Meroterpenes isolated from the endophytic fungus Guignardia mangiferae

Two new guignardones and one tricycloalternarene derivatives, named guignardone D, E (2-3) and tricycloalternarene F (4), and the known guignardone A (1) were isolated from Guignardia mangiferae, an endophytic fungus from the leaves of Viguiera arenaria (Asteraceae), after fermentation in Czapek medium. Structures were established on the basis of their spectroscopic data, including H-1 NMR, C-13 NMR, HMQC, HMBC and HRESI-MS. (C) 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Speziation mikrobiologisch alkylierter, leichtflüchtiger Selenverbindungen in Abhängigkeit der geochemischen Verfügbarkeit des Selens

Selen kann in verschiedenen Oxidationszuständen (+6, +4, ±0, -2) in unterschiedlichen Umweltkompartimenten auftreten. Verbundenen damit sind verschiedene Eigenschaften, wie z. B. die Wasserlöslichkeit, die in direktem Zusammenhang mit der Migrationsfähigkeit sowie der Bioverfügbarkeit steht. Im Rahmen der vorliegenden Arbeit wurden die Verfügbarkeit anorganischer Selenspezies und damit die Mobilisierbarkeit dieser in verschiedenen Laborexperimenten untersucht. Hierbei wurde an Goethit adsorbiertes Selenit sowohl mit einer Reinkultur des aktiv methylierenden Pilzes Alternaria alternata als auch mit einer angereicherten Umweltmischkultur inkubiert und die mikrobiologische Zugänglichkeit anhand der Bildung leichtflüchtiger, alkylierter Selenmetabolite wie z. B. Dimethylselenid und Dimethyldiselenid beobachtet. Zur Analyse dieser wurde eine cryotrapping-cryofocussing-GC-ICP-MS-Kopplung etabliert. Die Anteile der methylierten Selenverbindungen stiegen bei Verwendung von A. alternata mit der Inkubationszeit auf 10 % des gelösten Selens und 1 % des Gesamtselens an. Dieser Trend konnte während der Inkubation der Umweltmischkultur nicht beobachtet werden. Hier lagen die Anteile über den gesamten Untersuchungszeitraum bei ca. 0,5 % des gelösten bzw. 0,1 % des Gesamtselens, inklusive eines leichten Abwärtstrends, welcher wahrscheinlich durch die Nutzung der Alkylselenide als Kohlenstoffquelle hervorgerufen wurde. Weiterhin wurde das reduzierte Eisenselenidmineral Ferroselit eingesetzt, um dessen Stabilität gegenüber der Aktivität des sulfatreduzierenden Bakteriums Desulfovibrio gigas zu untersuchen. Mit zunehmender Inkubationszeit und damit verbundener, zunehmender Reduktion des im Nährmedium vorhandenen Sulfates konnte ein Anstieg leichtflüchtiger Organoselenverbindungen in der Gasphase der Kulturansätze festgestellt werden, die im unteren Nanogrammbereich lagen. Einhergehend damit wurde auch die Zunahme der Gehalte an gelöstem Selen und somit die biologisch bedingte Rücklösung aus der Mineral- in die Wasserphase beobachtet. Es konnte gezeigt werden, dass die Aktivität von Mikroorganismen einen deutlichen Einfluss auf die Stabilität von Oberflächenkomplexen des Selenits als auch von mineralischen Selenidspezies hat.

Stable isotope fractionation of selenium by biomethylation in soil

Selen ist in geringen Mengen ein essentielles Nährelement, das aber in höheren Gehalten toxisch wird. Der Se-Kreislauf in der Umwelt ist eng mit Redoxreaktionen wie der Reduktion von Se-Oxyanionen zu Methylselenid verknüpft. Flüchtige Methylselenide sind weit verbreitet und stellen einen wichtigen Se-Fluss in der Umwelt dar. Das übergeordnete Ziel meiner Dissertation war, die Stabilisotopenfraktionierung von Se durch Biomethylierung verschiedener oxidierter Se-Spezies (Se[IV] und Se[VI]) im Boden aufzuklären. Zunächst wurde eine Methode entwickelt, die es erlaubte flüchte Methylselenide quantitativ zurückzuhalten. Es zeigte sich, dass alkalische Peroxid-Lösung dafür geeignet war. Mit alkalischer Peroxid-Lösung wurde eine Wiederfindung von 95,6 ± Standardabweichung 5,4% in Verflüchtigungsexperimenten mit Methylselenid-Standards erreicht. Bei Einsatz von alkalischer Peroxid-Lösung in geschlossenen Mikrokosmos-Experimenten kam es zu keinen Se-Verlusten und ausgeglichenen Se-Isotopenbilanzen. Die massengewichteten δ82/76Se-Werte lagen für Se(IV) und Se(VI) am Ende der Mikrokosmos-Inkubationen bei -0,31 ± 0,05‰ (n = 3) und -0,76 ± 0,07‰ (n = 3) verglichen mit -0,20 ± 0,05‰ und -0,69 ± 0,07‰ im jeweils zugegebenen Se. Im zweiten Teil meiner Dissertation wurde die Pilzart Alternaria alternata mit Se(VI) und Se(IV) in geschlossenen Mikrokosmen für 11-15 und Se(IV) zusätzlich für 3-5 Tage bei 30°C inkubiert. In 11-15 Tagen wurden 2,9-11% des Se(VI) und 21-29% des Se(IV) und in 3-5 Tagen, 3-5% des Se(IV) methyliert. Die anfänglichen δ82/76Se-Werte von Se(VI) und Se(IV) lagen bei -0,69 ± 0,07‰, und -0,20 ± 0,05‰. Die δ82/76Se-Werte der Methylselenide unterschieden sich nach 11-15 Tagen Inkubation signifikant zwischen Se(VI) (-3,97 bis -3,25 ‰) und Se(IV) (-1,44 bis -0,16‰) als Quellen. Die δ82/76Se-Werte der Methylselenide zeigen also die Quellen der Biomethylierung von Se an. Die kürzere Inkubation von Se(IV) für 3-5 Tage führte zu einer ausgeprägten Se-Isotopenfraktonierung von mindestens -6‰, bevor ein Fließgleichgewicht erreicht wurde. Im dritten Teil bestimmte ich die Bindungsformen von Se mit drei operativ definierten sequentiellen Extraktionen und die δ82/76S-Werte des gesamten Selens in zehn urbanen Oberböden mit 0,09-0,52 mg/kg Se, die fünf verschiedene Landnutzungstypen repräsentierten (Überschwemmungsgrünland, Garten, Park, Straßenrand und Wald). Nur ein kleiner Teil des Seleniums lag in austauschbarer und damit direkt bioverfügbarer und in residualer, wenig reaktiver Form vor. Das meiste Se war an die organische Substanz und Fe-(Hydr-)Oxide gebunden (42-77% des gesamten Selens). Der mittlere δ82/76Se-Wert des gesamten Selens in den Oberböden lag mit -0,03 ± 0,38‰ nahe beim Mittelwert der gesamten Erde. Geringfügig niedrigere Se-Isotopensignale von -0,59 bis -0,35‰ v.a. in Waldböden und geringfügig höhere von 0,26 to 0,45‰ in Überschwemmungsgrünland wurden vermutlich durch Boden-Pflanze-Recycling und Se-Kontaminationen durch das Flusswasser verursacht. Der vierte Teil umfasste ein “Natural Attenuation”-Experiment und Mikrokosmos-Inkubationen von Bodenproben mit A. alternata. Die Equilibrierung von zum Boden gegebenem Se(IV) und Se(VI) für drei Tage führte zu abnehmenden wasserlöslichen Se-Gehalten um 32-44% bzw. 8-14, die mit kleinen Isotopenfraktionierung (ε = -0,045 bis -0,12 ‰ and -0,05 to -0,07‰ verbunden waren. In zwei der inkubierten Böden mit mäßig sauren pH-Werten wurden zwischen 9,1 und 30% des zugefügten Se(IV) und 1,7% des zugefügten Se(VI) methyliert während in einem stark sauren Boden keine Methylierung auftrat. Das aus Se(IV) entstandene Methylselenid war deutlich gegenüber dem zugegebenen Se-Standard (0,20‰) an 82Se verarmt (δ82/76Se = -3,3 bis -4,5‰). Meine Ergebnisse zeigen, dass die stabilen Isotopenverhältnisse von Se neue Einblicke in Se-Transformationsprozesse erlauben.rn

IgE-bearing cells in bronchoalveolar lavage fluid and allergen-specific IgE levels in sera from RAO-affected horses

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterized by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of immunoglobulin E antibodies (IgE) in the pathogenesis of RAO is unclear. We hypothesized that the number of cells with receptor-bound IgE in bronchoalveolar lavage fluid (BALF) and IgE levels in serum would be higher in RAO-affected than in healthy horses living in the same environment. Therefore, IgE-positive (+) cells were identified by immunocytochemistry on cytospins from BALF and counted. IgE levels against the mould extracts Aspergillus fumigatus (Asp. f.) and Alternaria alternata (Alt. a.) and the recombinant mould allergen Aspergillus fumigatus 8 (rAsp f 8) were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of seven RAO-affected and 22 clinically healthy mature horses housed in the same conventional stable environment. After correcting for the number of neutrophils, there were no significant differences in IgE+ cells on cytospins from BALF between both groups of horses (5% versus 7%, P > 0.1). Serum IgE levels against the mould extracts were significantly higher in RAO-affected than in clinically healthy horses [median = 119 versus 66 relative ELISA units (REU), P < 0.05]. Furthermore, significantly more RAO-affected than healthy horses had detectable serum IgE against the recombinant allergen rAsp f 8 (4/7 and 3/22, respectively, P < 0.05). Age had no significant effect on BALF cell ratios or on specific serum IgE levels. These results show that high IgE levels against mould antigens are associated with RAO under controlled environmental conditions but ranges of mould-specific serum IgE levels overlapped too much between diseased and clinically healthy animals to be of any diagnostic value. Further studies are needed to assess whether IgE-mediated reactions contribute to the pathogenesis of RAO.

Isotope fractionation of selenium by biomethylation in microcosm incubations of soil

The natural abundance of stable Se isotopes in methylselenides reflects sources and formation conditions of methylselenides. We tested the effects of (i) different inorganic Se species spiked to soils and (ii) different soil samples on the extent of fungal biomethylation of Se and the Se isotope ratios (δ82/76Se) in methylselenides. Furthermore, we assessed the decrease of dissolved, bioavailable Se during three days of equilibration of the soils with Se-enriched solutions. We conducted closed microcosm experiments containing soil spiked with Se(IV) or Se(VI), a growth medium, and the fungus species Alternaria alternata for 11 d. The concentrations and isotope ratios of Se were determined in all components of the microcosm with multicollector ICP-MS. The equilibration of the spiked Se(IV) and Se(VI) for 3 d resulted in a decrease of dissolved, bioavailable Se concentrations by 32 to 44% and 8 to 14%, respectively. Very little isotope fractionation occurred during this phase, and it can be attributed to mixing of the added Se with the pre-existing Se in the soils and minor Se(IV) reduction in one experiment. In two of the incubated soils – moderately acidic roadside and garden soils – between 9.1 and 30% of the supplied Se(IV) and 1.7% of the supplied Se(VI) were methylated while in a strongly acidic forest soil no Se methylation occurred. The methylselenides derived from Se(IV) were strongly depleted in 82Se (δ82/76Se = − 3.3 to − 4.5‰) compared with the soil (0.16–0.45‰) and the added Se(IV) (0.20‰). The methylselenide yield of the incubations with Se(VI) was too small for isotope measurements. Our results demonstrate that Se source species and soil properties influence the extent of Se biomethylation and that the produced methylselenides contain isotopically light Se.

Microbiota asociada a la enfermedad de la punta negra del trigo duro. Efectos del riego, el abonado nitrogenado y la variedad cultivada en la incidencia de la enfermedad

La enfermedad fúngica de la punta negra del trigo se caracteriza por provocar en las semillas afectadas un oscurecimiento de la zona del embrión que en ocasiones puede extenderse hacia la hendidura central dejando el grano completamente ennegrecido. Su incidencia en el cultivo de trigo es extremadamente variable y depende en gran medida de las condiciones ambientales, así pues, condiciones de alta humedad pueden incrementar a punta negra. Su presencia en el trigo duro repercute en el rendimiento semolero. Además la pasta elaborada a partir de semilla enferma presenta manchas negras y adquiere color y olor desagradable. Esta enfermedad es poco conocida a nivel europeo, sin embargo existen numerosos estudios para conocer su etiología y los factores que afectan a su aparición en países como Nueva Zelanda, Australia, Estados Unidos o Canadá. El presente trabajo pretende dar a conocer esta patología en España, determinando la influencia del riego, el abonado nitrogenado y la variedad cultivada en la incidencia de la enfermedad. Para ello se ha contado con un diseño experimental basado en 10 cultivares sembrados en parcelas con dos tratamientos de riego y dos de abonado nitrogenado. El análisis de las semillas infectadas en cámara húmeda y medios de cultivo PDA y K reveló 12 géneros fúngicos diferentes, de los cuales Alternaria alternata y Fusarium proliferatum estaban presentes en todas las muestras. El estudio del riego y abonado nitrogenado mostró diferencias significativas en la incidencia de punta negra pero fueron los 10 cultivares incluidos en el ensayo los que mayor importancia cobraron desde el punto de vista de la aparición de la enfermedad. El genotipo resultó determinante a la hora de establecer los niveles de afectación ya que las muestras encuadradas botánicamente como Triticum turgidum subsp. Turgidum convar. Turgidum presentaron una mayor susceptibilidad. las prueas de patogenicidd con los tres principales hongos asociados a la punta negra dieron resultados negativos para la germinación-nascencia de las plántulas de trigo duro inoculadas.

Roles for mannitol and mannitol dehydrogenase in active oxygen-mediated plant defense

Reactive oxygen species (ROS) are both signal molecules and direct participants in plant defense against pathogens. Many fungi synthesize mannitol, a potent quencher of ROS, and there is growing evidence that at least some phytopathogenic fungi use mannitol to suppress ROS-mediated plant defenses. Here we show induction of mannitol production and secretion in the phytopathogenic fungus Alternaria alternata in the presence of host-plant extracts. Conversely, we show that the catabolic enzyme mannitol dehydrogenase is induced in a non-mannitol-producing plant in response to both fungal infection and specific inducers of plant defense responses. This provides a mechanism whereby the plant can counteract fungal suppression of ROS-mediated defenses by catabolizing mannitol of fungal origin.

A comparison of the effects of ocular preservatives on mammalian and microbial ATP and glutathione levels

The aim of this study was to investigate the mechanism of action of the preservative sodium chlorite (NaClO2), and the relationship with intracellular glutathione depletion. A detailed comparison of the dose responses of two cultured ocular epithelial cell types and four species of microorganism was carried out, and comparisons were also made with the quaternary ammonium compound benzalkonium chloride (BAK), and the oxidant hydrogen peroxide (H2O2). The viability of mammalian and microbial cells was assessed in the same way, by the measurement of intracellular ATP using a bioluminescence method. Intracellular total glutathione was measured by reaction with 5,5'-dithiobis-2-nitrobenzoic acid in a glutathione reductase-dependent recycling assay. BAK and H2O2 caused complete toxicity to conjunctival and corneal epithelial cells at similar to25 ppm, in contrast to NaClO2 , where >100 ppm was required. The fungi Candida albicans and Alternaria alternata had a higher resistance to NaClO2 than the bacteria Staphyloccus aureus and Pseudomonas aeruginosa , but the bacteria were extremely resistant to H2O2 NaClO2 caused substantial depletion of intracellular glutathione in all cell types, at concentrations ranging from <10 ppm in Pseudomonas , 25-100 ppm in epithelial cells, to >500 ppm in fungal cells. The mechanisms of cytotoxicity of NaClO2 , H2O2 and BAK all appeared to differ. NaClO2 was found to have the best balance of high antibacterial toxicity with low ocular toxicity. The lower toxicity of NaClO2 to the ocular cells, compared with BAK and H2O2 , is in agreement with fewer reported adverse effects of application in the eye.

Production locality influences postharvest disease development and quality in mangoes

Postharvest disease management is one of the key challenges in commercial mango supply chains. Comprehensive investigations were made regarding the impact of geographic locality on postharvest disease development and other quality parameters in 'Sindhri' and 'Samar Bahisht (S.B.) Chaunsa' mangoes under ambient (33±1°C; 55-60% RH) and low temperature storage/simulated shipping (12±1°C; 80- 85% RH) conditions (28 or 35 days storage for 'Sindhri' and 21 or 28 days for 'S.B. Chaunsa'). Physiologically mature (days from fruit set were 95-100 and 110-115 for 'Sindhri' and 'S.B Chaunsa', respectively) 'Sindhri' and 'S.B. Chaunsa' fruits were harvested from five geographic localities and subjected to ambient and simulated shipping conditions. Under ambient conditions, no disease incidence was observed till fruit eating stage in 'Sindhri'. However, in 'S.B. Chaunsa', significant variation in different localities was observed with respect to disease incidence. Maximum and at par disease was exhibited by the fruit collected from district Vehari and Khanewal in 'S.B. Chaunsa'. Under simulated shipping conditions, disease development varied significantly with respect to different locations and storage durations. In 'Sindhri', fruit of M. Garh, while, 'S.B. Chaunsa' fruit of districts R.Y. Khan, M. Garh and Khanewal showed higher disease incidence. Fruit peel colour development was significantly reduced as storage days increased. Fruit firmness, skin shriveling, fresh weight loss, dry matter, biochemical and organoleptic attributes also varied significantly among the fruit sourced from different orchards of different localities. Analysis of N contents in leaves and fruit peel revealed that N contents of leaf and peel were positively correlated with disease severity in mango. Botryodiplodia spp., Phomopsis mangiferae, Alternaria alternata, Colletotrichum gloeosporioides were the pathogens isolated from fruits of all locations; however, the prevalence frequency varied with the geographic localities. In conclusion, the production locality, cultivar and nutrition (nitrogen content of fruit peel) had significant effect on fruit quality out-turn at ripe stage in terms of disease development so area specific disease management system needs to be implemented for better quality at retail.

UNDERSTANDING LATE SEASON FRUIT ROT PATHOSYSTEMS AND INSECT INTERACTIONS IN MID-ATLANTIC VINEYARDS

Fungal fruit rots and insect pests are among the most important problems negatively affecting the yield and quality of mid-Atlantic wine. In pathogenicity trials of fungi recovered from diseased Chardonnay and Vidal blanc grapes, Alternaria alternata, Pestalotiopsis telopeae, and Aspergillus japonicus were found to be unreported fruit rot pathogens in the region. Additionally, P. telopeae and A. japonicus had comparable virulence to the region’s common fruit rot pathogens. Furthermore, a timed-exclusion field study was implemented to evaluate vineyard insect-fruit rot relationships. It was found that clusters exposed to early-season insect communities that included Paralobesia viteana had a significantly greater incidence of sour rot than clusters protected from insects all season. These results were contrary to the current assumption that fall insects are the primary drivers of sour rot in the region. This research provides diagnostic tools and information to develop management-strategies against fungal and insect pests for mid-Atlantic grape growers.

Fungal endophytic communities associated to the phyllosphere of grapevine cultivars under different types of management

Fungal endophytes present in different asymptomatic grapevine plants (Vitis vinifera L.) located in different vineyards within Alentejo, a highly important viticulture region in Portugal, were identified in this study. Sampled grapevine plants included the three most representative cultivars in the region, Syrah, Cabernet Sauvignon, and Aragonez, growing under two different modes of management, conventional and biological. Sixteen fungal taxa were identified through sequencing of the internal transcribed spacer region. Total number of endophytic fungi isolated showed significant differences both in management mode and in cultivars, with higher numbers in grapevines under conventional mode and from Syrah cultivar. The composition of fungal endophytic communities did not show significant differences among cultivars, but differences were observed between fungal communities isolated from grapevines under biological or conventional modes. The most fungal taxa isolated from grapevines cultivated under biological mode were Alternaria alternata, Cladosporium sp., and Nigrospora oryzae, and under conventional mode Botrytis cinerea, Epicoccum nigrum, and Epicoccum sp. These differences suggest that the different products used in grapevine production have impacts in fungal endophytic composition. Further investigation of the identified fungi with respect to their antagonistic characteristics and potential use in plant protection to ensure food safety is now in course.

Análise por CLAE para determinação da biodegradabilidade do fungicida carbendazim em meio de cultura líquido.

Uma vez que as técnicas cromatográficas permitem a separação, quantificação e identificação dos compostos organicos parentais e dos produtos de degradação e, considerando-se que a maioria dos fungicidas benzimidazois absorvem fortemente a luz ultra-violeta, testou-se o método de Cromatografia Liquida de Alta Eficiência (CLAE), para quantificar a biodegradabilidade do fungicida carbendazim. Neste experimento foi utilizada uma linhagem de Alternaria alternata isolada de solos agrícolas suplementados com benomil. Os frascos com os microorganismos crescidos foram repicados para frascos contendo meios de cultura batata-dextrose (50%) + carbendazim (100mg/ml). Estes foram incubados sob agitacao a 28oC mais ou menos 1oC por períodos de 2,4,7,15 e 30 dias. Apos extração e purificação das amostras procedeu-se a analise cromatográfica. Para tanto utilizou-se uma coluna de troca cationica nas seguintes condições: temperatura da coluna: 40oC, fase móvel: fosfato de amônio 0,0125M com fluxo de 0,2ml por minuto e detector de absorbância operando a 280nm. Sob estas condições, o tempo de retenção de carbendazim e 2 amino-benzimidazol foi de aproximadamente 4,5 e 6,3 minutos, respectivamente. A quantificação foi feita utilizando-se regressão linear de curvas de calibração obtidas em soluções padrões de carbendazim versus a resposta (altura ou área do pico), obtida no cromatograma. A confirmação da identidade do pico com alto grau de confiança foi possível pela comparação do tempo de retenção e o espectro de absorbância em ultra-violeta do carbendazim. Este espectro, em diferentes condições de cultivo de A alternata, indicou índices de similaridade da molécula variando de 0,99 a 1 entre as diferentes amostras. Por este método foram obtidas recuperações acima de 70% da concentração inicial de carbendazim. A degradação de carbendazim foi de 66,4% aos dois dias de incubação.

Avaliação da população fúngica do solo e degradação do fungicida carbendazin.

Degradação acelerada esta geralmente associada a repetidas aplicações do mesmo ou de um pesticida estruturalmente relacionado. Os fungicidas sistêmicos do grupo dos benzimidazois se caracterizam por uma alta seletividade, agindo em poucos processos do metabolismo dos patógenos e seu uso indiscriminado podem causar sérios problemas de contaminação ambiental. Solos de três regiões agrícolas foram enriquecidos sucessivamente com 100ppm de benomil. Após incubação, isolamentos foram feitos em meio de cultura suplementado com o fungicida (100 ppm) e colonias individuais foram avaliadas quanto a resistência da população fúngica, com diferentes concentrações do ingrediente ativo. Treze isolados foram avaliados para observação da capacidade de degradação em meio de cultura liquido suplementado com carbendazin e nesta forma ele atua como fungicida. A extração de MBC foi feita com acetato de etila e acido clorídrico e a analise dos metabolitos efetuada através de HPLC. O fungo Alternaria alternata mostrou-se eficiente em degradar acima de 60% de MBC.

Pathogenic variation of Alternaria species associated with leaf blotch and fruit spot of apple in Australia

Four Alternaria species groups (A. longipes, A. arborescens, A. alternata/A. tenuissima and A. tenuissima/A. mali) are associated with leaf blotch and fruit spot of apple in Australia. There is no information on the variability of pathogenicity among the species and isolates within each species causing leaf blotch or fruit spot. We used a detached leaf assay and an in planta fruit inoculation assay to determine the pathogenicity and virulence of the four Alternaria species. Our results showed that isolates within the same species were not specific to either leaf or fruit tissue and showed great variability in pathogenicity and virulence, indicating cross-pathogenicity, which may be isolate dependent rather than species dependent. Generally, virulence of A. tenuissima and A. alternata isolates on leaf and fruit was higher than other species. Isolates of all species groups were pathogenic on leaves of different cultivars, but pathogenicity on fruit of different cultivars varied among isolates and species. Implications of our findings on prevalence of the diseases in different apple-producing regions in Australia and the development of targeted disease management of the diseases are discussed

Molecular characterisation of Alternaria linicola and its detection in linseed

Nucleotide sequences of the ribosomal DNA (rDNA) internal transcribed spacers (ITS) 1 and 2 and a 1068 bp section of the beta-tubulin gene divided seven designated species of Alternaria into five taxa. Stemphylium botryosum formed a sixth closely related taxon. Isolates of A. linicola possessed an identical ITS sequence to one group of A. solani isolates, and two clusters of A. linicola isolates, revealed from beta-tubulin gene data to show minor variation, were as genetically similar to isolates of A. solani as they were to each other. We suggest, therefore, that A. linicola falls within the species A. solani. Similar results suggest that A. lini falls within the species A. alternata. RAPD analysis of the total genomic DNA from the Alternaria spp. concurred with the nucleotide sequence analyses. An oligonucleotide primer (ALP) was selected from the rDNA ITS1 region of A. linicola/A. solani. PCR with primers ALP and ITS4 (from a conserved region of the rDNA) amplified a c. 536 bp fragment from isolates of A. linicola and A. solani but not from other Alternaria spp. nor from other fungi which may be associated with linseed. These primers amplified an identical fragment, confirmed by Southern hybridization, from DNA released from infected linseed seed and leaf tissues. These primers have the potential to be used also for the detection of A. solani in host tissues.