First report of Alternaria cinerariae causing leaf blight on Farfugium japonicum in South Carolina, USA.

Farfugium japonicum, commonly known as leopard plant, is a popular perennial used in landscapes in the Southeastern U.S. In March 2022, leaf blight was observed on 20 leopard plants at a landscape site in Georgetown Co., SC. Almost all leaves were infected. Symptoms included purple to brown necrotic leaf spots and blighted petioles. Large spots had concentric circles and coalesced causing entire leaves to blight. Leaf pieces surrounding necrotic spots were excised, sterilized in 10% bleach for 1 min, rinsed in sterile water, placed onto potato dextrose agar (PDA), and incubated at 25°C. A total of three Alternaria isolates, 22-094-A, 22-094-B, and 22-094-C were obtained by transferring hyphal tips to new plates. All isolates had identical morphological traits. Colonies on PDA were blackish at the center and brownish at the edge. Conidia were produced using a technique described by Shahin and Shepard (1979). Conidiophores were mostly short and unbranched. They were characterized by solitary conidia or short chains of two to three conidia. Conidia (n=30) were obpyriform to obclavate and averaged 88.5 ± 26.1 µm in body length, 118.4 ± 36.3 µm in total length, and 23.9 ± 5.9 µm in width. They had 3 to 7 transverse septa and 0 to 4 longitudinal septa. Beaks were broadly tapered. Sequence of the internal transcript spacer (ITS) region of isolate 22-094-A (GenBank Accession No. OP481973) had 100% homology to that of CBS 116495 (KC584190), a representative strain of A. cinerariae (Woudenberg et al. 2013). Based on the morphological and sequence characters, the casual fungus was identified as A. cinerariae. Pathogenicity confirmation was done in two separate assays. In a detached-leaf assay, mature leaves were collected from 5-year-old F. japonicum 'Gigantea' plants. Five leaves (abaxial surface) were sprayed with a mixture of conidial suspensions of the three isolates at 300 conidia per mL and 1.5 mL per leaf, while sterile water was used for a non-inoculated control leaf. Leaves were placed in a plastic tray with wet paper towels. The tray was placed at 22°C for an 8-h photoperiod and covered for 3 days to maintain moisture. Small purple to brown spots were visible on inoculated leaves 2 days after inoculation (DAI). More than 90% of inoculated leaf areas were blighted 10 DAI, whereas the control leaf remained asymptomatic. In a whole-plant assay, three F. japonicum 'Argenteo Marginata' plants grown in 10-inch pots were placed in a plastic tray and sprayed with a conidial suspension of 22-094-A onto both abaxial and adaxial surfaces at 300 conidia per mL and 40 mL per plant. The tray was maintained as described above. Sterile water was used for a non-inoculated control plant. Small leaf spots appeared on the inoculated plants 2 DAI. Large necrotic areas developed on leaves and girdled petioles causing aboveground tissues to collapse 4 DAI. All inoculated leaves were blighted 7 to 10 DAI, while the non-inoculated control plant remained healthy. Each assay was repeated once. Alternaria cinerariae, identified by distinct morphology traits (Nishikawa and Nakashima 2015), was consistently re-isolated from inoculated leaves in both assays. Leaf spot on F. japonicum caused by A. cinerariae has been reported in CA, USA (Woudenberg et al. 2013) and Japan (Sakoda et al. 2010). This is the first report in SC, USA. This fungus also infects at least 25 other hosts (Farr and Rossman 2022). This disease may pose a threat to leopard plants in nurseries and landscapes under conducive conditions. Disease management strategies are warranted.

Genome-Wide Analysis of Cytochrome P450s of Alternaria Species: Evolutionary Origin, Family Expansion and Putative Functions.

Cytochrome P450s are a group of monooxygenase enzymes involved in primary, secondary and xenobiotic metabolisms. They have a wide application in the agriculture sector where they could serve as a target for herbicides or fungicides, while they could function in the pharmaceutical industry as drugs or drugs structures or for bioconversions. Alternaria species are among the most commonly encountered fungal genera, with most of them living as saprophytes in different habitats, while others are parasites of plants and animals. This study was conducted to elucidate the diversity and abundance, evolutionary relationships and cellular localization of 372 cytochrome P450 in 13 Alternaria species. The 372 CYP proteins were phylogenetically clustered into ten clades. Forty (40) clans and seventy-one (71) cyp families were identified, of which eleven (11) families were found to appear in one species each. The majority of the CYP proteins were located in the endomembrane system. Polyketide synthase (PKS) gene cluster was the predominant secondary metabolic-related gene cluster in all the Alternaria species studied, except in A. porriof, where non-ribosomal peptide synthetase genes were dominant. This study reveals the expansion of cyps in these fungal genera, evident in the family and clan expansions, which is usually associated with the evolution of fungal characteristics, especially their lifestyle either as parasites or saprophytes, with the ability to metabolize a wide spectrum of substrates. This study can be used to understand the biology, physiology and toxigenic potentials of P450 in these fungal genera.

Alternaria spp. Associated with Leaf Blight of Maize in Heilongjiang Province, China.

Maize (Zea mays L.) is a major economic crop worldwide. Maize can be infected by Alternaria species causing leaf blight that can result in significant economic losses. In this study, 168 Alternaria isolates recovered from symptomatic maize leaves were identified based on morphological characteristics, pathogenicity, and multilocus sequence analyses of the genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the internal transcribed spacer of ribosomal DNA (rDNA ITS), the RNA polymerase II second largest subunit (RPB2), and histone3 (HIS3). Maize isolates grouped to four Alternaria species including Alternaria tenuissima, A. alternata, A. burnsii, and Alternaria sp. Notably, A. tenuissima (71.4%) was the most prevalent of the four isolated species, followed by A. alternata (21.5%), Alternaria sp. (4.1%), and A. burnsii (3.0%). Pathogenicity tests showed that all four Alternaria species could produce elliptic to nearly round, or strip, lesions on leaves of maize, gray-white to dry white in the lesion centers and reddish-brown at the edges. The average disease incidence (58.47%) and average disease index (63.55) of maize leaves inoculated with A. alternata were significantly higher than levels resulting from A. tenuissima (55.28% and 58.49), Alternaria sp. (55.34% and 58.75), and A. burnsii (56% and 55). Haplotype analyses indicated that there were 14 haplotypes of A. tenuissima and five haplotypes of A. alternata in Heilongjiang Province and suggested the occurrence of a population expansion. Results of the study showed that Alternaria species associated with maize leaf blight in Heilongjiang Province are more diverse than those that have been previously reported. This is the first report globally of A. tenuissima, A. burnsii, and an unclassified Alternaria species as causal agents of leaf blight on maize.

Alternaria host-specific (HSTs) toxins: An overview of chemical characterization, target sites, regulation and their toxic effects.

Alternaria causes pathogenic disease on various economically important crops having saprophytic to endophytic lifecycle. Pathogenic fungi of Alternaria species produce many primary and secondary metabolites (SMs). Alternaria species produce more than 70 mycotoxins. Several species of Alternaria produce various phytotoxins that are host-specific (HSTs) and non-host-specific (nHSTs). These toxins have various negative impacts on cell organelles including chloroplast, mitochondria, plasma membrane, nucleus, Golgi bodies, etc. Non-host-specific toxins such as tentoxin (TEN), Alternaric acid, alternariol (AOH), alternariol 9-monomethyl ether (AME), brefeldin A (dehydro-), Alternuene (ALT), Altertoxin-I, Altertoxin-II, Altertoxin-III, zinniol, tenuazonic acid (TeA), curvularin and alterotoxin (ATX) I, II, III are known toxins produced by Alternaria species. In other hand, Alternaria species produce numerous HSTs such as AK-, AF-, ACT-, AM-, AAL- and ACR-toxin, maculosin, destruxin A, B, etc. are host-specific and classified into different family groups. These mycotoxins are low molecular weight secondary metabolites with various chemical structures. All the HSTs have different mode of actions, biochemical reactions, and signaling mechanisms to causes diseases in the host plants. These HSTs have devastating effects on host plant tissues by affecting biochemical and genetic modifications. Host-specific mycotoxins such as AK-toxin, AF-toxin, and AC-toxin have the devastating effect on plants which causes DNA breakage, cytotoxic, apoptotic cell death, interrupting plant physiology by mitochondrial oxidative phosphorylation and affect membrane permeability. This article will elucidate an understanding of the disease mechanism caused by several Alternaria HSTs on host plants and also the pathways of the toxins and how they caused disease in plants.

Chemotaxonomy of Mycotoxigenic Small-Spored Alternaria Fungi - Do Multitoxin Mixtures Act as an Indicator for Species Differentiation?

Necrotrophic as well as saprophytic small-spored Alternaria (A.) species are annually responsible for major losses of agricultural products, such as cereal crops, associated with the contamination of food and feedstuff with potential health-endangering Alternaria toxins. Knowledge of the metabolic capabilities of different species-groups to form mycotoxins is of importance for a reliable risk assessment. 93 Alternaria strains belonging to the four species groups Alternaria tenuissima, A. arborescens, A. alternata, and A. infectoria were isolated from winter wheat kernels harvested from fields in Germany and Russia and incubated under equal conditions. Chemical analysis by means of an HPLC-MS/MS multi-Alternaria-toxin-method showed that 95% of all strains were able to form at least one of the targeted 17 non-host specific Alternaria toxins. Simultaneous production of up to 15 (modified) Alternaria toxins by members of the A. tenuissima, A. arborescens, A. alternata species-groups and up to seven toxins by A. infectoria strains was demonstrated. Overall tenuazonic acid was the most extensively formed mycotoxin followed by alternariol and alternariol mono methylether, whereas altertoxin I was the most frequently detected toxin. Sulfoconjugated modifications of alternariol, alternariol mono methylether, altenuisol and altenuene were frequently determined. Unknown perylene quinone derivatives were additionally detected. Strains of the species-group A. infectoria could be segregated from strains of the other three species-groups due to significantly lower toxin levels and the specific production of infectopyrone. Apart from infectopyrone, alterperylenol was also frequently produced by 95% of the A. infectoria strains. Neither by the concentration nor by the composition of the targeted Alternaria toxins a differentiation between the species-groups A. alternata, A. tenuissima and A. arborescens was possible.

A new high-resolution melting assay for genotyping Alternaria species causing citrus brown spot.

BACKGROUND: Alternaria brown spot is one of the most important diseases of tangerines and their hybrids worldwide. To set up effective control strategy, the accurate detection and identification of the species responsible for the diseases is crucial. However, characterization based on morphology and/or multilocus genetic approaches is time consuming, requires great expertise and sometimes is not conclusive. Therefore, the set-up of a rapid and efficient DNA-based assay might be of paramount importance. High-resolution melting (HRM) analysis represents an interesting tool for the uncovering of nucleotide variations as small as one base difference and, as such, relevant to species characterization. RESULTS: In the present investigation, an HRM assay based on the Alternaria barcoding region OPA1-3 was set up. Specimen strains of the main citrus-associated Alternaria species and morphotypes generated distinct and normalized profiles, allowing their differentiation when HRM-tested. Moreover, when the assay was used to screen an Alternaria collection from citrus fruit and leaves, it distributed the 180 isolates in three independent clusters, readily and consistently resolved. Isolates were identified as belonging to the species Alternaria alternata and the species complex A. arborescens. Within A. alternata, the morphotypes alternata (77% of the collection) and limoniasperae (17% of the collection) were present. CONCLUSIONS: Although further validation experiments will be performed to optimize the assay for a diagnostic use, this HRM approach might represent a rapid, sensitive and specific method for the detection and identification of Alternaria spp. responsible for citrus brown spot disease. © 2018 Society of Chemical Industry.

Alternaria Toxins: Potential Virulence Factors and Genes Related to Pathogenesis.

Alternaria is an important fungus to study due to their different life style from saprophytes to endophytes and a very successful fungal pathogen that causes diseases to a number of economically important crops. Alternaria species have been well-characterized for the production of different host-specific toxins (HSTs) and non-host specific toxins (nHSTs) which depend upon their physiological and morphological stages. The pathogenicity of Alternaria species depends on host susceptibility or resistance as well as quantitative production of HSTs and nHSTs. These toxins are chemically low molecular weight secondary metabolites (SMs). The effects of toxins are mainly on different parts of cells like mitochondria, chloroplast, plasma membrane, Golgi complex, nucleus, etc. Alternaria species produce several nHSTs such as brefeldin A, tenuazonic acid, tentoxin, and zinniol. HSTs that act in very low concentrations affect only certain plant varieties or genotype and play a role in determining the host range of specificity of plant pathogens. The commonly known HSTs are AAL-, AK-, AM-, AF-, ACR-, and ACT-toxins which are named by their host specificity and these toxins are classified into different family groups. The HSTs are differentiated on the basis of bio-statistical and other molecular analyses. All these toxins have different mode of action, biochemical reactions and signaling mechanisms to cause diseases. Different species of Alternaria produced toxins which reveal its biochemical and genetic effects on itself as well as on its host cells tissues. The genes responsible for the production of HSTs are found on the conditionally dispensable chromosomes (CDCs) which have been well characterized. Different bio-statistical methods like basic local alignment search tool (BLAST) data analysis used for the annotation of gene prediction, pathogenicity-related genes may provide surprising knowledge in present and future.

Identification of A. arborescens, A. grandis, and A. protenta as new members of the European Alternaria population on potato.

Alternaria species, primarily the small-spored Alternaria alternata and the large-spored Alternaria solani, are considered a serious threat to potato cultivation. To develop control strategies, it is important to gain insight into the Alternaria population. Based on the sequence analyses of the internal transcribed spacer region (ITS) and the glyceraldehyde-3-phosphate dehydrogenase gene, the small-spored and large-spored Alternaria isolates could be separated from each other. Sequence analyses of the calmodulin gene and the RNA polymerase second largest subunit showed that besides A. solani also A. grandis and A. protenta were present in the large-spored Alternaria population. Sequence analyses of the Alternaria major allergen gene Alt a 1 and the elongation factor-α revealed that both A. alternata and species belonging to the Alternaria arborescens species complex were present in the small-spored Alternaria population. Furthermore, according to the histone h3 sequence the members of the A. arborescens species complex could be subdivided into two groups. Concerning the fitness, it was concluded that the mycelium growth rate of the large-spored isolates was significantly lower compared to the growth rate of the small-spored isolates. In contrast, the spore-germinating capacity and early growth of the large-spored isolates was greater compared to those of the small-spored isolates. Within the groups of small-spored and large-spored isolates there were no significant differences in fitness between the species.

Alternaria Species and Their Associated Mycotoxins.

The genus Alternaria includes more than 250 species. The traditional methods for identification of Alternaria species are based on morphological characteristics of the reproductive structures and sporulation patterns under controlled culture conditions. Cladistics analyses of "housekeeping genes" commonly used for other genera, failed to discriminate among the small-spored Alternaria species. The development of molecular methods achieving a better agreement with morphological differences is still needed. The production of secondary metabolites has also been used as a means of classification and identification. Alternaria spp. can produce a wide variety of toxic metabolites. These metabolites belong principally to three different structural groups: (1) the dibenzopyrone derivatives, alternariol (AOH), alternariol monomethyl ether (AME), and altenuene (ALT); (2) the perylene derivative altertoxins (ATX-I, ATX-II, and ATX II); and (3) the tetramic acid derivative, tenuazonic acid (TeA). TeA, AOH, AME, ALT, and ATX-I are the main. Certain species in the genus Alternaria produce host-specific toxins (HSTs) that contribute to their pathogenicity and virulence. Alternaria species are plant pathogens that cause spoilage of agricultural commodities with consequent mycotoxin accumulation and economic losses. Vegetable foods infected by Alternaria rot could introduce high amounts of these toxins to the human diet. More investigations on the toxic potential of these toxins and their hazard for human consumption are needed to make a reliable risk assessment of dietary exposure.

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

AIMS: To identify the taxonomic differences between phytopathogenic small-spored Alternaria strains isolated from wheat kernels in Germany and Russia by a polyphasic approach. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: These results allow a reliable toxic risk assessment when detecting different Alternaria fungi on cereals.

Alternaria in Food: Ecophysiology, Mycotoxin Production and Toxicology.

Alternaria species are common saprophytes or pathogens of a wide range of plants pre- and post-harvest. This review considers the relative importance of Alternaria species, their ecology, competitiveness, production of mycotoxins and the prevalence of the predominant mycotoxins in different food products. The available toxicity data on these toxins and the potential future impacts of Alternaria species and their toxicity in food products pre- and post-harvest are discussed. The growth of Alternaria species is influenced by interacting abiotic factors, especially water activity (aw), temperature and pH. The boundary conditions which allow growth and toxin production have been identified in relation to different matrices including cereal grain, sorghum, cottonseed, tomato, and soya beans. The competitiveness of Alternaria species is related to their water stress tolerance, hydrolytic enzyme production and ability to produce mycotoxins. The relationship between A. tenuissima and other phyllosphere fungi has been examined and the relative competitiveness determined using both an Index of Dominance (ID) and the Niche Overlap Index (NOI) based on carbon-utilisation patterns. The toxicology of some of the Alternaria mycotoxins have been studied; however, some data are still lacking. The isolation of Alternaria toxins in different food products including processed products is reviewed. The future implications of Alternaria colonization/infection and the role of their mycotoxins in food production chains pre- and post-harvest are discussed.

Alternaria in Food: Ecophysiology, Mycotoxin Production and Toxicology

Alternaria species are common saprophytes or pathogens of a wide range of plants pre- and post-harvest. This review considers the relative importance of Alternaria species, their ecology, competitiveness, production of mycotoxins and the prevalence of the predominant mycotoxins in different food products. The available toxicity data on these toxins and the potential future impacts of Alternaria species and their toxicity in food products pre- and post-harvest are discussed. The growth of Alternaria species is influenced by interacting abiotic factors, especially water activity (a(w)), temperature and pH. The boundary conditions which allow growth and toxin production have been identified in relation to different matrices including cereal grain, sorghum, cottonseed, tomato, and soya beans. The competitiveness of Alternaria species is related to their water stress tolerance, hydrolytic enzyme production and ability to produce mycotoxins. The relationship between A. tenuissima and other phyllosphere fungi has been examined and the relative competitiveness determined using both an Index of Dominance (I(D)) and the Niche Overlap Index (NOI) based on carbon-utilisation patterns. The toxicology of some of the Alternaria mycotoxins have been studied; however, some data are still lacking. The isolation of Alternaria toxins in different food products including processed products is reviewed. The future implications of Alternaria colonization/infection and the role of their mycotoxins in food production chains pre- and post-harvest are discussed.

Antiangiogenetic effects of anthranoids from Alternaria sp., an endophytic fungus in a Thai medicinal plant Erythrina variegata.

Endophytic fungi are known as a prolific source for the discovery of structurally interesting and biologically active secondary metabolites, some of which are promising candidates for drug development. In the present study, three anthranoids were isolated from an Alternaria sp. endophytic fungus and evaluated for their antiangiogenic activity in a rat aortic sprouting assay, an ex vivo model of angiogenesis. Of these three compounds, altersolanol (2) was further characterized and found to show a promising activity in ex vivo, in vitro and in vivo angiogenesis asssays. Using human umbilical vein endothelial cells as an in vitro model, the angiogenic effect of 2 was found to occur via suppression of all three main functions of endothelial cells, namely proliferation, tube formation and migration.

A Case of Cutaneous Alternariosis / 대한의진균학회지

Alternaria species are common plant pathogens and saprophytic fungi. Cutaneous alternariosis is rare and an opportunistic infection in both healthy and immunosuppressed hosts. We report a case of cutaneous alternariosis developed in a 60-year-old female with iatrogenic Cushing syndrome. She had several various sized confluent suppurative ulcers on erythematous plaques on the both forearm clinically. Histology showed mixed-cell granulomatous infiltration with epithelioid cells, lymphocytes, neutrophils and a few plasma cells in the dermis. Many branching septate hyphae and spores were noted within the granulomatous tissue stained with PAS. A fungal culture from biopsy specimen revealed Alternaria species; pigmented hyphae and cornidia which had both transverse and longitudinal septae. The cutaneous lesions treated with terbinafine, 250 mg daily, for 3 months and 1 year later there is no evidence of recurrence.

A Case of Cutaneous Alternariosis / 대한의진균학회지

Alternaria species are common plant pathogens and saprophytic fungi. Cutaneous alternariosis is rare and an opportunistic infection in both healthy and immunosuppressed hosts. We report a case of cutaneous alternariosis developed in a 60-year-old female with iatrogenic Cushing syndrome. She had several various sized confluent suppurative ulcers on erythematous plaques on the both forearm clinically. Histology showed mixed-cell granulomatous infiltration with epithelioid cells, lymphocytes, neutrophils and a few plasma cells in the dermis. Many branching septate hyphae and spores were noted within the granulomatous tissue stained with PAS. A fungal culture from biopsy specimen revealed Alternaria species; pigmented hyphae and cornidia which had both transverse and longitudinal septae. The cutaneous lesions treated with terbinafine, 250 mg daily, for 3 months and 1 year later there is no evidence of recurrence.