Alternaria Section Research Articles

New Species of Large-Spored Alternaria in Section Porri Associated with Compositae Plants in China.

Alternaria is a ubiquitous fungal genus including saprobic, endophytic, and pathogenic species associated with a wide variety of substrates. It has been separated into 29 sections and seven monotypic lineages based on molecular and morphological data. Alternaria sect. Porri is the largest section, containing the majority of large-spored Alternaria species, most of which are important plant pathogens. Since 2015, of the investigations for large-spored Alternaria species in China, 13 species were found associated with Compositae plants based on morphological comparisons and phylogenetic analyses. There were eight known species and five new species (A. anhuiensis sp. nov., A. coreopsidis sp. nov., A. nanningensis sp. nov., A. neimengguensis sp. nov., and A. sulphureus sp. nov.) distributed in the four sections of Helianthiinficientes, Porri, Sonchi, and Teretispora, and one monotypic lineage (A. argyranthemi). The multi-locus sequence analyses encompassing the internal transcribed spacer region of rDNA (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), Alternaria major allergen gene (Alt a 1), translation elongation factor 1-alpha (TEF1), and RNA polymerase second largest subunit (RPB2), revealed that the new species fell into sect. Porri. Morphologically, the new species were illustrated and compared with other relevant large-spored Alternaria species in the study. Furthermore, A. calendulae, A. leucanthemi, and A. tagetica were firstly detected in Brachyactis ciliate, Carthamus tinctorius, and Calendula officinalis in China, respectively.

New molecular markers for distinguishing the main phylogenetic lineages within Alternaria section Alternaria

Several members of Alternaria section Alternaria are economically important plant-pathogenic fungi that cause disease on a wide range of host types and plant tissues. The production of Alternaria-derived mycotoxins can lead to significant post-harvest losses due to contamination of agricultural products. Multiple Alternaria species are listed as regulated organisms, which are monitored by international plant protection programmes. These taxa often share high levels of both morphological and phylogenetic similarity, and the establishment of molecular markers that are able to distinguish among them unambiguously has proven to be quite challenging. Previously, we examined fine-scale genome-wide phylogenetic patterns and proposed a list of candidate genes for development into informative markers that are diagnostic for the main section Alternaria lineages. Here, we design primer sets and sequence three new markers (ASA-05, ASA-10, ASA-19) in order to evaluate their diagnostic performance. Sequence data for 49 new independent test taxa were combined with existing data for 38 taxa, and phylogenetic analyses revealed that, in general, the new molecular markers consistently classified section Alternaria strains into one of the main lineages. A discrepancy among the three markers for lineage assignment was observed only for one strain. We also sequenced a commonly used marker in molecular phylogenetics of fungi (rpb2, RNA polymerase II second largest subunit) and found it was outperformed by all three of the new markers. We suggest that an ASA marker presented here could form the basis of a convenient one-locus test for rapid and routine diagnostic screening of unknown section Alternaria strains.

Distinction of Alternaria Sect. Pseudoalternaria Strains among Other Alternaria Fungi from Cereals.

Species of the genus Alternaria are ubiquitous and frequently isolated from various plants, including crops. There are two phylogenetically and morphologically close Alternaria sections: the relatively well-known Infectoriae and the rarely mentioned Pseudoalternaria. Currently, the latter includes at least seven species that are less studied and sometimes misidentified. To perform precise identification, two primers (APsF and APsR) were designed and a sect. Pseudoalternaria-specific PCR method was developed. Thirty-five Russian A. infectoria-like strains were then examined. Five strains were found to be the members of the sect. Pseudoalternaria. Additionally, specificity of the previously developed primer set (Ain3F and Ain4R) was checked. It was found to be highly specific for sect. Infectoriae and did not amplify sect. Pseudoalternaria DNA. Identification of strains of the sect. Pseudoalternaria was supported and refined by phylogenetic reconstruction based on analysis of two loci, the glyceraldehyde-3-phosphate dehydrogenase gene (gpd), and the plasma membrane ATPase gene (ATP). These fungi belonged to Alternaria kordkuyana and A. rosae, which were the first detection of those taxa for the Eastern Europe. Alternaria kordkuyana was isolated from cereal seeds and eleuthero leaves. Alternaria rosae was obtained from oat seed. All strains of sect. Pseudoalternaria were not able to produce alternariol mycotoxin, as well as the majority of A. sect. Infectoriae strains.

Characterization of New Small-Spored Alternaria Species Isolated from Solanaceae in Algeria.

Although large-spored Alternaria species of the section Porri are considered to be the major agents responsible for leaf spot and blight of Solanaceae, small-spored Alternaria species are also frequently isolated from symptomatic tissues. A survey of the north-western regions of Algeria during the 2017–2018 growing seasons revealed that amongst the 623 Alternaria isolates from tomato, potato, pepper, eggplant and black nightshade, 8% could not be morphologically assigned to either section Porri or section Alternaria. In order to more precisely determine the taxonomic position of these isolates, detailed morphological characterizations and multi-locus phylogenetic analyses were performed. Based on these analyses, the isolates were grouped into four main clades: section Ulocladioides, section Infectoriae, including two new species, section Embellisioides, and section Eureka, including one new species. These isolates were also characterized for their virulence under green-house conditions. They were able to produce leaf spot symptoms on tomato plants but with variable levels.

Contamination of grain in West Siberia by Alternaria fungi and their mycotoxins

The ubiquitous occurrence of Alternaria fungi belonging to sections Alternaria and Infectoriae was confirmed using
 real-time PCR in wheat, barley and oat grain grown in West Siberia in 2018‒2019. The DNA amount of Alternaria section
 Alternaria fungi varied from 53×10-4 to 21731×10-4 pg/ng and on average exceeded the DNA amount of Alternaria section
 Infectoriae fungi by 4.5‒14.6 times, depending on the crop and harvest year.The average DNA amount of Alternaria fungi
 belonging to both sections in the oat grain was lower than in wheat and barley grain. The grain samples from Altay region
 were the most infected with Alternaria fungi. The alternariol (AOH), alternariol monomethyl ether (AME), tentoxin
 (TEN), and tenuazonic acid (TeA) mycotoxins produced by Alternaria fungi were detected by HPLC-MS/MS in 23 %,
 6 %, 85 %, and 83 % of analyzed grain samples, respectively. The majority (61 %) of the samples contained two Alternaria
 mycotoxins in the grain (mainly TEN and TeA), 19 % of the samples three mycotoxins, and only one sample all four
 together. In the most of samples the content of Alternaria mycotoxins did not exceed 100 μg/kg, and only TeA content
 was higher (from 113 to 14963 μg/kg) than others. The significant differences in grain crops by the Alternaria mycotoxins
 content were revealed: more amounts of AOH, AME, and less amount of TEN were found in oat grain then in barley
 grain. A high positive significant correlation between the DNA amount of Alternaria section Alternaria fungi and TeA was
 established that indicates the role of these fungi as the main producers of TeA in the grain.

Phylogenomic analyses of Alternaria section Alternaria: A high-resolution, genome-wide study of lineage sorting and gene tree discordance

ABSTRACT The genus Alternaria contains a diversity of saprobic and pathogenic species that can be found in a wide range of environments. Alternaria is currently divided into 26 subgeneric sections, and the “small-spored” Alternaria section Alternaria includes many species that are economically important agricultural pathogens. Recognizing that a stable framework for systematics and species identification is essential for management and regulation purposes, this section has experienced much taxonomic debate and systematic revision in recent years. Molecular phylogenetic studies have challenged the reliability of using morphological characteristics to differentiate Alternaria species but have also suggested that commonly used molecular markers for fungal phylogenetics may not be sufficiently informative at this taxonomic level. To allow the assessment of molecular variation and evolutionary history at a genome-wide scale, we present an overview and analysis of phylogenomic resources for Alternaria section Alternaria. We review the currently available genomic resources and report five newly sequenced genomes. We then perform multiple comparative genomic analyses, including macrosynteny assessment and inference of phylogenetic relationships using a variety of data sets and analysis methods. Fine-scale, genome-wide phylogenetic reconstruction revealed incomplete lineage sorting and the genomic distribution of gene/species tree discordance. Based on these patterns, we propose a list of candidate genes that may be developed into informative markers that are diagnostic for the main lineages. This overview identifies gaps in knowledge and can guide future genome sequencing efforts for this important group of plant pathogenic fungi.

Natural Occurrence of Alternaria Fungi and Associated Mycotoxins in Small-Grain Cereals from The Urals and West Siberia Regions of Russia.

Alternaria fungi dominate the grain microbiota in many regions of the world; therefore, the detection of species that are able to produce mycotoxins has received much attention. A total of 178 grain samples of wheat, barley and oat obtained from the Urals and West Siberia regions of Russia in 2017–2019 were included in the study. Grain contamination with Alternaria fungi belonging to sections Alternaria and Infectoriae was analysed using qPCR with specific primers. The occurrence of four mycotoxins produced by Alternaria, AOH, AME, TEN, and TeA, was defined by HPLC-MS/MS. Alternaria DNA was found in all analysed grain samples. The prevalence of DNA of Alternaria sect. Alternaria fungi (range 53 × 10−4–21,731 × 10−4 pg/ng) over the DNA of Alternaria sect. Infectoriae (range 11 × 10−4‒4237 × 10−4 pg/ng) in the grain samples was revealed. Sixty-two percent of grain samples were contaminated by at least two Alternaria mycotoxins. The combination of TEN and TeA was found most often. Eight percent of grain samples were contaminated by all four mycotoxins, and only 3% of samples were free from the analysed secondary toxic metabolites. The amounts varied in a range of 2–53 µg/kg for AOH, 3–56 µg/kg for AME, 3–131 µg/kg for TEN and 9–15,000 µg/kg for TeA. To our knowledge, a new global maximum level of natural contamination of wheat grain with TeA was detected. A positive correlation between the amount of DNA from Alternaria sect. Alternaria and TeA was observed. The significant effects of cereal species and geographic origin of samples on the amounts of DNA and mycotoxins of Alternaria spp. in grain were revealed. Barley was the most heavily contaminated with fungi belonging to both sections. The content of AOH in oat grain was, on average, higher than that found in wheat and barley. The content of TEN in the grain of barley was lower than that in wheat and similar to that in oat. The content of TeA did not depend on the cereal crop. The effect of weather conditions (summer temperature and rainfall) on the final fungal and mycotoxin contamination of grain was discussed. The frequent co-occurrence of different Alternaria fungi and their mycotoxins in grain indicates the need for further studies investigating this issue.

Alternaria guilanica sp. nov., a new fungal pathogen causing leaf spot and blight on eggplant in Iran

Eggplant (Solanum melongena L.) is an economically important solanaceous crop in Iran with fruits used for food and traditional medicine. Despite the importance of Alternaria leaf spot and blight disease of solanaceous crops which is commonly seen in the fields, our knowledge about the causal agents on eggplant is limited. In this study, a set of large-spored Alternaria isolates was recovered from eggplant with leaf spot and blight symptoms in Somehsara region, Guilan province, Iran. All recovered isolates shared conspicuous morphological characteristics e.g. production of large, solitary conidia with several transverse disto- and eusepta and long tapering filamentous beak resemble those seen in the members of Alternaria section Porri. Multi-locus phylogenetic analyses based on the internal transcribed spacer region of nrDNA (ITS-rDNA) and parts of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), second largest subunit of RNA Polymerase II (RPB2), translation elongation factor 1-alpha (TEF1-α) and Alternaria major allergen (Alt a 1) gene sequences provided further evidence supporting not only their exact placement in Alternaria sect. Porri, but also in a distinct lineage representing a new species. The new species was named, described and illustrated herein as Alternaria guilanica sp. nov.. The phylogenetic and morphological comaprisions of the new species with other closely related species were also provided. Pathogenicity test conducted for the new strains revealed that they were capable to induce disease symptoms on eggplant leaves under greenhouse conditions, and re-isolation of the inoculated isolates confirmed Koch’s postulates.

Morphological and Molecular Characterization of Alternaria spp. Isolated from European Pears.

In a recent survey of post-harvest rot pathogens in European pear in Southern Oregon, Alternaria spp. were frequently isolated from orchard samples of pear flowers and fruits. Morphological differences were observed within the isolated cultures. A preliminary NCBI BLAST search analysis using sequences of the ATPase locus across 94 isolates of Alternaria spp. obtained from pear fruit rots revealed three major Alternaria sections: sect. Alternata, sect. Infectoriae, and sect. Ulocladioides. Thirteen isolates were selected based on their genetic and morphological diversity across three Alternaria sections and were subjected to multilocus phylogenetic analysis using sequences from plasma membrane ATPase, calmodulin, and Alt a1 loci. Within sect. Alternata, four Alternaria arborescens isolates and one A. destruens isolate were identified; within sections Infectoriae and Ulocladioides, one A. rosae isolate and two A. botrytis isolates were identified, respectively. The remaining five isolates could not be identified based on the available sequences for the three loci used in this study. In addition to the phylogenetic analysis, pathogenicity assays revealed differential responses to these isolates on four pear cultivars: Anjou, Bartlett, Comice, and Bosc. Inoculation of isolates within Alternaria sect. Alternata resulted in fruit lesions across all cultivars, with Bosc pear being significantly susceptible (P < 0.0001). Isolates within Alternaria sect. Ulocladioides caused rots on Anjou and Bosc pears, while isolates within Alternaria sect. Infectoriae developed rots on Bosc pear only. This study suggests that there is differential susceptibility of pear cultivars to Alternaria rots, and the severity of post-harvest rot depends on the type of Alternaria spp. and cultivar predominant in a region.

First Report of Moldy Core of Sweet Tango Apples from New Zealand Caused by Alternaria arborescens.

First Report of Moldy Core of Sweet Tango Apples from New Zealand Caused by Alternaria arborescens.

Alternaria in malting barley: Characterization and distribution in relation with climatic conditions and barley cultivars

Alternaria is one of the main fungal genera affecting the quality of barley grains. In this study, a polyphasic approach was carried out to characterise the Alternaria population infecting different cultivars of barley grains from the major producing regions of Argentina in the 2014 and 2015 seasons. Its relationship with Fusarium and correlations between predominant species, barley cultivars, and climatic conditions in the growing regions were evaluated.Alternaria incidence exceeded that of Fusarium in all the barley samples and was higher in the drier season (21% in 2014 and 42% in 2015 vs. 6% and 4%, respectively). The main Alternaria species-groups identified were present in both growing seasons in similar frequencies (A. tenuissima sp.-grp., 83.4% in 2014 and 81.7% in 2015; A. infectoria sp.-grp., 11.7% in 2014 and 11.3% in 2015). The dominant Alternaria species-group isolated and identified based on morphological characteristics, DNA sequencing, and metabolite profile was A. tenuissima (72.9%), followed by A. infectoria (14.6%). An association between their frequency and field temperature was observed; A. tenuissima sp.-grp. was more frequent in northern localities, where higher temperatures were registered, while the opposite was observed for A. infectoria sp.-grp. A smaller percentage of A. arborescens sp.-grp. (5%), A. alternata sp.-grp. (3.9%) and A. vaccinii (1.4%) were also identified.Both secondary metabolite profiles and phylogenetic analysis were useful to distinguish isolates from Alternaria section Alternaria and section Infectoriae. Regarding metabolite profiles, alternariol was the most frequent compound produced by isolates of the section Alternaria. Infectopyrones and novae-zelandins were produced by most of the isolates from section Infectoriae.The barley cultivars analysed in this study did not show a particular susceptibility regarding the Alternaria population composition, except for Andreia, which presented the highest frequency of contamination with A. tenuissima sp.-grp. The rest of the cultivars, when grown in different regions, showed different proportion of the Alternaria sp.-grps., suggesting that other factors were determinant in their distribution.The results obtained in the present study will be a valuable tool for health authorities to assess the need for regulations on Alternaria mycotoxins, given the high incidence of Alternaria spp. in barley and the diversity of metabolites that might contaminate the grains.

Novel species of Alternaria section Nimbya from Iran as revealed by morphological and molecular data

ABSTRACT In this study, the diversity of Alternaria species in section Nimbya associated with symptomatic plants in the Cyperaceae and Juncaceae families was assessed. Multilocus sequence analyses of the rDNA internal transcribed spacer (ITS) region and parts of Alternaria major allergen (Alt a 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), second largest subunit of RNA polymerase II (RPB2), and translation elongation factor 1-alpha (TEF1) genes revealed the presence of two previously known species, A. scirpivora and A. caricicola, and three new species, which are described here as A. cypericola, sp. nov., A. heyranica, sp. nov., and A. junci-acuti, sp. nov. These new species were characterized morphologically with respect to the dimensions of conidia, the number of pseudosepta in mature conidia, and the type of conidium apical beak. According to the results of phylogenetic analyses, the presence of long, filiform true beak is not a reliable morphological indicator for grouping species in sections Alternantherae and Nimbya and phylogenetic species recognition should be used. All identified species were described, illustrated, and their morphology and phylogenetic relationships with other species in Alternaria section Nimbya were discussed.

Mycotoxin Profile and Phylogeny of Pathogenic Alternaria Species Isolated from Symptomatic Tomato Plants in Lebanon.

The tomato is one of the most consumed agri-food products in Lebanon. Several fungal pathogens, including Alternaria species, can infect tomato plants during the whole growing cycle. Alternaria infections cause severe production and economic losses in field and during storage. In addition, Alternaria species represent a serious toxicological risk since they are able to produce a wide range of mycotoxins, associated with different toxic activities on human and animal health. Several Alternaria species were detected on tomatoes, among which the most important are A. solani, A. alternata, and A. arborescens. A set of 49 Alternaria strains isolated from leaves and stems of diseased tomato plants were characterised by using a polyphasic approach. All strains were included in the recently defined phylogenetic Alternaria section and grouped in three well-separated sub-clades, namely A. alternata (24 out of 49), A. arborescens (12 out of 49), and A. mali morpho-species (12 out of 49). One strain showed high genetic similarity with an A. limoniasperae reference strain. Chemical analyses showed that most of the Alternaria strains, cultured on rice, were able to produce alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT) and tenuazonic acid (TA), with values up to 5634, 16,006, 5156, and 4507 mg kg−1, respectively. In addition, 66% of the strains were able to co-produce simultaneously the four mycotoxins investigated. The pathogenicity test carried out on 10 Alternaria strains, representative of phylogenetic sub-clades, revealed that they were all pathogenic on tomato fruits. No significant difference among strains was observed, although A. alternata and A. arborescens strains were slightly more aggressive than A. mali morpho-species strains. This paper reports new insights on mycotoxin profiles, genetic variability, and pathogenicity of Alternaria species on tomatoes.

Rethinking of the Roles of Endophyte Symbiosis and Mycotoxin in Oxytropis Plants.

Plants in the Oxytropis genus can live with the endophytic fungi Alternaria sect. Undifilum. Swainsonine, the mycotoxin produced by the endophyte render the host plant toxic and this has been detrimental to grazing livestock in both China and U.S.A. Despite previous efforts, many questions remain to be solved, such as the transmission mode and life cycle of host–endophyte symbiont, the biosynthesis pathway of swainsonine, and in particular the ecological role and evolution of such symbiosis. In this review, we compile the literature to synthesize ideas on the diversity of the symbiosis and propagation of the endophyte. We further compare the previous work from both Alternaria sect. Undifilum and other swainsonine producing fungi to orchestrate a more comprehensive biosynthesis pathway of swainsonine. We also connect swainsonine biosynthesis pathway with that of its precursor, lysine, and link this to a potential role in modulating plant stress response. Based on this we hypothesize that this host–endophyte co-evolution originated from the needs for host plant to adapt for stress. Validation of this hypothesis will depend on future research on endophytic symbiosis in Oxytropis and help in better understanding the roles of plant–endophyte symbiosis in non-Poaceae species.

Draft Genome Sequence of Cumin Blight Pathogen Alternaria burnsii.

The fungal genus Alternaria consists of highly diverse species. They can be isolated readily from soil, water, and many plants, and even from animals and humans. Alternaria burnsii is a small-spored species of section Alternaria. It has been reported as a pathogen, an endophyte, and a saprophyte, and can also be found in indoor air. It causes cumin blight, a destructive disease on cumin (Cuminum cyminum), and also causes other serious diseases, such as pumpkin seed rot, date palm leaf spot, wheat leaf spot, and gray spot of Notopterygium incisum. In this study, we sequenced and assembled the first genome of A. burnsii isolate CBS 107.38. The draft genome can be used as a reference for the further study of related pathogens and comparative genomics of Alternaria species.

A Pilot Study on Baseline Fungi and Moisture Indicator Fungi in Danish Homes.

In many complaint cases regarding bad indoor environments, there is no evidence of visible fungal growth. To determine if the problems are fungi-related, dust sampling is the method of choice among building surveyors. However, there is a need to differentiate between species belonging to a normal, dry indoor environment and species belonging to a damp building envelope. The purposes of this pilot study were to examine which fungal species are present in problem-free Danish homes and to evaluate different detection and identification methods. Analyses showed that the fungal diversity outside was different from the diversity inside and that the composition of fungal species growing indoors was different compared to those found as spores, both indoors and outdoors. Common for most homes were Pseudopithomyces chartarum, Cladosporium allicinum and Alternaria sect. Infectoriae together with Botrytis spp., Penicillium digitatum and Pen. glabrum. The results show that ITS sequencing of dust samples is adequate if supported by thorough building inspections and that food products play as large a role in the composition of the baseline spora as the outdoor air and surrounding vegetation. This pilot study provides a list of baseline fungal species found in Danish homes with a good indoor environment.

Morphological and Molecular Characterization of Alternaria sect. Ulocladioides/Ulocladium Isolated from Citrus Fruits in Upper Egypt

Citrus is the most important crop in Upper Egypt. 150 infected samples were collected from citrus samples (Navel orange, Tangerine and Lemon) in Upper Egypt, 50 samples from each fruit. Twenty-two isolates representing three species of Alternaria belong to A. sect. Ulocladioides and A. sect. Ulocladium were isolated on dichloran chloramphenicol- peptone agar (DCPA) medium at 27°C. Tangerine samples were more contaminated with Alternaria followed by Navel orange and no Alternaria species appeared from Lemon samples. Based on the Alt a1 the phylogenetic analysis identified the isolates as Alternaria atra, Alternaria botrytis and Alternaria oudemansii. The pathogenicity of the isolates was tested by inoculation of healthy navel orange, the resulted data showed that all tested isolates were pathogenic to healthy navel orange with different degrees ranged from 31.5±1 - 20±1 mm and A. oudemansii had a low virulent effect. The mycotoxins ability of tested isolates indicated that about 83% of the isolates were TeA toxin producers with amount ranged from 1.54 - 18.47 ug/ml.

First Report of Leaf Spot Caused by Alternaria alternata on Phlox maculata in Italy

Phlox maculata (common name spotted phlox), Polemoniaceae family, is an herbaceous perennial plant with showy panicle inflorescences, commonly grown in mixed borders. During summer 2019, about twenty 5-year-old plants of P. maculata growing in a garden located in the Biella province (northern Italy) showed symptoms of a leaf spot, starting from the basal part of the foliage. Symptoms consisted in brown spots with not well-defined margins, irregularly circular to polygonal in shape, up to 2 to 3 mm in diameter, sometimes confluent. The internal part of spots dried. Chlorosis and yellowing surrounded the spots, involving the most part of affected leaf surfaces, due to the spread of the disease. The ornamental value of affected plants was significantly reduced. Several symptomatic leaves were washed in sterile water for 1 min. Isolations were carried out, taking several small fragments of tissues from the margins of the spots and placing them on potato dextrose agar. Fungal colonies that developed were dark green at the end of development. Successively, single-spore colonies were obtained, and pure cultures were transferred on potato carrot agar (PCA, Simmons 2007) under a light/dark regime of 11 h/13 h. On this medium, colonies formed branched conidiophores with brown, multicellular conidia, ovoid to obclavate in shape, containing one to four (average 2.4, n = 50) transverse septa and zero to one (average 0.1, n = 50) longitudinal septa. Conidia measured 10.8 to 29.3 × 5.3 to 11.2 µm (average 18.7 × 7.6 µm, n = 50). If present, the beak was 2.1 to 11.0 µm (average 5.5 µm) long. These morphological characteristics permitted identifying the fungus isolated from P. maculata as Alternaria section alternata (Woudenberg et al. 2015). The fungal DNA from the 19/33 isolate was extracted using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany), and PCR was performed using the ITS (White et al. 1990), the rpb2, and the endoPG primers (Woudenberg et al. 2015). Three sequences with 529 bp (ITS), 889 bp (rpb2), and 452 bp (endoPG) (GenBank accession nos. MN871825, MN871979, and MN871978, respectively) were produced. A BLASTn analysis of these sequences exhibited 100% identity with the reference strain CBS 117.44 of A. alternata (Fries) Keissler in rpb2 and endoPG portions (accession nos. KP124772 and KP124001, respectively) and 99.6% identity in ITS region (accession no. KP124303). One P. maculata fungal isolate (19/33) was grown on PCA. A spore suspension obtained from 15-day-old cultures was sprayed at the concentration of 10⁵ CFU/ml onto leaves of three healthy plants of P. maculata. Three control plants were treated with sterile water. All plants were closed in moist plastic bags for 7 days and maintained at temperatures ranging from 15 to 28°C. The first symptoms of leaf spot appeared 10 days after the inoculation, and A. alternata was reisolated from affected leaves. Control plants remained symptomless. The pathogenicity test was repeated once using the isolate 19/40, obtaining the same results. To our knowledge, this is the first report of A. alternata on P. maculata in Italy as well as worldwide. The spread of A. alternata on P. maculata and the difficulty to manage this disease, particularly in public gardens, may discourage the use of this ornamental in Italy.

Molecular Characterization of a Fungal Ketide Synthase Gene Among Swainsonine-Producing Alternaria Species in the USA

Locoweeds are toxic leguminous plants in Astragalus and Oxytropis genera that contain fungal endophytes of Alternaria section Undifilum species. These fungi produce swainsonine, an alkaloid α-mannosidase inhibitor that causes a neurological syndrome, locoism in grazing animals. A SWN gene cluster has been identified in many swainsonine-producing fungi. The swnK gene, which is an essential component of the swainsonine biosynthetic pathway, encodes a polyketide synthase-nonribosomal peptide synthase (PKS-NRPS). To determine if swnK was conserved among Alternaria section Undifilum endophytes of locoweed, the sequence of the KS region of swnK was compared between various swainsonine-producing fungi. The internal transcribed spacer (ITS), andglyceraldehyde-3-phosphate dehydrogenase (GPD)regions from the same fungi were also assessed. Sequences were examined at the nucleotide and protein levels. Alternaria oxytropis, A. fulva, A. cinerea, and Alternaria sp. from Swainsona species produced distinct clades for all multigene data sets. swnK-KS sequence did not differ among fungi isolated from Astragalus mollissimus varieties or A. lentiginosus varieties. The swnK-KS amino acid sequence was essentially identical among all swainsonine-producing Alternaria sp. Two low swainsonine-producing fungi, Alternaria bornmuelleri and A. gansuense, clustered together, as did non-pathogen Alternaria endophytes. The swnK-KS sequence comparisons were effective in identifying swainsonine production capability and differentiating among swainsonine-producing fungal species.

Molecular Identification and Mycotoxin Production by Alternaria Species Occurring on Durum Wheat, Showing Black Point Symptoms.

Black point is a fungal disease of wheat, mainly associated with mycotoxigenic Alternaria species. Affected wheat kernels are characterized by dark brown discolouration of the embryo region and reduction of grain quality. Potential risk is the possible accumulation of Alternaria mycotoxins, alternariol (AOH), alternariol-monomethyl ether (AME), tenuazonic acid (TA), and altenuene (ALT), provided by haemato-toxic, genotoxic, and mutagenic activities. One hundred and twenty durum wheat samples belonging to 30 different genotypes grown in Bologna and Modena areas, in Italy, showing black point symptoms, were analyzed for Alternaria species and their mycotoxin contamination. Alternariol was selected as an indicator of the capability of the Alternaria species to produce mycotoxin in vivo in field conditions. The data showed that Alternaria species occurred in 118 out of 120 wheat kernels samples, with the incidence of infected kernels ranging between 1% and 26%. Moreover, AOH was detected by using a HPLC with a diode array detector (LC-DAD) in 98 out of 120 samples with values ranging between 24 and 262 µg Kg−1. Ninety-two Alternaria representative strains, previously identified morphologically, were identified at species/section level using gene sequencing, and therefore were analyzed for their mycotoxin profiles. Eighty-four strains, phylogenetically grouped in the Alternaria section, produced AOH, AME, and TA with values up to 8064, 14,341, and 3683 µg g−1, respectively, analyzed by using a LC-DAD. On the other hand, eight Alternaria strains, included in Infectoriae Section, showed a very low or no capability to produce mycotoxins.