Determinación de perfiles de producción de metabolitos secundarios característicos de especies del género Alternaria aisladas de tomate

BackgroundMany Alternaria species have been studied for their ability to produce bioactive secondary metabolites, such as tentoxin (TEN), some of which have toxic properties. The main food contaminant toxins are tenuazonic acid, alternariol (AOH), alternariol monomethyl ether (AME), altenuene, and altertoxins i, ii and iii.AimsTo determine the profiles of secondary metabolites characteristic of Alternaria strains isolated from tomato for their chemotaxonomic classification.MethodsThe profiles of secondary metabolites were determined by HPLC MS.ResultsThe Alternaria isolates obtained from spoiled tomatoes belong, according to their morphological characteristics, to the species groups Alternaria alternata, Alternaria tenuissima and Alternaria arborescens, with A. tenuissima being the most frequent. The most frequent profiles of secondary metabolites belonging to the species groups A. alternata (AOH, AME, TEN), A. tenuissima (AOH, AME, TEN, tenuazonic acid) and A. arborescens (AOH, AME, TEN, tenuazonic acid) were determined, with some isolates of the latter being able to synthesize AAL toxins.ConclusionsSecondary metabolite profiles are a useful tool for the differentiation of small spored Alternaria isolates not easily identifiable by their morphological characteristics.     

Phylogenetic,toxigenic and virulence profiles of <Emphasis Type="Italic">Alternaria</Emphasis> species causing leaf blight of tomato in Egypt

Species of Alternaria are serious plant pathogens, causing major losses on a wide range of crops. Leaf blight symptoms were observed on tomato leaves, and samples were collected from various regions. Isolation was done from symptomatic tomato leaves, and 15 representatives were selected from a collection of 65 isolates of Alternaria species. The virulence of Alternaria isolates was investigated on detached leaves (DL) and whole plants of tomato cv. Super strain B. A phylogenetic analysis was performed based on three partial gene regions, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the RNA polymerase second largest subunit (RPB2) and the Alternaria major allergen gene (Alt a 1). The potentiality of Alternaria isolates to produce toxins was also investigated on the basis of thin-layer chromatography (TLC). Our investigations revealed that Alternaria isolates showed different levels of virulence either on tomato plants or DL. Based on the phylogeny of three genes, Alternaria isolates encompassed two species of small-spored morphospecies: A. alternata (14 isolates) and A. arborescens (single isolate). The produced toxins varied among Alternaria isolates with tenuazonic acid (TeA) being the most abundant mycotoxin produced by most isolates. This study highlighted on other Alternaria species in Egypt that might represent a serious concern for tomato producers as causal agents of leaf blight over other species, i.e. A. solani.     

Influence of dietary fibre on plasma and urinary levels of zearalenone and metabolites in swine

The genusAlternaria is responsible for different plant diseases such as tobacco brown spot, tomato blight, and citrus seedling chlorosis but can also be present during storage of grain. The objective of the present paper is to summarize the knowledge concerning regulation of secondary metabolism inAlternaria, particularA alternata (A tenuis). The paper mainly deals with regulation of polyketide biosynthesis, one of the major pathways leading to the biosynthesis of mycotoxins inAlternaria. The mostly studiedAlternaria mycotoxins are dibenzopyrones such as alternariol (AOH) and alternariol monomethyl ether (AME) and altenuene along with the tetramic acid tenuazonic acid. The biosynthesis ofAlternaria mycotoxins has been reviewed by Stinson (12). Most information is available for the biosynthesis of the polyketides AOH / AME while a few biosynthetic studies have been accomplished for tenuazonic acid (11).     

Mycotoxin-producing potential of fungi isolated from red kidney beans

The predominant fungi present in samples of reject and retail red kidney beans were Aspergillus glaucus, Penicillium spp. and Alternaria spp. Together with A. ochraceus, A. flavus, Fusarium spp., and Trichoderma, these isolates from the reject beans were screened for numerous mycotoxins by TLC. The most consistently produced mycotoxins were penicillic acid (from A. ochraceus and Penicillium spp.) and Alternaria toxins (tenuazonic acid and alternariol). A. glaucus strains were tested for cytotoxicity in three tissue culture cell lines with positive results.     

Alternaria mycotoxins associated with grape berries in vitro and in situ

The aim of this study was to assess the production of secondary metabolites of Alternaria species isolated from grape berries and determined the occurrence of Alternaria mycotoxins in vitro and in dried berries. Direct morphological examination on different media was used for identification and HPLC/MS method for quantification of Alternaria mycotoxins. We isolated A. alternata and A. tenuissima and randomly selected strains in vitro and in dried berries. In vitro we identified the production of Alternaria metabolites, included mycotoxins: alternariol, alternariol methylether, tenuazonic acid. Beside that one in dry berries were in measurable concentrations: macrosporin A, tentoxin and altenuete (I, II).     

<Emphasis Type="Italic">Alternaria</Emphasis> toxins in South African sunflower seeds: cooperative study

Sunflower seed samples (N = 80) from different sunflower cultivars originating from different localities in South Africa were analyzed for 15 toxins produced by fungi of the genus Alternaria by means of a simple one-step extraction dilute-and-shoot HPLC-MS/MS approach. References for valine-tenuazonic acid (Val-TeA), altenusin (ALTS), and altenuisol (ALTSOH) were isolated from fungal culture extracts and spectroscopically characterized. Additionally, valine-tenuazonic acid was tested regarding its cytotoxicity in comparison with tenuazonic acid (TeA) and showed less activity on HT-29 cells. Furthermore, alternariol monomethyl ether-3-O-ß-D-glucoside (AME-3G) was produced by fermentation of alternariol monomethyl ether (AME) with the fungus Rhizopus oryzae. The seed samples were analyzed both with and without hulls. The method covers the AAL toxins TA₁ and TA₂, altenuene (ALT) and iso-altenuene (iso-ALT), altenuisol, altenusin, altertoxin I (ATX-I) and altertoxin II (ATX-II), alternariol (AOH) and alternariol monomethyl ether, alternariol monomethyl ether-3-O-ß-D-glucoside, tenuazonic acid, allo-tenuazonic acid (allo-TeA) and valine-tenuazonic acid, and tentoxin (TEN). More than 80% of the samples were positive for one or more analytes above the respective limit of detection (0.2–23 μg/kg). Alternariol, its monomethyl ether, tentoxin, tenuazonic acid, altenuisol, and valine-tenuazonic acid were found in quantifiable amounts. The highest prevalences were found for tentoxin (73% positive, mean content 13.2 μg/kg, maximum level 130 ± 0.9 μg/kg) followed by tenuazonic acid (51% positive, mean content 630 μg/kg, maximum level 6300 ± 560 μg/kg). The obtained data were further analyzed statistically to identify quantitative or qualitative relationships between the levels of Alternaria toxin in the samples.     

Survey of <Emphasis Type="Italic">Alternaria</Emphasis> toxin contamination in food from the German market,using a rapid HPLC-MS/MS approach

A HPLC-MS/MS-based method for the quantification of nine mycotoxins produced by fungi of the genus Alternaria in various food matrices was developed. The method relies on a single-step extraction, followed by dilution of the raw extract and direct analysis. In combination with an analysis time per sample of 12 min, the sample preparation is cost-effective and easy to handle. The method covers alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), altenuene (ALT), iso-altenuene (isoALT), tentoxin (TEN), altertoxin-I (ATX-I), and the AAL toxins TA₁ and TA₂. Some Alternaria toxins which are either not commercially available or very expensive, namely AOH, AME, ALT, isoALT, and ATX-I, were isolated as reference compounds from fungal cultures. The method was extensively validated for tomato products, bakery products, sunflower seeds, fruit juices, and vegetable oils. AOH, AME, TeA, and TEN were found in quantifiable amounts and 92.1 % of all analyzed samples (n?=?96) showed low level contamination with one or more Alternaria toxins. Based on the obtained results, the average daily exposure to Alternaria toxins in Germany was calculated.     

Effect of pesticides inAlternaria alternata mycotoxins production

The effect of pesticides onAlternaria mycotoxins was evaluated both in culture media and in sunflower seeds. Different behaviour was observed depending on whether insecticides or fungicides were considered. When Captan, Lindaphor and Dichlorvos were evaluated in sunflower seeds their effects were dependent on the toxin being evaluated (alternariol, alternariol monomethyl ether and tenuazonic acid). A full spectrum of effects was observed, ranging from no effect, stimulation, to inhibition.     

Incidence of Alternaria Species Associated with Watermelon Leaf Blight in Korea

Alternaria leaf blight is one of the most common diseases in watermelon worldwide. In Korea, however, the Alternaria species causing the watermelon leaf blight have not been investigated thoroughly. A total of 16 Alternaria isolates was recovered from diseased watermelon leaves with leaf blight symptoms, which were collected from 14 fields in Korea. Analysis of internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and RNA polymerase II second largest subunit (RPB2) were not competent to differentiate the Alternaria isolates. On the contrary, analysis of amplicon size of the histone H3 (HIS3) gene successfully differentiated the isolates into three Alternaria subgroups, and further sequence analysis of them identified three Alternaria spp. Alternaria tenuissima, A. gaisen, and A. alternata. Representative Alternaria isolates from three species induced dark brown leaf spot lesions on detached watermelon leaves, indicating that A. tenuissima, A. gaisen, and A. alternata are all causal agents of Alternaria leaf blight. Our results indicate that the Alternaria species associated watermelon leaf blight in Korea is more complex than reported previously. This is the first report regarding the population structure of Alternaria species causing watermelon leaf blight in Korea.     

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 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.     

Aspergillus bertholletius sp. nov. from Brazil Nuts

During a study on the mycobiota of brazil nuts (Bertholletia excelsa) in Brazil, a new Aspergillus species, A. bertholletius, was found, and is described here. A polyphasic approach was applied using morphological characters, extrolite data as well as partial β-tubulin, calmodulin and ITS sequences to characterize this taxon. A. bertholletius is represented by nineteen isolates from samples of brazil nuts at various stages of production and soil close to Bertholletia excelsa trees. The following extrolites were produced by this species: aflavinin, cyclopiazonic acid, kojic acid, tenuazonic acid and ustilaginoidin C. Phylogenetic analysis using partial β-tubulin and camodulin gene sequences showed that A. bertholletius represents a new phylogenetic clade in Aspergillus section Flavi. The type strain of A. bertholletius is CCT 7615 ( = ITAL 270/06 = IBT 29228).     

Natural products of Alternaria sp., an endophytic fungus isolated from Salvadora persica from Saudi Arabia

This study is to evaluate the potential of endophytic fungi of Salvadora persica for the production of bioactive compounds against pathogenic bacteria and fungi. Forty-two fungal isolates were obtained from 135 young and old stem and 125 root segments. Those 42 isolates representing ten fungi include: Trichoderma sp. (the most common), two species of Alternaria, Rhizopus arrhizus and 6 sterile mycelia. The ten fungi were grown in liquid culture and their crude extracts were tested against pathogenic bacteria and fungi. Nine crude extracts gave positive reactions against pathogenic bacteria of which Alternaria sp. (A8) was chosen further study. The fungal isolate was growing as sterile mycelium and was identified by phylogenetic analyses based on LSU rDNA sequence data and it might represent undescribed species of Alternaria. Sixty-two bioactive chemical compounds were identified from the ethyl acetate crude extracts of Alternaria sp., of which the following were recorded as major compounds in the active sub-fractions. These compounds showed strong antibacterial activity in combination.     

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.     

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.     

Characterization,pathogenicity, and fungicide sensitivity of Alternaria isolates associated with preharvest fruit drop in California citrus

Alternaria rot has been recently described as an emerging fungal disease of citrus causing significant damage in California groves. A survey was conducted to determine latent infections on fruits, twigs, and leaves and investigate their seasonal patterns during 2019 and 2020. On fruits, latent infections were more associated with the stem end than with the stylar end, except during spring when a significantly high percentage of flowers (86%) had latent infections. Latent infections on twigs varied markedly between years (28% in 2019 and 9.5% in 2020), while Alternaria spp. were also recovered from citrus leaves. Alternaria isolates collected during the survey were identified based on multigene sequence analysis, confirming that Alternaria alternata and Alternaria arborescens are the two species associated with infections of citrus fruits. Of the 23 isolates, 19 were identified as A. alternata and demonstrated the dominance of this species over A. arborescens. Isolates representing populations of these two species were selected as representative isolates for physiological and morphological studies. A. alternata and A. arborescens showed similar conidial dimensions but differed in the number of conidia produced. Growth rates demonstrated that A. alternata grows faster than A. arborescens at all the temperatures evaluated, except at 25 and 35 °C. The growth patterns were similar for both species. The sporulation rate of the Alternaria isolates was influenced differently by temperature. This parameter also influenced conidial germination and appressorium formation, and no significant differences were observed between Alternaria species. Pathogenicity and aggressiveness tests on detached fruit demonstrated the ability of A. alternata and A. arborescens to cause internal lesions and produce fruit drop in the orchards with no quantitative differences between them (disease severity indexes of 58 and 68%, respectively). The fungicide sensitivity tests showed that DMI fungicides are the most effective fungicides in reducing mycelial growth. The SDHI fungicides had intermediate activity against the mycelial growth but also suppressed spore germination. The spore germination assay suggested that some of the isolates included in this study might have some level of resistance to QoI and SDHI fungicides. The findings of this study provide new information about the pathogens associated with the excessive fruit drop recently observed in some California citrus groves.     

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.     

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.     

Action of tenuazonic acid,a natural phytotoxin,on photosystem II of spinach

Tenuazonic acid (TeA) is a putative phytotoxin obtained from Alternaria alternata, the organism that can cause brown leaf spot disease of Crofton weed (Eupatorium adenophorum). It is demonstrated here that the tenuazonic acid inhibits the activity of photosystem II (PSII); the I₅₀-value is 48 μg mL^?1. Evidences from chlorophyll fluorescence show that tenuazonic acid interrupts electron transport between Q_A and Q_B on the acceptor side of PSII. It does not have an effect on the antenna pigments, the oxygen-evolving complex (OEC) at the donor side of PSII. On the basis of the fluorescence induction kinetics and competition experiments with [¹⁴C]atrazine, it is shown that tenuazonic acid does not share the same binding environment with atrazine despite their common action target: the Q_B-site. It is concluded that tenuazonic acid is a member of a novel class of PSII inhibitors.     

Cholinesterase inhibitory potential of different Alternaria spp. and their phylogenetic relationships

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors are used for the treatment of various disorders related to decline in acetylcholine levels in the brain by inhibiting the activity of the neurotransmitter AChE. The present study reports the potential of endophytic Alternaria spp. for their potential to produce cholinesterase inhibitors active against both acetylcholine and butyrylcholine. Twenty-nine isolates belonging to Alternaria spp. were isolated from different plants and screened. Variation with respect to inhibitor production was observed in different isolates. Out of 29 cultures screened, good cholinesterase (both AChE and BChE) inhibitory activity in range of 70–85% was observed in three isolates, whereas three showed only AChE inhibition. No correlation was observed in AChE and BChE inhibitor production. TLC bioautography for the inhibitor in the selected cultures evinced different Rf values of inhibitors indicating different nature of the compounds produced. In order to analyze evolutionary relationships between producer and non-producer strains, phylogenetic analysis of six producer and five non-producer strains was carried out using amplified ITS-I-5.8SrDNA-ITS-II region. Phylogenetic analysis revealed majority of the non-producer strains to be present on different clades indicating different evolutionary origins. The dual cholinesterase inhibitory activity and the diversity in the inhibitors produced by different isolates could prove to be novel sources of pharmaceutical as well as agriculturally important biomolecules after purification and characterization.