DNA fingerprinting analysis of Petromyces alliaceus (Aspergillus section Flavi)

The objective of this study was to evaluate the ability of the Aspergillus flavus pAF28 DNA probe to produce DNA fingerprints for distinguishing among genotypes of Petromyces alliaceus (Aspergillus section Flavi), a fungus considered responsible for the ochratoxin A contamination that is occasionally observed in California fig orchards. P. alliaceus (14 isolates), Petromyces albertensis (one isolate), and seven species of Aspergillus section Circumdati (14 isolates) were analyzed by DNA fingerprinting using a repetitive sequence DNA probe pAF28 derived from A. flavus. The presence of hybridization bands with the DNA probe and with the P. alliaceus or P. albertensis genomic DNA indicates a close relationship between A. flavus and P. alliaceus. Twelve distinct DNA fingerprint groups or genotypes were identified among the 15 isolates of Petromyces. Conspecificity of P. alliaceus and P. albertensis is suggested based on DNA fingerprints. Species belonging to Aspergillus section Circumdati hybridized only slightly at the 7.0-kb region with the repetitive DNA probe, unlike the highly polymorphic hybridization patterns obtained from P. alliaceus and A. flavus, suggesting very little homology of the probe to Aspergillus section Circum dati genomic DNA. The pAF28 DNA probe offers a tool for typing and monitoring specific P. alliaceus clonal populations and for estimating the genotypic diversity of P. alliaceus in orchards, vineyards, or crop fields.     

Common genotypes (RFLP) within a diverse collection of yellow-green aspergilli used to produce traditional Oriental fermented foods

 DNA fingerprinting was performed on 72 strains of Aspergillus oryzae and 9 strains of Aspergillus sojae isolated from chu (China) or koji (Japan) mold inoculum used in the production of traditional Oriental fermented beverages or foods including soy sauce, miso, and sake. The cultures were deposited with the ARS Culture Collection (NRRL) between 1909 and 2001. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. All strains of A. sojae that we examined produced an identical DNA fingerprint and belong to the same DNA fingerprint group (GTAo-9). Strains of A. oryzae were distributed among 41 DNA fingerprint groups, including GTAo-12 represented by 11 strains, GTAo-19 represented by 5 strains, GTAo-5 and GTAo-15 each represented by 4 strains, and GTAo-8, GTAo-17, and GTAo-24 each represented by 3 strains. Thirty-three single strain isolates of A. oryzae produced unique fingerprints. Our data offer evidence suggesting that (1) the successful domestication of A. parasiticus genotypes yielding A. sojae occurred far less frequently than among genotypes of A. flavus var. oryzae; and (2) some Aspergillus genotypes employed in different fermentations and regions were derived from a common ancestral clonal population. Received: February 18, 2002 / Accepted: April 22, 2002     

Diversity of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> genotypes (RFLP) isolated from traditional soy sauce production within Malaysia and Southeast Asia

DNA fingerprinting was performed on 64 strains of Aspergillus oryzae and 1 strain of Aspergillus sojae isolated from soy sauce factories within Malaysia and Southeast Asia that use traditional methods in producing “tamari-type” Cantonese soy sauce. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. Strains of A. oryzae were distributed among 32 genotypes (30 DNA fingerprint groups). Ten genotypes were recorded among 17 A. oryzae isolates from a single soy sauce factory. Genotypes Ao-46 and GTAo-47, represented by 8 and 5 strains, respectively, were isolated from a soy sauce factory in Kuala Lumpur and factories in two Malaysian states. Four strains of GTAo-49, isolated from three soy sauce factories in Malaysia; each produced sclerotia. Two strains were found to be naturally occurring color mutants of NRRL 32623 (GTAo-49) and NRRL 32668 (GTAo-52). Only two fingerprint matches were produced with the 43 DNA fingerprint groups in our database, representing A. oryzae genotypes from Japan, China, and Taiwan. Aspergillus sojae NRRL 32650 produced a fingerprint matching GTAo-9, the only known genotype representing koji strains of A. sojae. No aflatoxin was detected in broth cultures of these koji strains as determined by TLC.     

Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus section Flavi.

The Aspergillus parasiticus aflR gene, a gene that may be involved in the regulation of aflatoxin biosynthesis, encodes a putative zinc finger DNA-binding protein. PCR and sequencing were used to examine the presence of aflR homologs in other members of Aspergillus Section Flavi. The predicted amino acid sequences indicated that the same zinc finger domain, CTSCASSKVRCTKEKPACARCIERGLAC, was present in all of the Aspergillus sojae, Aspergillus flavus, and Aspergillus parasiticus isolates examined and in some of the Aspergillus oryzae isolates examined. Unique base substitutions and a specific base deletion were found in the 5' untranslated and zinc finger region; these differences provided distinct fingerprints. A. oryzae and A. flavus had the T-G-A-A-X-C fingerprint, whereas A. parasiticus and A sojae had the C-C-C-C-C-T fingerprint at the corresponding positions. Specific nucleotides at positions -90 (C or T) and -132 (G or A) further distinguished A. flavus from A. oryzae and A. parasiticus from A. sojae, respectively. A sojae ATCC 9362, which was previously designated A. oryzae NRRL 1988, was determined to be a A. sojae strain on the basis of the presence of the characteristic fingerprint, A-C-C-C-C-C-C-T. The DNAs of other members of Aspergillus Section Flavi, such as Aspergillus nomius and Aspergillus tamarii, and some isolates of A. oryzae appeared to exhibit low levels of similarity to the A. parasiticus aflR gene since low amounts of PCR products or no PCR products were obtained when DNAs from these strains were used.     

Properties of Aspergillus tamarii,A. caelatus and related species from acidic tea field soils in Japan

Fungi in Aspergillus section Flavi include both aflatoxin producers and non-producers. Aspergillus caelatus is a recently described non-aflatoxigenic species in this section, which has some common characteristics with A. tamarii, such as yellowish brown color and double walled spores. In contrast to the morphological similarities, all of the A. caelatus isolates tested produced no cyclopiazonic acid whereas most isolates of A. tamarii produce this compound. There are six nucleotide differences that distinguish the DNA sequences of these two species in the regions of ITS1, ITS2, 5.8S rDNA and 28S rDNA and this is a consistent difference. Both species were isolated from acidified field soils, but A. tamarii isolates were more common than A. caelatus in highly acidic soils. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of aflatoxigenic and non-aflatoxigenic <Emphasis Type="Italic">Aspergillus flavus</Emphasis> isolates from pistachio

Pistachio is a popular snack food. Aflatoxin contamination of pistachio nuts is a serious problem for many producing countries. The development of biological control methods based on ecological parameters is an environmentally friendly approach. Thirty-eight Aspergillus flavus isolates collected from a pistachio orchard in California (CA) were analyzed for production of aflatoxin (AF), cyclopiazonic acid (CPA), vegetative compatibility groups (VCGs), and mating types. All aflatoxigenic isolates produced both AFB₁ and CPA. The most toxigenic one was CA28 which produced 164 μg AFB₁ per 5 ml PDA fungal culture and small sclerotia (S strain, sclertoium size less than 400 μm). The other aflatoxigenic strains produce AFB₁ ranging from 1.2 μg to 80 μg per 5 ml fungal culture. Twenty-one percent of the CA isolates produced AFB₁, 84% produced CPA and half formed sclerotia on at least one of three tested media. The 38 CA isolates formed 26 VCGs, 6 of which had two or more isolates and 20 contained single isolates. The S strain isolates belong to 4 different VCGs. Genomic profiling by a retrotransposon DNA probe revealed fingerprint patterns that were highly polymorphic. The predicted VCGs (Pred-VCGs) based on a similarity coefficient >80% matched the VCGs of multiple isolates determined by complementation. All isolates within a VCG had the same mating-type gene of either MAT1-1 or MAT1-2. Uncorrected and VCG-corrected MAT1-1 and MAT1-2 among the isolates were equally distributed.     

Clonality and genetic variation in Amylostereum areolatum and A. chailletii from northern Europe

Genetic variation within and between vegetative compatibility groups (VCGs) of Amylostereum areolatum (Fr.) Boid. and Amylostereum chailletii (Pers.: Fr.) Boid. isolates was investigated. DNA fingerprints were made using the M13 core sequence as a primer. A total of 53 isolates of A. areolatum and 57 isolates of A. chailletii from Lithuania, Sweden, Denmark and Great Britain were studied. In all cases isolates belonging to the same VCG showed identical DNA banding patterns, suggesting that VCGs correspond to clones. In A. areolatum the vast majority of the isolates (spread by Sirex juvencus L.) were assigned to dispersive clones, that have wide geographical distribution (i.e. the same genotypes were detected in Lithuania, Sweden and Denmark), with low genetic variation between the different clones. By contrast, A. chailletii population structure was consistent with the spread of airborne basidiospores produced by outcrossing. Only a small fraction of A. chailletii isolates studied, could be assigned to dispersal clones with a local distribution, spread by Urocerus gigas L. Overall, M13 fingerprinting detected low genetic differentiation in both species in the samples we studied.     

Genetic diversity within and among vegetative compatibility groups of Stereum sanguinolentum determined by arbitrary primed PCR

Genetic variation within and among vegetative compatibility groups (VCGs) of Stereum sanguinolentum isolates was investigated with various geographical distances. DNA fingerprints were made using the M13 core sequence as a primer. A total of 113 isolates from 12 plots in Sweden, Finland and Lithuania were studied. Each VCG produced a discrete group of banding patterns. Among 20 isolates from the largest VCG, G1, the incidence of identical banding patterns was 24% within a sample plot, 8% among sample plots within a country, and 0% among countries. In the other 15 VCGs that comprised two and more isolates, corresponding percentages were 42%, 30% and 0%. Average band-sharing indices (ABSIs) within VCGs decreased with increasing geographical distance: for isolates from VCG G1, ABSI was 91.3% within sample plots, 85.4% among sample plots within a country, and 79.2% among countries. Corresponding figures for the other 15 VCGs were 97.0%, 92.7% and 80.4%, respectively. Among VCGs similarities were significantly lower ( P < 0.001) and the trend of decreasing similarity with larger geographical distances was less pronounced: ABSI within sample plots was 73.7%, among sample plots within countries 73.8%, and among countries 71.1%. However, the similarity of populations as compared within and between countries differed significantly ( P < 0.001), suggesting geographical differentiation between S. sanguinolentum populations separated by 400 km and the Baltic sea. Calculations of G _ST clearly indicated geographical population subdivision within the large VCG but not among the total sample. In summary, the results show population structure of inbred lines within VCGs containing closely related strains in local populations and more distant relationships among populations.     

Aspergillus bombycis genotypes (RFLP) from silkworm cultivation

 Eighteen isolates of Aspergillus bombycis from samples of dust, insect frass, and soil collected from eight silkworm rearing facilities in Japan, as well as single silkworm rearing facilities in Indonesia and Malaysia, were subjected to DNA fingerprinting. PstI digests of total genomic DNA from each isolate were probed using the pAF 28 repetitive sequence. Among 18 isolates analyzed, 7 distinct DNA fingerprint groups were identified, including GTAb-2 isolated from rearing facilities in four prefectures of Japan. Aspergillus bombycis isolates share several features in common with domesticated yellow-green aspergilli, the koji molds used in traditional Oriental food fermentations, including a loose and deep colony texture, smooth-walled stipes, and the absence of sclerotia. Although aflatoxin is unknown from koji molds, all isolates of A. bombycis produced B and G aflatoxins. Aflatoxin has been linked to increased virulence in Aspergillus disease of silkworms, and there should be strong selection for aflatoxin production among clonal populations of A. bombycis adapted to silkworm cultivation. A hypothesis is offered that A. bombycis isolates from silkworm cultivation represent highly adapted forms of yet to be discovered “wild” populations that may infect Bombyx mandarina. Received: December 24, 2002 / Accepted: March 10, 2003 RID="*" ID="*" Disclaimer: Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the products, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable Acknowledgments We thank Dr. Akira Nakagiri, Institute for Fermentation, Osaka, for providing Aspergillus cultures isolated from diseased silkworms.     

Analysis of single nucleotide polymorphisms in three genes shows evidence for genetic isolation of certain Aspergillus flavus vegetative compatibility groups

Genetic exchange by asexual filamentous fungi is presumed to be limited to isolates in the same vegetative compatibility group (VCG). To evaluate genetic isolation of Aspergillus flavus due to vegetative incompatibility, three gene regions were chosen that contained closely spaced nucleotides that were polymorphic among some of the six VCGs examined. A member of each VCG was collected from five regions across the southern United States. Isolates belonging to the same VCG had similar sets of single nucleotide polymorphisms regardless of isolate origin. The six VCGs formed four genetically distinct groups. Although recombination between certain pairs of VCGs could not be excluded, none was found for YV36, the VCG that includes the atoxigenic A. flavus isolate currently used to mitigate aflatoxin contamination in cotton in Arizona.     

Restriction fragment length polymorphisms in isolates of Aspergillus fumigatus probed with part of the intergenic spacer region from the ribosomal RNA gene complex of Aspergillus nidulans

Differences in restriction fragment length polymorphisms (RFLPs) have been detected in isolates of Aspergillus fumigatus. Genomic DNA from 11 isolates was digested with EcoRI, separated by electrophoresis, Southern blotted and probed with DNA from the intergenic spacer or non-transcribed spacer region of the rRNA gene complex of Aspergillus nidulans. Three distinct RFLP patterns were detected which differed from the control patterns observed with A. nidulans, Aspergillus flavus and Aspergillus niger hybridized with the same probe. Furthermore, the differences in RFLP patterns in the A. fumigatus isolates were not detected when probed with DNA coding for the rRNA complex in Saccharomyces cerevisiae. These findings may be of use in the study of the epidemiology and pathogenesis of infections caused by A. fumigatus.     

Species identification of Aspergillus section Flavi isolates from Portuguese almonds using phenotypic, including MALDI-TOF ICMS, and molecular approaches

AIMS: Section Flavi is one of the most significant sections in the genus Aspergillus. Taxonomy of this section currently depends on multivariate approaches, entailing phenotypic and molecular traits. This work aimed to identify isolates from section Flavi by combining various classic phenotypic and genotypic methods as well as the novel approach based on spectral analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF ICMS) and to evaluate the discriminatory power of the various approaches in species identification. METHODS AND RESULTS: Aspergillus section Flavi isolates obtained from Portuguese almonds were characterized in terms of macro- and micromorphology, mycotoxin pattern, calmodulin gene sequence and MALDI-TOF protein fingerprint spectra. For each approach, dendrograms were created and results were compared. All data sets divided the isolates into three groups, corresponding to taxa closely related to Aspergillus flavus, Aspergillus parasiticus and Aspergillus tamarii. In the A. flavus clade, molecular and spectral analyses were not able to resolve between aflatoxigenic and nonaflatoxigenic isolates. In the A. parasiticus cluster, two well-resolved clades corresponded to unidentified taxa, corresponding to those isolates with mycotoxin profile different from that expected for A. parasiticus.     

Development and evaluation of a real-time quantitative PCR assay for Aspergillus flavus

Aspergillus flavus is a ubiquitous mold and the most common mold contaminating foodstuffs. Many strains of A. flavus produce aflatoxins. In addition it is an allergen and an opportunistic pathogen of animals and plants. A. flavus often is underestimated in traditional culture analyses due to the expertise required and the cost associated with speciating members of the genus Aspergillus. The goal of this study was to develop and validate a primer and probe set for the rapid detection and quantitation of A. flavus in pure culture using real-time quantitative polymerase chain reaction (QPCR) amplification. Unique DNA regions were located in the genome of the target organism by sequence comparison with the GenBank database, and several candidate oligonucleotides were identified from the scientific literature for potential use with the TaqMan QPCR technology. Three primer and probe sets were designed and validated for specificity and sensitivity in laboratory experiments. Initial screening to test for sensitivity was performed with seven A. flavus isolates and selected nontarget fungi. Specificity testing was conducted with the selected primer and probe set, which amplified all nine A. flavus isolates tested, including an aflatoxin producing strain. The primers did not amplify DNA extracted from 39 other fungal species (comprising 16 genera), including 18 other Aspergillus species and six Penicillium species. No amplification of human or bacterial DNA was observed; however cross-reactivity was observed with Aspergillus oryzae. PCR analysis of DNA dilutions and the use of an internal positive control demonstrated that 67% of the fungal DNA samples assayed contained PCR inhibitors. The assay validated for the target organism is capable of producing PCR results in less than 1 h after DNA extraction. The results of this research demonstrate the capabilities of QPCR for the enhanced detection and enumeration of fungi of significance to human health.     

An evaluation of ERIC PCR and AP PCR fingerprinting for discriminating Salmonella serotypes

PCR fingerprints of 89 Salmonella isolates belonging to 22 serotypes were obtained using ERIC PCR (enterobacterial repetitive intergenic consensus PCR) and AP PCR (arbitrarily primed PCR) to evaluate the ability of different fingerprinting methods to differentiate or identify serotypes and subtypes. Fingerprints were scored and comparisons were made using a computer program. ERIC PCR produced a unique, complex fingerprint for almost every isolate, but these fingerprints did not identify serotypes. One AP PCR primer also produced complex fingerprints that discriminated among isolates, but again did not identify serotypes. A second AP PCR primer produced simple patterns, including one pattern shared by 35 isolates from 12 different serotypes. In general, the three sets of PCR fingerprints distinguished isolates, but were not correlated with serotypes. Matching fingerprints from different gels by computer was difficult, since similarities were based on both intense and faint bands. In addition, this study suggests that dendrograms created from PCR fingerprints should be viewed with caution.     

Twenty-four microsatellite markers for the aflatoxin-producing fungus Aspergillus flavus

Aspergillus flavus infects both plants and humans and contaminates diverse agricultural crops with aflatoxins, highly carcinogenic fungal metabolites. We describe 24 microsatellite markers developed to assess genetic diversity and recombination within and between three vegetative compatibility groups (VCGs) of Aspergillus flavus. These loci are polymorphic within at least one VCG or between VCGs. For loci polymorphic across all three VCGs, the number of alleles ranged from two to 19. These markers will be useful for genetic studies of this economically important pathogen.     

DNA Fingerprinting and Analysis of Population Structure in the Chestnut Blight Fungus, Cryphonectria Parasitica

We analyzed DNA fingerprints in the chestnut blight fungus, Cryphonectria parasitica, for stability, inheritance, linkage and variability in a natural population. DNA fingerprints resulting from hybridization with a dispersed moderately repetitive DNA sequence of C. parasitica in plasmid pMS5.1 hybridized to 6-17 restriction fragments per individual isolate. In a laboratory cross and from progeny from a single perithecium collected from a field population, the presence/absence of 11 fragments in the laboratory cross and 12 fragments in the field progeny set segregated in 1:1 ratios. Two fragments in each progeny set cosegregated; no other linkage was detected among the segregating fragments. Mutations, identified by missing bands, were detected for only one fragment in which 4 of 43 progeny lacked a band present in both parents; no novel fragments were detected in any progeny. All other fragments appeared to be stably inherited. Hybridization patterns did not change during vegetative growth or sporulation. However, fingerprint patterns of single conidial isolates of strains EP155 and EP67 were found to be heterogenous due to mutations that occurred during culturing in the laboratory since these strains were first isolated in 1976-1977. In a population sample of 39 C. parasitica isolates, we found 33 different fingerprint patterns with pMS5.1. Most isolates differed from all other isolates by the presence or absence of several fragments. Six fingerprint patterns each occurred twice. Isolates with identical fingerprints occurred in cankers on the same chestnut stems three times; isolates within the other three pairs were isolated from cankers more than 5 m apart. The null hypothesis of random mating in this population could not be rejected if the six putative clones were removed from the analysis. Thus, a rough estimate of the clonal fraction of this population is 6 in 39 isolates (15.4%).     

Synnema and sclerotium production in Aspergillus caelatus and the influence of substrate composition on their development in selected strains

The ability of Aspergillus caelatus, a species in Aspergillus section Flavi, to produce synnemata and sclerotia was investigated. Forty-eight isolates of A. caelatus differed widely in their production of synnemata and sclerotia; 83% of the isolates produced varying numbers of synnemata and sclerotia, and 17% produced neither sclerotia nor synnemata. Most strains produced synnemata and mostly sessile and few stipitate sclerotia on the same Czapek agar (CZA) plate. Two strains of A. caelatus were selected for further study because of the contrasting morphology of their synnemata and sclerotia. Those strains are NRRL 25528, the type species and a representative of the synnema- and black sclerotium-forming isolates, and NRRL 26119, considered an atypical strain that produced numerous synnemata and few slightly melanized or tan sclerotia. The induction and maturation of sclerotia in A. caelatus were affected greatly by the type of media as well as the kind and concentration of the carbon and nitrogen sources. CZA induced synnema and sclerotium production in both strains, whereas other media did not. Production of abundant synnemata and sclerotia also occurred when the carbon source in CZA is replaced with dextrose, xylose, cellobiose, melibiose and trehalose. CZA amended with serine, threonine, KNO(3) and NaNO(3) induced the production of numerous sclerotia and synnemata. For both strains, the optimal levels of sucrose and NaNO(3) for maximum production of synnemata or sclerotia were 3 and 0.9%, respectively. The production of synnemata and stipitate/sessile sclerotia by several wild-type strains of A. caelatus further substantiates previous suggestions for an evolutionary link between Aspergillus section Flavi and synnematal species A. togoensis, which also produces stipitate sclerotia.     

Sample size, library composition, and genotypic diversity among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution

A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from E. coli strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep. HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting. Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification. However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification. The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm. Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in E. coli was very great and is most likely accounting for our inability to correctly classify many environmental E. coli isolates. Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods.     

Primer R108 performs best in the RAPD strain typing of threeAspergillus species frequently isolated from patients

We evaluated the suitability of various primers for the RAPD (random amplified polymorphic DNA) accurate species identification and strain typing of Aspergillus clinical isolates. Five primers described previously were tested for their discriminatory power in three Aspergillus species (A. fumigatus, A. niger agg. and A. flavus - 23 clinical isolates and 2 reference strains). Clustering of RAPD fingerprints corresponded well with the identification based on morphological features. All isolates were resolved as different strains using the primer R108 and the RAPD protocol optimized for a Robocycler thermal cycler. RAPD with the primer R108 thus can be considered to be a valuable, simple and powerful tool for identification and strain delineation of Aspergillus spp.