis> and <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

The effect of crop rotation and monocropping on the occurrence of bacteria with antagonistic activity toward Pythium debaryanum and Fusarium oxysporum was shown. Arthrobacter spp., fluorescent Pseudomonas spp. and actinomycetes were isolated from winter rape, sugar beet and winter barley rhizosphere and bulk soil from the plots of a long-term crop rotation experiment (18 years). The occurrence of mycoantagonistic isolates and their antibiosis level exhibited specificity for the site, crop and crop rotation. Mycoantagonistic activity was common among actinomycetes and fluorescent Pseudomonas spp. and less frequent among Arthrobacter spp. Antibiosis of fluorescent Pseudomonas spp. and Arthrobacter spp. was in general stronger against P. debaryanum than F. oxysporum. The highest percentage of antagonistic Pseudomonas spp. against P. debaryanum was in the plots of barley crop, while plots of winter rape showed higher frequency of antagonists against F. oxysporum. The highest antibiosis activity of Arthrobacter spp. against both pathogens occurred in isolates from barley and winter rape monoculture, and there were no F. oxysporum antagonists among these bacteria in sugar beet monoculture. Most of actinomycete isolates strongly inhibited growth of P. debaryanum and F. oxysporum. The percentage of mycoantagonistic actinomycetes and their antibiosis level were the highest in the 6-year crop rotation system.