Tenuazonic acid production by Alternaria alternata and Alternaria tenuissima isolated from cotton

Cultures of Alternaria alternata (three isolates) and Alternaria tenuissima (three isolates) obtained from cottonseeds and bolls were toxigenic when cultured on various laboratory media. A mycotoxin was isolated and identified as tenuazonic acid by using solvent partition, thin-layer chromatography, and instrument analyses. Toxicity was monitored with brine shrimp and chicken embryo bioassays. All cultures except A. alternata 938 produced tenuazonic acid when grown on cottonseed and on yeast extract-sucrose broth. The most toxin (266 mg/kg) was produced by A. tenuissima 843 on cottonseed.     

Tenuazonic Acid, a Toxin Produced by Alternaria alternata

Fifty-seven of 87 isolates of Alternaria alternata (Fr) Keissler grown on autoclaved, moist corn-rice substrate and fed to rats were lethal. The major toxin produced was isolated and characterized as tenuazonic acid. Twenty of 23 toxigenic Alternaria isolates examined produced tenuazonic acid. No tenuazonic acid could be detected in either of the field samples of sorghum or blackeyed peas, which were heavily invaded by Alternaria.     

Toxins of molds from decaying tomato fruit.

Among 27 mold isolates from decaying tomatoes, culture filtrates or ethyl acetate extracts of 8 isolates grown in yeast extract-sucrose medium were markedly toxic (mortality, greater than 50%) to brine shrimp larvae. The toxicity of six of these isolates could be attributed to the presence of citrinin, tenuazonic acid, or T-2 toxin. Ethyl acetate extracts of five Alternaria isolates and one Fusarium isolate were mutagenic for Salmonella typhimurium strains. In ripe tomatoes inoculated with toxin-producing isolates and incubated at 25 degrees C, one Alternaria alternata isolate produced tenuazonic acid in seven of seven tomatoes at levels of up to 106 micrograms/g and alternariol methyl ether in one of the seven tomatoes at 0.8 microgram/g. Another A. alternata isolate produced tenuazonic acid or alternariol methyl ether at much lower levels in only three of seven tomatoes. Patulin and citrinin were produced by a Penicillium expansum isolate at levels of up to 8.4 and 0.76 microgram/g, respectively. In tomatoes incubated at 15 degrees C, a Fusarium sulphureum isolate produced T-2 toxin, HT-2 toxin, and neosolaniol at levels of up to 37.5, 37.8 and 5.6 micrograms/g, respectively. If these mycotoxins are thermostable, they may occur at detectable levels in tomato products whenever partially moldy tomatoes are used as raw material.     

Toxins of molds from decaying tomato fruit.

Among 27 mold isolates from decaying tomatoes, culture filtrates or ethyl acetate extracts of 8 isolates grown in yeast extract-sucrose medium were markedly toxic (mortality, greater than 50%) to brine shrimp larvae. The toxicity of six of these isolates could be attributed to the presence of citrinin, tenuazonic acid, or T-2 toxin. Ethyl acetate extracts of five Alternaria isolates and one Fusarium isolate were mutagenic for Salmonella typhimurium strains. In ripe tomatoes inoculated with toxin-producing isolates and incubated at 25 degrees C, one Alternaria alternata isolate produced tenuazonic acid in seven of seven tomatoes at levels of up to 106 micrograms/g and alternariol methyl ether in one of the seven tomatoes at 0.8 microgram/g. Another A. alternata isolate produced tenuazonic acid or alternariol methyl ether at much lower levels in only three of seven tomatoes. Patulin and citrinin were produced by a Penicillium expansum isolate at levels of up to 8.4 and 0.76 microgram/g, respectively. In tomatoes incubated at 15 degrees C, a Fusarium sulphureum isolate produced T-2 toxin, HT-2 toxin, and neosolaniol at levels of up to 37.5, 37.8 and 5.6 micrograms/g, respectively. If these mycotoxins are thermostable, they may occur at detectable levels in tomato products whenever partially moldy tomatoes are used as raw material.     

Toxicity of mycotoxins produced by four Alternaria species toArtemia salina larvae

22 isolates ofAlternaria alternata, A raphani, A consortiale, andA chartarum were examined for the production of alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), altertoxin I (ATX I), and tenuazonic acid (TA) on wheat grain and for toxicity of culture extracts toArtemia salina larvae. The total amount of 5 toxins produced under laboratory conditions ranged from 5 mg/kg to 11.112mg/kg. The toxic extracts showed EC50 values in the range of 3.3 to 144.5 mg/mL. There was no correlation between toxicity of extracts toArtemia salina and the amount of mentioned mycotoxins in culture.     

Putative mycotoxin-producing fungi isolated from alpataco (Prosopis flexuosa) fruits

Fungi contaminant of alpataco (Prosopis flexuosa) fruits from La Pampa province (Argentina) were identified. Alternaria alternata and Sphaeropsis sapinea were the dominant species. Phoma sp., Nigrospora sp., Preussia minima, Cladosporium sp., Pithomyces chartarum, Epicoccum nigrum, Aspergillus niger and Aspergillus speluneus were also isolated but with less frequency. Twelve strains of Alternaria alternata, the toxigenic species with higher incidence, were screened for alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) production. Since one isolate was able to produce AME, six isolates produced AOH and AME and two isolates produced AOH, AME and TA, these results indicate a potential risk of contamination with Alternaria toxins in this substrate.     

河北鸭梨病果上链格孢分离系的形态及分子特性研究

A total of 123 Alternaria isolates were obtained from roting fruits of Pyrus bretschneideri "Ya Li" collected in Hebei Province, China. The isolates, according to morphological characteristics of conidia and sporulation patterns, were segregated into four groups: A. alternata group, A. tenuissima group, A. yaliinficiens group and Alternaria sp. group. Molecular characteristics of part of the isolates were determined using random amplified polymorphism DNA (RAPD) analysis. Based on cluster analysis of RAPD data, large-and small-spored Alternaria spp. were evidently distinguished, and the small-spored species can form five clusters that fundamentally paralleled the morphological groupings.     

Sensitivity of the Alternaria infectoria species-group 1 to cycloheximide

Aims:  A possibility of using cycloheximide tolerance and/or sensitivity as an additional diagnostic tool for distinguishing morphologically related species within common small-spored Alternaria has been tested during this study.
Methods and Results:  A total of 33 strains from four Alternaria species-groups, namely Alternaria alternata , Alternaria arborescens , Alternaria infectoria and Alternaria tenuissima were tested for their growth response to 100 μg m⁻¹ cycloheximide in potato carrot agar. All A. infectoria strains were completely inhibited, showing no growth at all even after prolonged incubation. In contrast, all other strains representing the remnant three species exhibited a high resistance to this antibiotic.
Conclusions:  Cycloheximide sensitivity represents a further important physiological character for distinguishing A. infectoria from the three similar species.
Significance and Impact of the Study:  The relevance of these findings corresponds with the potential ability of the Alternaria species produce mycotoxins. Cycloheximide may be in future used in the design of selective media for the isolation of some potentially toxigenic food-borne Alternaria species such as A. alternata , A. tenuissima and/or A. arborescens , for example in screening cereals for toxigenic Alternaria spp. and for their direct separation from nontoxigenic representatives of A. infectoria species-group.     

Incidence and mycotoxin production by Alternaria tenuis in decayed apples

Alternaria tenuis was the main species of Alternaria which produced post-harvest decay in apples. Alternariol (AOH) and alternariol monomethyl ether (AME) were the major mycotoxins produced in Alternaria -decayed apples at 25°C and 2°C. Only the strain Alternaria tenuis CMTA 65 at 25°C produced tenuazonic acid (TA). Altertoxin-I (ATX-I) and altertoxin-II (ATX-II) were not detected in any of the apple samples.
A large percentage of strains of A. tenuis studied produced TA (97%) and AT-I (82·3%) when grown in a yeast extract sucrose medium (YES) at 25°C. A much smaller percentage of strains produced AOH and AME and none were found to produce detectable levels of ATX-II.     

Molecular systematics of citrus-associated Alternaria species

The causal agents of Alternaria brown spot of tangerines and tangerine hybrids, Alternaria leaf spot of rough lemon and Alternaria black rot of citrus historically have been referred to as Alternaria citri or A. alternata. Ten species of Alternaria recently were described among a set of isolates from leaf lesions on rough lemon (Citrus jambhiri) and tangelo (C. paradisi × C. reticulata), and none of these isolates was considered representative of A. alternata or A. citri. To test the hypothesis that these newly described morphological species are congruent with phylogenetic species, selected Alternaria brown spot and leaf spot isolates, citrus black rot isolates (post-harvest pathogens), isolates associated with healthy citrus tissue and reference species of Alternaria from noncitrus hosts were scored for sequence variation at five genomic regions and used to estimate phylogenies. These data included 432 bp from the 5' end of the mitochondrial ribosomal large subunit (mtLSU), 365 bp from the 5' end of the beta-tubulin gene, 464 bp of an endopolygalacturonase gene (endoPG) and 559 and 571 bp, respectively, of two anonymous genomic regions (OPA1-3 and OPA2-1). The mtLSU and beta-tubulin phylogenies clearly differentiated A. limicola, a large-spored species causing leaf spot of Mexican lime, from the small-spored isolates associated with citrus but were insufficiently variable to resolve evolutionary relationships among the small-spored isolates from citrus and other hosts. Sequence analysis of translation elongation factor alpha, calmodulin, actin, chitin synthase and 1, 3, 8-trihydroxynaphthalene reductase genes similarly failed to uncover significant variation among the small-spored isolates. Phylogenies estimated independently from endoPG, OPA1-3 and OPA2-1 data were congruent, and analysis of the combined data from these regions revealed nine clades, eight of which contained small-spored, citrus-associated isolates. Lineages inferred from analysis of the combined dataset were in general agreement with described morphospecies, however, three clades contained more than one morphological species and one morphospecies (A. citrimacularis) was polyphyletic. Citrus black rot isolates also were found to be members of more than a single lineage. The number of morphospecies associated with citrus exceeded that which could be supported under a phylogenetic species concept, and isolates in only five of nine phylogenetic lineages consistently were correlated with a specific host, disease or ecological niche on citrus. We advocate collapsing all small-spored, citrus-associated isolates of Alternaria into a single phylogenetic species, A. alternata.     

Two cases of cutaneous phaeohyphomycosis by Alternaria alternata and Alternaria tenuissima

Two cases of cutaneous phaeohyphomycosis, one with a nodular appearance and the other with an erythematous infiltrating patch, are reported in immunocompromised patients. Diagnosis was based on histological examination, which revealed hyphae and round-shaped fungal cells in a granulomatous dermal infiltrate, and on identification of the moulds when biopsy fragments were cultured on Sabouraud-dextrose agar without cycloheximide. The pathogens were Alternaria tenuissima in the first case and A. alternata in the second. The fungi were examined by scanning electron microscopy. The patients were checked for bone and lung involvement and were then treated with surgical excision and itraconazole, and itraconazole only, respectively, with clinical and mycological resolution. This revised version was published online in August 2006 with corrections to the Cover Date.     

小孢子链格孢endoPG基因核苷酸序列分析及系统发育研究

对供试小孢子链格孢菌株的内聚半乳糖醛酸酶(endoPG)基因进行扩增,大部分菌株都可获得PCR产物。核苷酸和氨基酸序列比较表明:不同种小孢子链格孢endoPG基因核苷酸序列存在明显差异,甚至表现在氨基酸水平,这些差异可以作为一些种如梨黑斑链格孢、长柄链格孢区分的分子性状。利用邻近结合法构建系统发育树,所有菌株被分为8个聚类组。在系统发育树上,链格孢的一些不同分离物被聚在不同组中,而细极链格孢、链格孢的部分菌株、苹果链格孢、柑橘链格孢、粗柠檬褐斑链格孢、橘树链格孢被聚为一组,显示根据形态学特征划分的这些种与分子性状的不一致性。endoPG基因核苷酸序列富于变化,为小孢子链格孢系统发育研究提供了一种有用的手段。     

Production of a Host-Specific Toxin by Germinating Spores of Alternaria tenuissima Causing Leaf Spot of Pigeon Pea

Production of a host-specific toxin by Alternaria tenuissima , the cause of pigeon pea leaf spot, was investigated in spore-germination fluids (SGF). The SGF selectively induced necrosis on pigeon pea leaves in a deteched leaf assay. Necrotic lesions were observed when a toxin from SGF was applied onto detached young leaves of the pigeon pea cultivar Bahar at concentration as low as 5 ng/ml. The resistant line Tanzania and nonhosts tolerated at least 20,000 times higher concentration of the toxin. The differential activity of the toxin on hosts and nonhosts of the fungus, as well as on susceptible and resistant cultivars or lines, suggested host-specific property of the toxin. At a concentration of 10 ng/ml, the toxin induced susceptibility of pigeon pea leaves to a non-pathogenic isolate of Alternaria alternata. The toxin possibly plays a role as a disease determinant of A. tenuissima , because the toxin was released from germinating spores as early as 3 h of incubation andthe, amount detected within 9 h was about 6 times of the concentration required for necrotic toxicity.     

Blue light inhibits mycotoxin production and increases total lipids and pigmentation in Alternaria alternata

Light inhibits production of the mycotoxins alternariol and alternariol monomethyl ether, both polyketids produced by Alternaria alternata. This effect seems to be general because seven isolates of A. alternata with different alternariol- and alternariol monomethyl ether-producing abilities all respond to continuous light with reduced levels of alternariol and alternariol monomethyl ether when the mycotoxins were calculated on a microgram-per-milligram (dry weight) basis. Blue light inhibited alternariol and alternariol monomethyl ether production 69 and 77%, respectively. Red light gave no reduction of toxin levels. Total lipids were increased 25% when mycelium was grown in blue light as compared with red light or darkness. In white or blue light, but not in red light or darkness, a red-brown pigment accumulated by the mycelium.     

Blue light inhibits mycotoxin production and increases total lipids and pigmentation in Alternaria alternata.

Light inhibits production of the mycotoxins alternariol and alternariol monomethyl ether, both polyketids produced by Alternaria alternata. This effect seems to be general because seven isolates of A. alternata with different alternariol- and alternariol monomethyl ether-producing abilities all respond to continuous light with reduced levels of alternariol and alternariol monomethyl ether when the mycotoxins were calculated on a microgram-per-milligram (dry weight) basis. Blue light inhibited alternariol and alternariol monomethyl ether production 69 and 77%, respectively. Red light gave no reduction of toxin levels. Total lipids were increased 25% when mycelium was grown in blue light as compared with red light or darkness. In white or blue light, but not in red light or darkness, a red-brown pigment accumulated by the mycelium.     

Viruslike particles in tentoxin-producing strains of Alternaria alternata.

Double-stranded (ds) RNAs associated with viruslike particles have been found in six isolates of Alternaria alternata which produce tentoxin. Isolates had from one to three dsRNAs ranging in size from 1.0 to 5.1 kilobase pairs. In two isolates the dsRNAs were associated with 30-nm particles. No dsRNA was detected in any of six other tentoxin-producing isolates or nine isolates which did not produce tentoxin.     

一种来源于链格孢菌的MAPK激酶AtPBS2基因的克隆及功能分析

通过遗传学手段,构建了细极链格孢菌（Alternaria tenuissima）cDNA酵母表达文库并筛选获得MAPK激酶AtPBS2基因,该基因全长2 492bp,编码683个氨基酸。AtPbs2p与来源于烟曲霉（Aspergillus fumigatus）的AtPbs2p（XP_752961）、粗糙脉孢菌（Neurospora crassa）的NcPbs2p（XP_965727）、稻瘟菌（Magnaporthe grisea）MGCH7（XP_001522946）及酵母（Saccharomyces cerevisiae）ScPbs2p（EDN63254）序列分别具有52%、52%、49%和47%的相似性。在高渗环境下,AtPBS2基因与酵母的ScPBS2基因功能相同,具有耐高渗透压环境的能力。细极链格孢菌（A.tenuissima）中存在HOG通路信号途径,AtPBS2基因可能与链格孢菌（A.tenuissima）的逆境适应性调节密切相关。     

Phytotoxicity of pathogenic fungi and their mycotoxins to cereal seedling viability

Aspergillus flavus, Alternaria alternata and Fusarium oxysporum were found to be the pathogenic fungi mostly reducing cereal (barley, sorghum and wheat) seedlings. The pathogens have the ability to produce aflatoxin B1 and G1, diacetoxyscirpenol, kojic acid and tenuazonic acid that reduced seedling viability. The inhibition dose for 50% reduction (LD50) was recorded by aflatoxins at 0.83 mg L-1 for barley, 1.74 mg L-1 for wheat and 2.75 mg L-1 for sorghum. Diacetoxyscirpenol produced its inhibition at 1.26 mg L-1 for barley, 3.98 mg L-1 for wheat and 10 mg L-1 for sorghum. Kojic acid induced 50% inhibition at 63 mg L-1 for barley, 105 mg L-1 for wheat and 251 mg L-1 for sorghum. However, tenuazonic acid was less toxic where, the toxicity was ranged between 79-550 mg L-1. The inhibition in germination was more pronounced in barley followed by wheat and negligible in sorghum to all tested mycotoxins. This inhibition attributed to the reduction in seedling amylase activity. Amylase was also reduced in the same trend: barley > wheat > sorghum. Grain treatment with carboxin-captan and thiophanatemethyl-thiram at 1 g kg-1 grain increased seedlings vigour of wheat in sterilized soil by 45 and 22%, barley by 24 and 33% and sorghum by 15 and 30%, respectively. These fungicides have also a positive effect on cereal when soil was inoculated with A. flavus, A. alternata and F. oxysporum.     

Dicarboximide resistance in field isolates of Alternaria alternata is mediated by a mutation in a two-component histidine kinase gene

Isolates of Alternaria alternata collected from a field site which had previously been treated with the dicarboximide fungicide iprodione were found to demonstrate a high level of resistance to iprodione and the phenylpyrrole fungicide, fludioxonil in plate assays. In order to determine the genetic basis for this fungicide resistance a partial length clone of a two-component histidine kinase (HK) was isolated from genomic DNA of a fungicide-sensitive A. alternata isolate using degenerate primers by PCR. Analysis of the AaHK1 gene structure indicates the presence of six 90 amino acid repeat domains upstream of a kinase domain as found in the homologous HK genes from other fungal species. Comparison of nucleic acid sequences from the fungicide-sensitive and fungicide-resistant A. alternata isolates confirmed the presence of mutations leading to premature termination of the translated HK protein. The possible role of the two-component HK in the development of dicarboximide resistance in A. alternata is discussed.     

Endopolygalacturonase is essential for citrus black rot caused by Alternaria citri but not brown spot caused by Alternaria alternata

Alternaria citri, the cause of Alternaria black rot, and Alternaria alternata rough lemon pathotype, the cause of Alternaria brown spot, are morphologically indistinguishable pathogens of citrus: one causes rot by macerating tissues and the other causes necrotic spots by producing a host-selective toxin. To evaluate the role of endopolygalacturonase (endoPG) in pathogenicity of these two Alternaria spp. pathogens, their genes for endoPG were mutated by gene targeting. The endoPGs produced by these fungi have similar biochemical properties, and the genes are highly similar (99.6% nucleotide identity). The phenotypes of the mutants, however, are completely different. An endoPG mutant of A. citri was significantly reduced in its ability to cause black rot symptoms on citrus as well as in the maceration of potato tissue and could not colonize citrus peel segments. In contrast, an endoPG mutant of A. alternata was unchanged in pathogenicity. The results indicate that a cell wall-degrading enzyme can play different roles in the pathogenicity of fungal pathogens. The role of a cell wall-degrading enzyme depends upon the type of disease but not the taxonomy of the fungus.