Combined molecular and biochemical approach identifies Aspergillus japonicus and Aspergillus aculeatus as two species

We examined nine Aspergillus japonicus isolates and 10 Aspergillus aculeatus isolates by using molecular and biochemical markers, including DNA sequences of the ITS1-5.8S rRNA gene-ITS2 region, restriction fragment length polymorphisms (RFLP), and secondary-metabolite profiles. The DNA sequence of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene could not be used to distinguish between A. japonicus and A. aculeatus but did show that these two taxa are more closely related to each other than to other species of black aspergilli. Aspergillus niger pyruvate kinase (pkiA) and pectin lyase A (pelA) and Agaricus bisporus 28S rRNA genes, which were used as probes in the RFLP analysis, revealed clear polymorphism between these two taxa. The A. niger pkiA and pelA probes placed six strains in an A. japonicus group and 12 isolates in an A. aculeatus group, which exhibited intraspecific variation when they were probed with the pelA gene. The secondary-metabolite profiles supported division of the isolates into the two species and differed from those of other black aspergilli. The strains classified as A. japonicus produced indole alkaloids and a polar metabolite, while the A. aculeatus isolates produced neoxaline, okaramins, paraherquamidelike compounds, and secalonic acid. A. aculeatus CBS 114.80 showed specific RFLP patterns for all loci examined. The secondary-metabolite profile of strain CBS 114.80 also differed from those of A. japonicus and A. aculeatus. Therefore, this strain probably represents a third taxon. This study provides unambiguous criteria for establishing the taxonomic positions of isolates of black aspergilli, which are important in relation to industrial use and legal protection of these organisms.     

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.Abbreviations kb kilobase pair - mtDNA mitochondrial DNA - RAPD random amplified polymorphic DNA - rDNA ribosomal RNA gene cluster - RFLP restriction fragment length polymorphisms     

Recombination of mitochondrial DNAs following transmission of mitochondria among incompatible strains of black Aspergilli

Successful intra- and interspecific mitochondrial transfers were performed by polyethylene glycol (PEG)-induced protoplast fusion among incompatible strains belonging to the Aspergillus niger species aggregate. The mitochondrial DNAs (mtDNAs) of the strains examined were of three main types based on their restriction fragment length polymorphism (RFLP) profiles. mtDNA types 1 and 2 correspond to A. niger and A. tubingensis species, respectively, while type 3 is represented by some Brazilian wild-type isolates (possibly a distinct species or subspecies). mtDNA types 1 and 2 could be further divided into several subgroups (1a–1e and 2a–2f?). All these strains, representing different RFLP groups or subgroups, were fully incompatible with respect to nuclear complementation. The transfer experiments were carried out under selection pressure, using a mitochondrial oligomycin-resistant mutant of mtDNA type 1a as donor. Following fusion mitochondrial oligomycin-resistant progenies were recovered in the presence of oligomycin by selecting for the nuclear phenotypes of the oligomycin-sensitive recipient strains. All attempted transfers were successful, and resulted in different varieties of resistant recombinant mitochondrial progenies at various frequencies. Within the group of strains of mtDNA type 1, the transfer of oligomycin-resistant mitochondria resulted in the appearance of a single recombinant type of RFLP profile in each case. The recombination events were more complex when the transfer of oligomycin resistance occurred between strains representing different species (mtDNA groups 1a→2 and 1a→3). A great variety of recombinant mtDNA RFLP profiles appeared. Explanation for this phenomenon are discussed on the basis of preliminary physical mapping data.     

Recombination of mitochondrial DNAs following transmission of mitochondria among incompatible strains of black Aspergilli

Successful intra- and interspecific mitochondrial transfers were performed by polyethylene glycol (PEG)-induced protoplast fusion among incompatible strains belonging to the Aspergillus niger species aggregate. The mitochondrial DNAs (mtDNAs) of the strains examined were of three main types based on their restriction fragment length polymorphism (RFLP) profiles. mtDNA types 1 and 2 correspond to A. niger and A. tubingensis species, respectively, while type 3 is represented by some Brazilian wild-type isolates (possibly a distinct species or subspecies). mtDNA types 1 and 2 could be further divided into several subgroups (1a–1e and 2a–2f ). All these strains, representing different RFLP groups or subgroups, were fully incompatible with respect to nuclear complementation. The transfer experiments were carried out under selection pressure, using a mitochondrial oligomycin-resistant mutant of mtDNA type 1a as donor. Following fusion mitochondrial oligomycin-resistant progenies were recovered in the presence of oligomycin by selecting for the nuclear phenotypes of the oligomycin-sensitive recipient strains. All attempted transfers were successful, and resulted in different varieties of resistant recombinant mitochondrial progenies at various frequencies. Within the group of strains of mtDNA type 1, the transfer of oligomycin-resistant mitochondria resulted in the appearance of a single recombinant type of RFLP profile in each case. The recombination events were more complex when the transfer of oligomycin resistance occurred between strains representing different species (mtDNA groups 1a→2 and 1a→3). A great variety of recombinant mtDNA RFLP profiles appeared. Explanation for this phenomenon are discussed on the basis of preliminary physical mapping data. Received: 16 July 1996 / Accepted: 2 December 1996     

Molecular analysis of variability within the toxigenic Aspergillus ochraceus species

Genetic variability of Aspergillus ochraceus was examined at the DNA level. Based on the HaeIII-Bg/II generated mitochondrial DNA restriction profiles, most isolates could be classified into two distinct groups. These two groups could also be distinguished by the random amplified polymorphic DNA technique, and with telomeric PCR amplifications. Phylogenetic analysis of sequences of the intergenic transcribed spacer region of some of the strains resulted in a dendrogram with the same topology as that based on mitochondrial DNA and amplified DNA data. None of the isolates with type 2 mtDNA profiles produce ochratoxins. Some strains (e.g., A. ochraceus ICMP 939) displayed strain-specific mitochondrial DNA patterns, and their amplified DNA profiles were also different from all other A. ochraceus strains examined.     

Intron mobility results in rearrangement in mitochondrial DNAs of heterokaryon incompatible Aspergillus japonicus strains after protoplast fusion.

Mitochondrial transmission was carried out under selective conditions between incompatible Aspergillus japonicus strains always using an oligomycin-resistant mitochondrial donor and selecting for recipient nuclei and oligomycin-resistant mitochondria. All attempted intraspecific mitochondrial transmissions were successful, but the transmission between closely related A. japonicus and A. aculeatus failed. Under selection pressure, resistant progeny harbor the mitochondrial DNA (mtDNA) of the donor strain, which may remain unchanged or may be modified by the introns of the recipient mitochondrial genome. Detailed analysis of a certain strain harboring rearranged mtDNA suggests that the mtDNA profiles of recombinant-like progeny are strongly influenced by the characteristics and mobility of introns of both parental mtDNAs. Both intron loss and intron acquisition play a role in the rearrangement of mtDNA. In certain parental combinations, a particular intron was lost very frequently.     

Effect of gentian violet on the growth of the N and T RFLP types of the Aspergillus niger aggregate

Taxa included in the Aspergillus niger aggregate are difficult to distinguish by phenotypic characterization. In this work, the effect of gentian violet on the growth of the N and T RFLP types of A. niger aggregate strains has been investigated. In total, 105 strains from different sources and origins, including reference cultures and field isolates were studied. Type N and T RFLP patterns, ochratoxin A production and the effect of different concentrations of gentian violet on the growth were determined in these strains. Forty nine strains belonged to the N type and 56 strains to the T type. Sixteen out of the 105 strains assayed were OTA producers. All the OTA-producing species belonged to the RFLP type N and none of the T type strains was able to produce OTA. Approximately 90% of the N type strains grew in the presence of 25 ppm of gentian violet. Only five N type strains did not grow on this medium. One of these strains was A. niger ATCC 22343, a well documented induced mutant strain and the remaining four strains belonged to the new species A. brasiliensis. On the contrary, all the T type strains failed to grow on this medium after 3 days of incubation (sensitivity 89.79%; specificity 100%). The use of growth in gentian violet as an additional character for classification and identification purposes in this taxonomic group may be useful because no phenotypic methods have yet been found that can distinguish between these species.     

Acanthamoeba: keratopathogenicity of isolates from domestic tap water in Korea

In a previous study, we reported on the contamination rate of free living amoeba, including Acanthamoeba, isolated from contact lens storage cases (CLSC) and domestic tap water in Korea. In an effort to evaluate the potential kerato-pathogenicity of 5 isolates from CLSC and 17 isolates from domestic tap water, we have conducted an investigation into the morphological features, mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) phenotypes, 18S rDNA sequences, and drug sensitivities of these isolates, and have compared the results with those of 20 amoebic keratitis (AK) isolates from Korea, as well as 14 reference strains. Cysts from 22 isolates obtained from CLSC and domestic tap water showed typical characteristics of morphological group 2. A total of three and five mtDNA RFLP patterns generated by EcoRI were found in 5 of the isolates from CLSC and 17 of the isolates from domestic tap water, respectively. The mtDNA RFLP patterns of four of the five isolates from the CLSC were found to be identical to those of the isolates from domestic tap water of students who had contaminated CLSC. The majority had mtDNA RFLP patterns identical to those of AK isolates in Korea. The results of 18S rDNA sequencing analysis were also shown to coincide with the results of mtDNA RFLP analysis. KA/WP12 was determined to be profoundly sensitive to chlorhexidine (MCC; 6.25microg/ml), and KAWP2 was the most sensitive strain to polyhexamethylene biguanide (PHMB) (MCC; 4.69microg/ml). Some difference in the cytopathic effects of isolates against human corneal epithelial cells was observed according to their mtDNA genotypes. In conclusion, domestic tap water may constitute a source of Acanthamoeba contamination of CLSC, and most isolates from CLSC and domestic tap water appear to be potentially keratopathogenic.     

Genetic variability among <Emphasis Type="Italic">Pholiota aurivella</Emphasis> isolates from a small natural population

Genetic differences between 36 Pholiota aurivella wild isolates collected from 13 decayed logs of Salicaceae trees distributed along about 1200 m of a streambed in a forest were characterized by somatic incompatibility and mating tests, and by restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA). There was a perfect correlation between somatic incompatibility and mating type groups, and isolates could be divided into 15 genets (genetically identical clones). Because the mtDNAs of the 36 wild isolates have 14 different EcoRI RFLP patterns, they likely originated from at least 14 distinct wild strains, indicating that multiple wild strains with distinct genetic compositions coexist in the forest investigated in this study. mtDNA variation of P. aurivella is apparently very high despite the close proximity of sample collection sites within the forest. The territories of single P. aurivella genets within a host log are apparently larger than other nonpathogenic wood-decaying basidiomycetes reported previously, such as Flammulina velutipes and Lentinula edodes.     

Interpretation of mtDNA RFLP variability among <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> isolates

Aspergillus tubingensis isolates collected from distant geographic areas were earlier classified into six groups on the basis of the mtDNA RFLP variability they exhibited (mtDNA types 2a-2f). In the present work, we investigated the reason for the intraspecific mtDNA variability and we describe here how this fungus, with a relatively small mitochondrial genome, can display intraspecific polymorphism due to intron acquisition and also sporadic point mutations affecting the recognition motifs of the restriction enzymes employed in the RFLP analysis. Three different LAGLI-DADG type group I introns were identified in the cox1 gene amongst the six mtDNA RFLP types. MtDNAs of types 2b and 2d contain all of the three introns, mtDNA of type 2f carries only one, and the other mtDNA types contain two introns each. Comparative analysis showed that the first and second introns of mtDNAs of types 2b and 2d are well distributed among fungi, indicating their active horizontal transfer capacity. The third intron occurs rarely among fungi and is restricted to a limited number of fungal species, namely to A. tubingensis and the yeast Candida stellata. It is interesting that this intron is present in a small mitochondrial genome such as that of A. tubingensis and, considering its rarity, its presence amongst black Aspergillus isolates is recommended to be considered as a tool to establish taxonomical unit(s) or to track down evolutionary divergence of closely related taxonomical units.     

Physiological, chemical, morphological, and plant infectivity characteristics of Frankia isolates from Myrica pennsylvanica: correlation to DNA restriction patterns.

The filter exclusion method was used to isolate Frankia strains from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. A total of 16 isolates from five locations were cultured. The isolates were characterized by morphological, chemical, physiological, and plant infectivity criteria and compared with genomic DNA restriction pattern data, which were used to assign the isolates into gel groups (see accompanying paper). The isolates from M. pennsylvanica evaluated in this study were characteristic of Frankia physiological group B strains and were indistinguishable on the basis of whole-cell wall chemistry and diaminopimelic acid isomer analysis. Distinct differences in the spectrum of utilized organic acids and carbohydrates were observed among the isolates and were the only phenotypic criteria by which the isolates could be separated and assigned into separate groups. In general, isolates within a restriction pattern gel group had identical utilization patterns, whereas intragroup isolates had different utilization patterns. Correlation of these phenotypic characteristics with the results of molecular analysis revealed an exclusive carbohydrate and organic acid utilization pattern for each gel group as established by restriction pattern analysis.     

Mitochondrial DNA polymorphism of the yeast Saccharomyces bayanus var. uvarum

Genetic relationships among forty-one strains of Saccharomyces bayanus var. uvarum isolated in different wine regions of Europe and four wild isolates were investigated by restriction analysis (RFLP) of mitochondrial DNA (mtDNA) with four restriction endonucleases, AluI, DdeI, HinfI and RsaI. No clear correlation between origin and source of isolation of S. bayanus var. uvarum strains and their mtDNA restriction profiles was found. On the whole, the mtDNA of S. bayanus var. uvarum is much less polymorphic than that of S. cerevisiae. This observation is in good agreement with results obtained by electrophoretic karyotyping. Unlike wine S cerevisiae, strains of S. bayanus var. uvarum display a low level of chromosome length polymorphism.     

Physiological, chemical, morphological, and plant infectivity characteristics of Frankia isolates from Myrica pennsylvanica: correlation to DNA restriction patterns

The filter exclusion method was used to isolate Frankia strains from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. A total of 16 isolates from five locations were cultured. The isolates were characterized by morphological, chemical, physiological, and plant infectivity criteria and compared with genomic DNA restriction pattern data, which were used to assign the isolates into gel groups (see accompanying paper). The isolates from M. pennsylvanica evaluated in this study were characteristic of Frankia physiological group B strains and were indistinguishable on the basis of whole-cell wall chemistry and diaminopimelic acid isomer analysis. Distinct differences in the spectrum of utilized organic acids and carbohydrates were observed among the isolates and were the only phenotypic criteria by which the isolates could be separated and assigned into separate groups. In general, isolates within a restriction pattern gel group had identical utilization patterns, whereas intragroup isolates had different utilization patterns. Correlation of these phenotypic characteristics with the results of molecular analysis revealed an exclusive carbohydrate and organic acid utilization pattern for each gel group as established by restriction pattern analysis.     

Genetic diversity of Acanthamoeba isolated from ocean sediments

Genetic diversity of 18 Acanthamoeba isolates from ocean sediments was evaluated by comparing mitochondrial (mt) DNA RFLP, 18S rDNA sequences and by examining their cytopathic effects on human corneal epithelial cells versus reference strains. All isolates belonged to morphologic group II. Total of 16 restriction phenotypes of mtDNA from 18 isolates demonstrated the genetic diversity of Acanthamoeba in ocean sediments. Phylogenetic analysis using 18s rDNA sequences revealed that the 18 isolates were distinct from morphological groups I and III. Fifteen isolates showed close relatedness with 17 clinical isolates and A. castellanii Castellani and formed a lineage equivalent to T4 genotype of Byers group. Two reference strains from ocean sediment, A. hatchetti BH-2 and A. griffini S-7 clustered unequivocally with these 15 isolates. Diversity among isolates was also evident from their cytopathic effects on human corneal cells. This is the first time describing Acanthamoeba diversity in ocean sediments in Korea.     

Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Morocco

AIMS: To determine the biodiversity of rhizobial strains nodulating Cicer arietinum L. in representative soils from various areas of Morocco. METHODS AND RESULTS: Symbiotic traits, utilization of 49 carbohydrate sources, resistance to antibiotics and heavy metals, tolerance to salinity, to extreme temperatures and pH were studied as phenotypic markers. In addition, restriction fragment length polymorphism (RFLP) of PCR-amplified 16S rDNAs were compared with those of reference strains. Numerical analysis of the phenotypic characteristics showed that the 48 strains studied fell into three distinct groups. RFLP analysis of 16S rRNA genes revealed an additional heterogeneity and four ribotypes were identified. CONCLUSIONS: Chickpea rhizobia isolated from Moroccan soils are both phenotypically and genetically diverse. Most of these rhizobia belong to the Mesorhizobium genus. However, some strains originating from a particular soil appeared to have 16S rRNA genes similar to Sinorhizobium as well as very distinct auxanographic characteristics compared with Mesorhizo- bium isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: A well characterized collection of chickpea-nodulating rhizobia in representative soils of Morocco has been generated, which can be used to develop efficient inoculants for this crop. This is the first report evidencing that chickpeas may be nodulated by bacteria from the Sinorhizobium genus.     

Genomic and phenotypic heterogeneity of Acidithiobacillus spp. strains isolated from diverse habitats in China

The genetic variability among 32 Chinese Acidithiobacillus spp. environmental isolates and four reference strains representing three recognized species of the genus Acidithiobacillus was characterized by using a combination of molecular methods, namely restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers, repetitive element PCR, arbitrarily primed PCR and 16S rRNA gene sequence analyses. 16S rRNA gene sequences revealed that all Acidithiobacillus spp. strains could be assigned to seven groups, three of which encompassed the Acidithiobacillus ferrooxidans strains from various parts of the world. A comparative analysis of the phylogenetic Group 1 and 2 was undertaken. Restriction fragment length polymorphism results allowed us to separate the 35 Acidithiobacillus strains into 15 different genotypes. An integrated phenotypic and genotypic analysis indicated that the distribution of A. ferrooxidans strains among the physiological groups were in agreement with their distribution among the genomic groups, and that no clear correlation was found between the genetic polymorphism of the Acidithiobacillus spp. strains and either the geographic location or type of habitats from which the strains were isolated. In addition, five unidentified sulfur-oxidizing isolates may represent one or two novel species of the genus Acidithiobacillus. The results showed that the Chinese Acidithiobacillus spp. isolates exhibited a high degree of genomic and phenotypic heterogeneity.     

The global genetic structure of the wheat pathogen Mycosphaerella graminicola is characterized by high nuclear diversity,low mitochondrial diversity,regular recombination,and gene flow

A total of 1673 Mycosphaerella graminicola strains were assayed for DNA fingerprints and restriction fragment length polymorphism (RFLP) markers in the nuclear and mitochondrial genomes. The isolates were collected from 17 wheat fields located in 11 countries on five continents over a six year period (1989-1995). Our results indicate that genetic diversity in the nuclear genome of this fungus was high for all but three of the field populations surveyed and that populations sampled from different continents had similar frequencies for the most common RFLP alleles. Hierarchical analysis revealed that more than 90% of global gene diversity was distributed within a wheat field, while approximately 5% of gene diversity was distributed among fields within regions and approximately 3% was distributed among regions on different continents. These findings suggest that gene flow has occurred on a global scale. On average, each leaf was colonized by a different nuclear genotype. In contrast, only seven mtDNA haplotypes were detected among the 1673 isolates and the two most common mtDNA haplotypes represented approximately 93% of the world population, consistent with a selective sweep. Analysis of multilocus associations indicated that all field populations were in gametic equilibrium, suggesting that sexual recombination is a regular occurrence globally.     

Mitochondrial DNAs and plasmids as taxonomic characteristics in Trichoderma viride.

Mitochondrial DNA (mtDNA) was purified from 12 isolates of the Trichoderma viride aggregate and found to be, on the average, 32.7 kb in size. Plasmids were present in the mtDNA preparations from 8 of 12 strains of T. viride examined. Plasmids in four of the strains produced ladderlike banding patterns on gels, and these plasmids were studied in detail. The ladderlike patterns were produced by single molecules that were supercoiled to various degrees. Plasmids from two of the strains do not have homology with the mtDNA but do have a limited amount of homology with each other. No phenotype could be associated with the presence of a plasmid. Restriction endonuclease digestion of the mtDNAs produced patterns in which the presence or absence of certain fragments correlated with the classification of the strains into T. viride group I or II. Phenetic cluster analysis and parsimony analysis of the fragment patterns produced groups that corresponded to T. viride groups I and II. The fragment patterns were very diverse, with nearly all strains having a unique pattern. However, two strains of T. viride group I from widely different geographical locations did have identical restriction patterns for all the enzymes used in this study. This result indicates that it may not be possible to use mtDNA restriction patterns alone to identify Trichoderma strains.     

Analysis of mitochondrial DNA restriction fragment length polymorphisms for examining genetic variability among isolates of Phellinus linteus

 Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNAs (mtDNAs) from nine Japanese wild isolates of Phellinus linteus was carried out to examine their genetic variability. BamHI and EcoRI digests of mtDNAs from these isolates produced four and five distinct RFLP patterns, respectively. By combining the RFLP patterns obtained with the two endonucleases, mtDNAs from the nine isolates could be assigned to five different genotypes, but no mtDNA variation was detected among the isolates collected from a small area. Distance values calculated among all pairs of mtDNA genotypes, based on the presence or absence of comigrating restriction fragments, were clearly smaller than those among the mtDNA genotypes of Lentinula edodes and Pleurotus ostreatus samples collected worldwide, suggesting the necessity of collecting P. linteus wild isolates for genetic resources from geographically wider areas. Received: June 27, 2002 / Accepted: August 19, 2002 Correspondence to:T. Nakamura     

Restriction Enzyme Analysis of Mitochondrial DNA of Acanthamoeba Strains in Japan

ABSTRACT. Eight isolates, identified as either Acanthamoeba castellanii or A. polyphaga from human eye infections, contact lens containers, and soil in Japan, were characterized by restriction fragment length polymorphisms (RFLP) of mitochondrial DNA (mtDNA). Mitochondrial DNA was digested with either Bgl II, Eco R I, Hind III, Hpa I, Sca I or Xba I, electrophoresed in agarose gels, and stained with ethidium bromide. Four distinct RFLP phenotypes that refer to the collection of six fragment size patterns obtained for a single strain with six enzymes, were discovered among the eight strains used in this study. Three strains morphologically classified as A. polyphaga share a single RFLP phenotype with the Ma strain of A. castellanii. The interspecific sequence differences of 7.06–12.74% in DNA nucleotide were estimated from the proportion of DNA fragments shared by each pair of mtDNA.