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.     

Mycotoxin production by some IndianAlternaria species

A total of seven species ofAlternaria: A. alternata (Fr.) Keissler;A. capsici-annui Savul & Sandu;A. citri Ellis & Pierce;A. porri (Ellis) Clfferi;A. radicina Meler, Drechsler & Eddy;A. tenuissima (Kunze: Pers) Wiltshire andA. tomato (Cooke) Jones were screened on rice culture medium for their ability to elaborate five majorAlternaria mycotoxins viz. tenuazonlc acid (TA), alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT) and altertoxin-l (ATX-I). All the species produced mycotoxins in varying concentrations. A.capsici-annul was recorded as the mycotoxin producer for the first time. ALT byA. citri andA. tomato; ALT, and ATX-I byA. tenuissima; ALT, TA and AME byA. porri and TA byA. radicina are the new additions to the list of mycotoxins produced by the respective species ofAlternaria.     

Species-specific banding patterns of restriction endonuclease-digested mitochondrial DNA from the genusPythium

Restriction endonuclease-digested mitochondrial DNA from 29Pythium spp. showed distinctly different species-specific electrophoretic banding patterns. Numerical comparisons among species were conducted by calculating the percentage of restriction fragments having the same apparent molecular size. The greatest interspecific similarity in banding patterns (67%) was observed betweenHindIII digests ofPythium heterothallicum andP. sylvaticum. However, comparisons among other species generally revealed similarities of less than 50%, and often less than 30%. The lack of similarity of restriction banding patterns was observed even with several species that share many common morphological features:P. arrhenomanes vsP. graminicola (20%),P. myriotylum vsP. aristosporum (28%), andP. torulosum vsP. vanterpoolii (32%). In contrast to the fragment size heterogeneity among different species, isolates of the same species have highly conserved restriction patterns. Ten isolates ofP. oligandrum, collected from the United States, South Africa, and Czechoslovakia, had a minimum of 86% similarity inHindIII banding patterns. Similar results were observed with eight isolates ofP. ultimum, five ofP. acanthicum, six ofP. spinosum, five ofP. sylvaticum, and eight ofP. irregulare. However, two isolates ofP. irregulare exhibited a higher degree of heterogeneity and shared only 64 to 76% comigrating bands with the eight other isolates of this species.     

Polyphasic classification of Alternaria isolated from hazelnut and walnut fruit in Europe

Brown apical necrosis of English walnut and grey necrosis of hazelnut are destructive fruit diseases caused by a complex of opportunistic fungi including several small-spored catenulate Alternaria taxa. Thirty Alternaria isolates recovered from walnut and hazelnut fruit that were pathogenic on their respective host were compared along with type or representative isolates of A. alternata, A. tenuissima, A. arborescens, and A. infectoria using morphological and molecular criteria. Morphological examination using standardized procedures separated the walnut and hazelnut isolates into three morphological groups: the A. alternata group, the A. tenuissima group, and the A. arborescens group based upon common characteristics of the conidium and the sporulation apparatus. To evaluate genetic relationships among these groups, AFLP markers, inter simple sequence repeat (ISSR) markers, and histone gene sequence data were compared. Based upon AFLP data, the A. alternata and A. tenuissima groups comprised a single lineage, and the A. arborescens group comprised a separate lineage. ISSR data supported the grouping by AFLP data except for three isolates of the A. alternata group that clustered with the A. arborescens group. Base substitution of the H4 gene supported the discrimination of the A. arborescens group from the A. alternata and A. tenuissima groups. Tests of hypotheses based upon groupings derived from the various data sets supported the discrimination of the A. arborescens group but did not support the discrimination of the A. alternata group from the A. tenuissima group.     

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

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

Accumulation of sugar alcohols by filamentous fungi

Five hundred isolates of different xerophilic and non-xerophilic fungi belonging to 10 genera and 74 species were screened for alditol (sugar alcohol) accumulation. Ninety-two of the isolates failed to grow on a salt medium, most of the isolates (408) produced alditols; 348,44 and 16 of them produced low, moderate and high levels of alditols, respectively. The high alditol producers belonged to five species ofAspergillus, six species ofEurotium andFennellia flavipes. Glycerol andd-mannitol were the main constituents of alditol pools of the 16 high alditol producers.d-Arabinitol andmeso-erythritol were also formed but at low concentrations by several of the tested isolates.     

Laboratory culture and taxonomy of two species ofDerbesia (Class Chlorophyceae) in Japan

Two species ofDerbesia (Class Chlorophyceae),D. marina andD. tenuissima, have been studied for the purpose of obtaining a better understanding of their morphological details and life histories, using preserved and living specimens as well as laboratory cultures. The life histories of both species were completed in the laboratory, starting from both zoospores and zygotes. Specimens were collected at Asamushi, Aomori-ken, and Shimoda, Shizuoka-ken. Their life history types are fundamentally identical, zoospores giving rise upon germination toa Halicystis-phase, while zygotes grow into aDerbesia-phase. The thallus of theHalicystis-phase which alternates withD. marina is the same as that ofH. ovalis which grows in the northern regions of Japan. On the other hand, the thallus of theHalicystis-phase alternating withD. tenuissima is the same as that ofH. parvula known to occur in the temperate to subtropical regions of Japan. These results coincide with those obtained withD. marina andD. tenuissima in Europe, where the type localities of both species are located. Specimens assignable to these two species were collected at several localities in Japan and, as a result of detailed examination of the morphology, they are believed to be identical with eitherD. marina orD. tenuissima.     

Taxonomic classification of asexual isolates ofPythium ultimum based on cultural characteristics and mitochondrial DNA restriction patterns

Taxonomic characteristics of 8 isolates ofPythium ultimum and 11 isolates ofPythium in the spherical hyphal swelling (HS) group of Van der Platts-Niterink were compared. Isolates in the two groups had identical temperature growth responses and morphological features of hyphal swellings. All isolates were pathogenic on sugar beet. Attempts to cross HS isolates among themselves and with opposite mating types ofP. heterothallicum andP. sylvaticum failed. HS isolates were not induced to form antheridia when paired with isolates ofP. ultimum using a polycarbonate membrane sandwich technique. In single culture, some HS isolates formed low numbers of spherical structures encompassed by swollen hyphal masses resembling antheridia. Comparisons of restriction banding patterns ofHindIII-digested mitochondrial DNA revealed that all but 1 isolate ofP. ultimum were identical; however, the variable isolate shared 80% of the bands in common with the others. Seven of 11 HS isolates had banding patterns identical to the predominantP. ultimum pattern and 1 isolate shared 96% comigrating bands. On the basis of the number of shared characteristics, these isolates appear to beP. ultimum which have lost the ability to reproduce sexually.     

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.     

Filamentous fungi quiescent in seeds and culm nodes of weedy and forage grass species endemic to the Palouse Region of Washington and Idaho

Asymptomatic seeds of forage and weedy grasses from germplasm accessions and from uncultivated sites in eastern Washington and western Idaho were assayed for the presence of quiescent filamentous fungi. Asymptomatic culm nodes of the same species were similarly assayed. The predominant taxa isolated were strains of dematiaceous hyphomycetes, principally strains of Alternaria and Cladosporium. A. infectoria was the species most frequently isolated from seed. A. tenuissima was common and A. alternata was rare. Aspergillus, Penicillium and Fusarium were very rare or absent in stored germplasm. Pathogenic coelomycetes were common in asymptomatic node samples, occasionally present in asymptomatic field seed, but were not detected in germplasm accessions. Previous reports of frequent occurrence of A. alternata in grass seed are probably the results of misidentification of various small-spored Alternarias, especially A. infectoria, as A. alternata.This revised version was published online in October 2005 with corrections to the Cover Date.     

A polyphasic approach for the characterization of endophytic Alternaria strains isolated from grapevines

A polyphasic approach was set up and applied to characterize 20 fungal endophytes belonging to the genus Alternaria, recovered from grapevine in different Italian regions.Morphological, microscopical, molecular and chemical investigations were performed and the obtained results were combined in a pooled cluster analysis. Following morphological analyses, all strains were grouped according to their three-dimensional sporulation pattern on PCA and to the colony characteristics on different substrates. After DNA extraction, all strains were analyzed by RAPD-PCR and the resulting profiles were subjected to cluster analysis. The metabolites extracted from the 20 Alternaria endophytes were analyzed by a HPLC and the resulting metabolite profiles were subjected to multivariate statistic analyses. In comparison with reference ‘small-spored’ Alternaria species, the 20 strains were segregated into two morphological groups: one belonging to the A. arborescens species-group and a second to the A. tenuissima species-group. RAPD analysis also showed that grapevine endophytes belonged to either the A. arborescens or the A. tenuissima species-group and that they were molecularly distinct from strains belonging to A. alternata. Chemotaxonomy gave the same grouping: the grapevine endophytic strains belong to A. arborescens or A. tenuissima species-groups producing known metabolites typical of these species-groups. Interestingly, the 20 grapevine endophytes were able to produce also a number of unknown metabolites, whose characterization could be useful for a more precise segregation of the two species-groups.The results show how complementary morphological, molecular and chemical data can clarify relationships among endophyte species-groups of low morphological divergence.     

Insecticidal and nematicidal properties of microbial metabolites

Summary Metabolites from 942 microbial isolates were screened for insecticidal and nematicidal properties. The isolates included 302 streptomycetes, 502 novel actinomycetes including representatives of 18 genera, 28 unidentified aerobic actinomycetes, 70 fungi and 40 bacteria other than actinomycetes. When diluted 10-fold, the metabolites from 55 isolates caused nearly 100% mortality in mosquito larvae (Aedes aegypti) within 24 h. These isolates included 27 isolates ofStreptomyces, four ofActinoplanes, three isolates each ofActinomadura andStreptoverticillium, two isolates each ofMicromonospora, Bacillus andPaecilomyces, and one isolate each ofMicropolyspora, Nocardiopsis, Streptosporangium, Oerskovia, Thermomonospora, Chainia, Pseudomonas, Fusarium, Monilia andSyncephalestrum. Two fungal isolates could not be identified to the generie level. Extracts from the culture broth of 18 isolates caused 100% mortality in mosquito larvae within 15 min to 24 h at a concentration of 1000 ppm. The LC₅₀ of partially purified products from two isolates was 1–2.5 ppm and that of the semipurified preparations from four other isolates was 50 ppm. Valinomycin was identified as an active component in the culture broth from one isolate. The culture broth from 15 isolates of aerobic actinomycetes and 4 of fungi were toxic toPanagrellus redivivus (Nematoda); these included 12 isolates with selective nematicidal properties.Michigan Agriculture Experiment Station, Journal Article No. 12321.     

Screening for Fusarin C production by European isolates of Fusarium species

A total of 137Fusarium isolates were screened forin vitro production of the mutagenic metabolite fusarin C, using a simple thin layer chromatographic method. It has been proven thatFusarium species (F. culmorum, F. graminearum, F. crookwellense, F. sporotrichioides, F. poae, F. tricinctum, andF. Avenaceum) isolated from European agricultural crops and soils are able to produce fusarin C. No fusarin C production was detected among isolates ofF. arthrosporioides, F. acuminatum, or F. equiseti. Results obtained by High-Performance Liquid Chromatography (HPLC) analyses of fungal extracts show that up to 26 chromatographic peaks having UV spectra similar to that of fusarin C are produced. It is not known if any of these metabolites are as mutagenic as fusarin C.     

Absorption,translocation, and metabolism of14C-clomazone in soybean (Glycine max) and threeAmaranthus weed species

The patterns of clomazone (2-[(2-chlorophenyl) methyl-4,4-dimethyl-3-isoxazolidinone) absorption, translocation, and metabolism and their contribution to the plant selectivity of this herbicide were studied in tolerant soybean [Glycine max (L.) Merr.] andAmaranthus hybridus and in susceptibleA. retroflexus andA. lividus. Differential root absorption appeared to play a significant role in the differential response of these four plant species to clomazone. Absorption of root-applied¹⁴C-clomazone was greater by the two sensitiveAmaranthus weeds than by the tolerant soybean andA. hybri-dus. Following application of¹⁴C-clomazone to roots, most of the absorbed radioactivity was translocated to the leaves of all four species. Approximately 50% of the absorbed¹⁴C-clomazone was metabolized by all four plant species as early as 12 h after treatment. Thin layer Chromatographic (TLC) analysis of plant tissue extracts from all four species revealed the formation of two major metabolites of clomazone. These unidentified metabolites had Rf values of 0.4 and 0.8, respectively, in a butanol∶acetic acid∶water (12∶3∶5, vol/vol/vol) developing system. The Rf value of unaltered clomazone in this system was 0.95. Differential metabolism or differential rate of metabolism of clomazone was not observed in this study and did not seem to account for the tolerance of soybean andA. hybridus or the suceptibility ofA. retroflexus andA. lividus to this herbicide.     

Gust J, a salt-insensitive mutant ofDrosophila melanogaster

A set of 340 P insert lines on the X chromosome and the autosomes were screened for altered responses of the labellar chemoreceptors to salts and sucrose. A mutant linegustJ was isolated in which the electrophysiological response of the salt-sensitive neuron to Na⁺ in the sensilla of the proboscis is reduced. The responses to KC1 and sucrose are unaffected. In feeding tests,gustJ flies have Na⁺-specific defects. Heterozygotes ofgustJ with two other salt mutantsgustE andBE1323 are normal. Multiple alleles ofgustJ have been obtained by excision of the original P element. All mutants have defects in Na⁺ sensing specifically, thus defining a new gene that affects Na⁺ response of the fly.