Infectoria Species-group Research Articles

Phenotypic and phylogenetic segregation of Alternaria infectoria from small-spored Alternaria species isolated from wheat in Germany and Russia

To identify the taxonomic differences between phytopathogenic small-spored Alternaria strains isolated from wheat kernels in Germany and Russia by a polyphasic approach. Ninety-five Alternaria (A.) strains were characterized by their colony colour, their three-dimensional sporulation patterns, mycotoxin production and phylogenetic relationships based on sequence variation in translation elongation factor 1-α (TEF1-α). The examination of toxin profiles and the phylogenetic features via TEF1-α resulted in two distinct clusters, in each case containing Alternaria infectoria isolates (92 and 96% respectively) in the first and the Alternaria alternata, Alternaria arborescens and Alternaria tenuissima isolates (77 and 79% respectively) in the other combined cluster. The production of Alternariol, Altertoxin and Altenuene has not been reported previously in the A.infectoria species group. The isolates from Germany and Russia differ slightly in species composition and mycotoxin production capacity. We identified that the A.infectoria species group can be differentiated from the A.alternata, A.arborescens and A.tenuissima species group by colour, low mycotoxin production and by the sequence variation in TEF1-α gene. These results allow a reliable toxic risk assessment when detecting different Alternaria fungi on cereals.

Characterization of Alternaria isolates from the infectoria species-group and a new taxon from Arrhenatherum, Pseudoalternaria arrhenatheria sp. nov.

The infectoria species-group within the genus Alternaria was originally conceived by Simmons in 1993 and was based upon common morphological characteristics that included the development of conidial chains with primary, secondary, and tertiary branching resulting in substantial three-dimensional complexity. These characters can overlap to varying degrees with numerous taxa in another Alternaria group, the alternata species-group, making species-group differentiation difficult. However, members of the infectoria species-group are also distinguished from other small-spored Alternaria species based upon colony characteristics that typically include white or nearly white floccose colonies on DRYES medium and clumps of sporulation islands on low sugar media such as V8 agar, PCA, and weak PDA. In addition, the infectoria species-group contains representatives that are known to produce teleomorphs (Lewia), whereas the members of the alternata species-group and other Alternaria species-groups are strictly asexual. In this study, an assemblage of isolates recovered from varied hosts from the west coast of the United States were examined based upon morphological characters and compared to previously described members of the infectoria species-group. These isolates and members of the infectoria species-group typically produce arachnoid vegetative hyphae with multiple primary conidiophores, whereas other small-spored Alternaria species produce primary conidiophores predominately directly from the agar surface. Additionally, molecular phylogenetic analyses resolved these isolates and members of the infectoria species-group as distinctly nested amongst other sexual taxa in Allewia (Embellisia anamorph) and Macrospora (Nimbya anamorph) and phylogenetically distant to asexual lineages of Alternaria. One taxon among these isolates was novel and clustered with the asexual A. rosae in a distinct clade basal to all other members of the infectoria species-group. A new genus is proposed, Pseudoalternaria gen. nov. and a new taxon is described, Pseudoalternaria arrhenatheria sp. nov.. Moreover, a second taxon is reclassified, Pseudoalternaria rosae comb. nov.

The sections of Alternaria: formalizing species-group concepts

The systematics of Alternaria and allied genera traditionally has been based on the characteristics of conidia and the sporulation apparatus. This emphasis on morphology in the reconstruction of organismal relationships has resulted in taxonomic uncertainty and flux for a number of taxa in Alternaria and the related genera Stemphylium, Embellisia, Nimbya and Ulocladium. The present study used a molecular phylogenetic approach for systematic resolution and incorporated extensive taxon sampling (n = 176 species) representing 10 genera and analyses of 10 protein-coding loci. Phylogenetic analyses based on five of these genes revealed eight distinct asexual lineages of Alternaria that cluster as the sister group to the asexual paraphyletic genus Ulocladium, while taxa with known teleomorphs currently circumscribed as Alternaria (the infectoria species-group) cluster among genera that also have representatives with known teleomorphs. This work proposes to elevate the eight well supported asexual lineages of Alternaria to the taxonomic rank of section. Evolutionary relationships among Alternaria and closely related genera are discussed.

PCR detection and identification of Alternaria species-groups in processed foods based on the genetic marker Alt a 1

In this study, a PCR method based on oligonucleotide primers targeting the Alt a 1 gene has been developed for the rapid detection of DNA from Alternaria spp. and for identification of Alternaria alternata, Alternaria porri, Alternaria radicina, and Alternaria infectoria species-groups in raw materials and processed food products. The assay design consists of two steps. First, a duplex PCR using primers Dir5cAlta1–Inv4Alta1 (that amplifies a specific DNA fragment of approximately 195 bp in all Alternaria spp.) and 18Sfweu–18Srveu (that amplifies a conserved 99 bp fragment on all the eukaryotic species), allows detection of Alternaria spp. DNA in foodstuffs with a high sensitivity and specificity. As a second step, identification of Alternaria species-groups is obtained through a seminested PCR method, without the need for sequencing PCR products. The specificity of the primer pairs designed was verified by PCR analysis of DNA from various Alternaria cultures, and also from several non target species. The detection limit of the method was approximately 10 2 CFU/ml, either in viable culture, heat inactivated culture or inoculated tomato pulp. Nevertheless, a sensitivity of 10 3 CFU/ml was obtained for tomato pulp inoculated with A. alternata or A. porri cultures heat inactivated at 90 °C for 5 min. PCR analysis of commercial foodstuff samples demonstrated the presence of DNA from A. alternata species-group in 100% of spoiled tomato samples, and 8% of tomato products, whilst 36.4% of cereal based infant food samples analyzed contained DNA from A. infectoria species-group.

A polyphasic approach to the taxonomy of the Alternaria infectoria species–group

Different taxa in the species–group of Alternaria infectoria (teleomorph Lewia spp .) are often isolated from various cereals including barley, maize and wheat grain, ornamental plants and skin lesions from animals and humans. In the present study we made a polyphasic characterization of 39 strains morphologically identifiable as belonging to the A. infectoria species–group together with 12 strains belonging to closely related species: Alternaria malorum (syn. Cladosporium malorum), Chalastospora cetera (syn. Alternaria cetera) and Embellisia abundans. Morphological examination separated the 51 strains in three groups based on conidial appearance and arrangement: the A. infectoria species–group, E. abundans and a group containing C. cetera and A. malorum. The metabolite analyses on three different media showed two clusters, one containing all 39 A. infectoria species–group strains and one containing 10 strains of E. abundans, C. cetera and A. malorum. One E. abundans strain and one A. malorum strain were not included due to insufficient metabolite production. The separation of the A. infectoria species–group from E. abundans, C. cetera and A. malorum resulted mainly from the ability to produce altertoxins and novae-zelandins. The metabolite analyses also showed that all 51 strains were able to produce infectopyrones. The metabolite profiles of C. cetera and A. malorum were very similar with several metabolites of unknown structure in common. This is the first time that E. abundans, C. cetera and A. malorum have been reported as producers of infectopyrones. Sequence analyses of the internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase ( gpd) and translocation elongation factor 1α ( tef-1α) showed two clades: one with the 39 strains from the A. infectoria species–group and one with the 12 strains of E. abundans, C. cetera and A. malorum. The polyphasic approach in this study suggests that A. malorum var. polymorpha and the eight A. malorum strains do not belong in Alternaria, but in Chalastospora, however, as several distinct species. Splits Tree alignment of gpd sequences of 38 strains belonging to the A. infectoria species–group indicates that only three strains showed signs of recombination, while the remaining strains appeared to be clonal. Long term incubation at 7 °C in the dark showed that 12 out of 33 tested strains from the A. infectoria species–group were able to produce proascomata in axenic culture, but with no mature ascospores after 6 months. These findings suggest that Lewia/ A. infectoria species–group must, at least in part, be homothallic. The results presented in this study show that ITS, tef-1α and gpd do not reflect ecology, secondary metabolism or morphology of the A. infectoria species–group and that molecular cladification and phylogeny cannot predict pathogenicity, host specificity or mycotoxin production.

Alternaria infectoria species‐group associated with black point of wheat in Argentina

Regional surveys are being conducted in Argentina to assess the presence of wheat (Triticum aestivum) pathogens on grains across the main cropping area. During 2001 and 2002, grain samples with a dark brown or blackish discoloration around the embryo end, known as black point, were observed on several cultivars across the wheat region of Buenos Aires Province. Seed analysis by blotter and agar tests (Neergaard, 1979) showed up to 55% of prevalence (number of samples infected over the total) of Alternaria spp. and a mean natural infection of 37%. Morpho-cultural studies were carried out on potato carrot agar. The isolates were typified as belonging to the A. infectoria species-complex according to their sporulation pattern and the production of long secondary conidiophores (Andersen et al., 2002). Pathogenicity tests were carried out in Petri dishes following two standard methods: blotter test and agar test using 2% potato dextrose agar, inoculating wheat seeds with a conidial suspension (2 × 105 conidia mL−1) of 20 selected isolates. Seeds were incubated for seven days at 20 ± 2°C in a growth chamber under 12 h fluorescent plus near ultraviolet photoperiod. No disease symptoms were observed in the controls. Infected seeds became totally or partially black and seedlings from them appeared weak with reduced vigour. Black point is known to affect grain quality adversely, impairing flour, semolina and their products (Mathur & Cunfer, 1993). Simmons (1992) organized the genus Alternaria into 14 species-groups based on characteristics of conidia and chain formation. The A. infectoria species-group comprises nine known species and an unknown number of distinct taxa yet to be described. In Europe, North America and Australia studies have shown that grain samples are infected with A. alternata and A. infectoria species-groups ranging from low levels to 100%. In Argentina, previous records of Alternaria spp. refer to A. alternata associated with black point in wheat. However, in this study the vast majority of Alternaria strains conformed to the A. infectoria complex. The incidence levels of this group are gaining importance and have increased in recent years probably due to changes in cropping systems in most of the different agroclimatic zones of Argentina. This is the first published report of Alternaria infectoria species-complex causing black point in wheat grains in Argentina. This highlights the necessity to better understand the relationship of this group with the deterioration of wheat sub-products and the risk of harmful mycotoxins production.

Lewia hordeicola sp. nov. from barley grain

Lewia hordeicola with Alternaria anamorph was isolated from barley grains in Norway. The fungus is homothallic. It produces fertile ascomata on synthetic nutrient agar (SNA) after long incubation at 4 C in the dark. On PCA its anamorph resembles members of the A. infectoria species group. On SNA L. hordeicola differs from the latter in the shape and size of ascospores, the conidial sporulation patterns, and the shape, size, septation and roughness of conidia. A key to currently known Lewia species is included.

Lewia hordeicola sp. nov. from barley grain

Lewia hordeicola with Alternaria anamorph was isolated from barley grains in Norway. The fungus is homothallic. It produces fertile ascomata on synthetic nutrient agar (SNA) after long incubation at 4 C in the dark. On PCA its anamorph resembles members of the A. infectoria species group. On SNA L. hordeicola differs from the latter in the shape and size of ascospores, the conidial sporulation patterns, and the shape, size, septation and roughness of conidia. A key to currently known Lewia species is included.

Novel Chemotaxonomic Markers of the Alternaria infectoria Species-Group

Two new structurally related metabolites, novae-zelandin A (1) and novae-zelandin B (2), as well as the novel metabolite 4Z-infectopyrone (3) were purified from extracts of filamentous fungi belonging to the Alternaria infectoria species-group. The structures were elucidated by a combination of 1D and 2D NMR spectroscopic data and MS data. 1-3 are important chemotaxonomic markers of the A. infectoria species-group and exhibit structures similar to those of known biologically active compounds, suggesting that they could be potential phytotoxins.

Cutaneous Phaeohyphomycosis due to Alternaria Infectoria

Here we report a case of cutaneous alternariosis in a 74-year-old man treated by corticotherapy for myasthenia, and presenting with papular, crusted lesions on the left elbow and the right knee. Histological examination of the biopsy specimens showed fungal hyphae associated with round-shaped cells which were highly suggestive of alternariosis. Mycological culture allowed the isolation of a dematiaceous fungus which was identified as a member of the Alternaria infectoria species-group. This was confirmed by PCR amplification and sequencing of the internal transcribed spacer domain of the gene encoding nuclear ribosomal DNA and of the mitochondrial small subunit ribosomal DNA domain. The fungus was therefore referred to the Scientific Institute of Public Health where it was identified as Alternaria infectoria, on the basis of its very small 1 or 2-celled conidia often arranged in long chains and presenting with very long secondary conidiophores. Corticotherapy was stopped and a local antifungal treatment with ketoconazole was initiated, allowing the stabilisation of the cutaneous lesions within 2 months.

Molecular characterization of Embellisia and Nimbya species and their relationship to Alternaria, Ulocladium and Stemphylium

DNA sequences from rDNA and protein-coding regions were determined for six Embellisia and two Nimbya spp. and were compared to those from Alternaria, Ulocladium and Stemphylium spp. Sequences determined included rDNA from the nuclear internal transcribed-spacer region (ITS1/5.8S/ITS2) and the mitochondrial small-subunit (mt SSU) and a portion of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene. Phylogenetic analyses were performed on each dataset separately and then combined for total evidence analysis using methods of maximum parsimony and maximum likelihood. Results revealed that Embellisia and Nimbya clustered within a large monophyletic Alternaria-Nimbya-Embellisia-Ulocladium clade with Stemphylium as the sister taxon. Members of the infectoria species-group were the most basal group in this large polygeneric clade. Embellisia and Nimbya were sister taxa of the remaining Alternaria and Ulocladium spp. and were related more closely to Alternaria than was Stemphylium. Four Embellisia spp. formed a monophyletic clade. However, E. allii clustered with the two Nimbya spp. and E. indefessa clustered with Alternaria and Ulocladium spp., revealing that Embellisia, as currently circumscribed, is polyphyletic. Potential revisions of taxonomy for all genera are discussed.

Infectopyrone, a Potential Mycotoxin from Alternaria infectoria.

A new metabolite, infectopyrone (1), has been isolated from the filamentous fungus Alternaria infectoria. The structure of 1 was elucidated by analysis of 2D NMR spectroscopic data. Compound 1 is an α-pyrone resembling known toxins, and is a useful phenotaxonomic marker for the A. infectoria species-group. Infectopyrone (1) was also produced by species within Stemphyllium and Ulocladium, and found in mouldy food.

Infectopyrone, a potential mycotoxin from Alternaria infectoria

A new metabolite, infectopyrone ( 1 ), has been isolated from the filamentous fungus Alternaria infectoria. The structure was elucidated by analysis of 2D NMR spectroscopic data. Infectopyrone is an α-pyrone resembling known toxins, and is a useful phenotaxonomic marker for the A. infectoria species-group. Infectopyrone was also produced by species within Stemphyllium and Ulocladium, and found in mouldy food.

Characterisation of Alternaria species-groups associated with core rot of apples in South Africa

Alternaria core rot of red apple cultivars is a serious post-harvest disease in South Africa. Thirty isolates of Alternaria spp. previously isolated from apple, together with reference isolates of A. alternata and A. infectoria, were characterised and grouped according to their sporulation patterns and conidial morphology. Isolates were identified as belonging to A. arborescens, A. infectoria and A. tenuissima species-groups. The isolates were also analysed for production of mycotoxins and other secondary metabolites and their cultural characteristics on DRYES medium were recorded. Apple fruit were artificially inoculated with the thirty-two Alternaria isolates and the resulting lesion types were recorded. A data matrix was constructed using all these characters and subjected to cluster analysis to show the similarity between different isolates. Isolates classified as A. infectoria species-group based on sporulation patterns, cultural and biochemical data could be easily differentiated from isolates classified as A. arborescens and A. tenuissima species-groups, which clustered close together. Isolates were further subjected to DNA sequence analysis of the internal transcribed spacers (ITS) 1 and 2 of the nuclear rRNA gene. A phylogeny estimated from the ITS data set delineated two clades, one being typified by the A. infectoria species-group, and the other representing both A. tenuissima and A. arborescens species-groups. The ITS data set clearly separated isolates of A. infectoria species-group from the other species-groups, as all isolates had a distinction of 35 base pair insertions and 6 base pair deletions in the ITS regions. The results obtained in the present study showed that the major pathogens associated with core rot disease of Top Red apples in South Africa belong to the A. tenuissima species-group.

Morphological, Pathogenic, and Molecular Characterization of Alternaria Isolates Associated with Alternaria Late Blight of Pistachio

ABSTRACT Alternaria isolates were obtained from various pistachio tissues collected in five orchards in California. For all isolates, morphological characteristics of the colony and sporulation apparatus were determined and compared with those of representative isolates of A. alternata, A. tenuissima, A. arborescens, and A. infectoria. A selection of the pistachio isolates and the representative Alternaria isolates were evaluated for pathogenicity to pistachio. Molecular characteristics of these isolates were determined using random amplified polymorphism DNA (RAPD) analysis, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of nuclear intergenic spacer rDNA, and sequence analysis of nuclear internal transcribed spacer (ITS) rDNA. Based on morphological characteristics, the pistachio isolates were grouped as identical or very similar to either A. alternata, A. tenuissima, A. arborescens, or A. infectoria. Isolates from the alternata, tenuissima, and arborescens species-groups were pathogenic to pistachio and no significant differences in pathogenicity were observed. Isolates from the infectoria species-group were only weakly pathogenic to pistachio. Based on cluster analysis of RAPD and PCR-RFLP data, three distinct clusters were evident; the infectoria cluster, the arborescens cluster, and a combined alternata/tenuissima cluster. Based on analysis of ITS sequence data, the infectoria species-group was phylogenetically distinct from the other species-groups. Isolates of the alternata, tenuissima, and arborescens species-groups comprised a monophyletic clade in which the three species-groups could not be further resolved.

Chemical and morphological segregation of Alternaria arborescens, A. infectoria and A. tenuissima species-groups

Correct morphological identification of Alternaria is important and demands a combination of modern standardised methods and up-to-date literature. The production of secondary metabolites has previously been used as a means of identification and classification. In this study, 153 fungal isolates belonging to the genus Alternaria were examined. They were grown under standardised conditions and subjected to morphological and chemical examination. All isolates were grouped according to their three-dimensional sporulation pattern on potato carrot agar and their colony colour on dichloran rose bengal yeast extract sucrose agar (DRYES). After extraction, all isolates were analysed by a high performance liquid chromatograph equipped with a diode array detector and the resulting metabolite profiles were subjected to multivariate statistic analyses. The analyses of metabolite profiles showed that the isolates could be divided into three major species-groups that were morphologically identifiable as the A. infectoria species-group, the A. arborescens species-group and the A. tenuissima species-group. The A. infectoria species-group is chemically very different from both the A. arborescens and the A. tenuissima species-groups with only a few metabolites in common. None of the 35 A. infectoria species-group isolates produced any known metabolites and all had white or greyish white colonies on DRYES. The A. arborescens species-group and the A. tenuissima species-group, shared most of the known metabolites and had colonies of various shades of green on DRYES. One cluster of isolates belonging to the A. tenuissima species-group was able to produce tentoxin, which has not been reported previously from any A. tenuissima isolate. The results suggest that each species-group contains several taxa and these taxa need to be formally described before species specific metabolite profiles can be established.