Microsatellite-primed PCR Research Articles

Phylogenetic analysis and genetic diversity of the xylariaceous ascomycete Biscogniauxia\xa0mediterranea from cork oak forests in different bioclimates

Cork oak is a tree species with ecological importance that contributes to economic and social development in the Mediterranean region. Cork oak decline is a major concern for forest sustainability and has negative impacts on cork oak growth and production. This event has been increasingly reported in the last decades and seems to be related with climate changes. Biscogniauxia mediterranea is an endophytic fungus of healthy cork oak trees that turns into a pathogen in trees weaken by environmental stress. Understanding the drivers of B. mediterranea populations diversity and differentiation is expected to allow a better control of cork oak decline and preserve forest sustainability. Endophyte isolates from different cork oak forests were identified as B. mediterranea and their genetic diversity was evaluated using phylogenetic and microsatellite-primed PCR analyses. Genetic diversity and variability of this fungus was correlated with environmental/phytosanitary conditions present in forests/trees from which isolates were collected. High genetic diversity and variability was found in B. mediterranea populations obtained from different forests, suggesting some degree of isolation by distance. Bioclimate was the most significant effect that explained the genetic variability of B. mediterranea, rather than precipitation or temperature intensities alone or disease symptoms. These findings bring new implications for the changing climate to cork oak forests sustainability, cork production and quality.

Diversity of fungi associated with macroalgae from an estuarine environment and description of Cladosporium rubrum sp. nov. and Hypoxylon aveirense sp. nov.

Fungal communities associated with macroalgae remain largely unexplored. To characterize algicolous fungal communities using culture dependent methods, macroalgae were collected from different sampling sites in the Ria de Aveiro estuary, Portugal. From a collection of 486 isolates that were obtained, 213 representative isolates were selected through microsatellite-primed PCR (MSP-PCR) fingerprinting analysis. The collection yielded 33 different genera, which were identified using the ITS region of the rDNA. The results revealed that the most abundant taxa in all collections were Acremonium-like species: Alternaria, Cladosporium, Leptobacillium and Penicillium. The fungal community composition varied with macroalgae species. Through multilocus phylogenetic analyses based on ITS, tub2, tef1-α and actA sequences, in addition to detailed morphological data, we propose Cladosporium rubrum sp. nov. (type strain=CMG 28=MUM 19.39) and Hypoxylon aveirense sp. nov. (type strain=CMG 29=MUM 19.40) as novel species.

Vinification without Saccharomyces: Interacting Osmotolerant and "Spoilage" Yeast Communities in Fermenting and Ageing Botrytised High-Sugar Wines (Tokaj Essence).

The conversion of grape juice to wine starts with complex yeast communities consisting of strains that have colonised the harvested grape and/or reside in the winery environment. As the conditions in the fermenting juice gradually become inhibitory for most species, they are rapidly overgrown by the more adaptable Saccharomyces strains, which then complete the fermentation. However, there are environmental factors that even Saccharomyces cannot cope with. We show that when the sugar content is extremely high, osmotolerant yeasts, usually considered as “spoilage yeasts“, ferment the must. The examination of the yeast biota of 22 botrytised Tokaj Essence wines of sugar concentrations ranging from 365 to 752 g∙L−1 identified the osmotolerant Zygosaccharomyces rouxii, Candida (Starmerella) lactis-condensi and Candida zemplinina (Starmerella bacillaris) as the dominating species. Ten additional species, mostly known as osmotolerant spoilage yeasts or biofilm-producing yeasts, were detected as minor components of the populations. The high phenotypical and molecular (karyotype, mtDNA restriction fragment length polymorphism (RFLP) and microsatellite-primed PCR (MSP-PCR)) diversity of the conspecific strains indicated that diverse clones of the species coexisted in the wines. Genetic segregation of certain clones and interactions (antagonism and crossfeeding) of the species also appeared to shape the fermenting yeast biota.

Revealing the hidden diversity of marine fungi in Portugal with the description of two novel species, Neoascochyta fuci sp. nov. and Paraconiothyrium salinum sp. nov.

Fungi are ubiquitous organisms with a wide distribution in almost all ecosystems, including marine environments. Coastal and estuarine ecosystems remain poorly unexplored as fungal habitats, potentially harbouring a hidden diversity with important ecological roles. During an extensive survey of marine fungi in coastal and estuarine Portuguese environments, a collection of 612 isolates was obtained from water, algae, sponges and driftwood. From these, 282 representative isolates were selected through microsatellite-primed PCR (MSP-PCR) fingerprinting analysis, which were identified based on DNA sequence data. The collection yielded 117 taxa from 38 distinct genera, which were identified using DNA sequence analysis. Overall, fungal community composition varied with host/substrate, but the most abundant taxa in the collection were Cladosporium cladosporioides, Penicillium terrigenum, Penicillium brevicompactum and Fusarium equiseti/incarnatum complex. The occurrence of a high fungal diversity harbouring novel species was disclosed. Through a multilocus phylogeny based on ITS, tub2 and tef1-α sequences, in conjunction with morphological and physiological data, we propose Neoascochyta fuci sp. nov. and Paraconiothyrium salinum sp. nov.

Diversity and pathogenicity of Lasiodiplodia and Neopestalotiopsis species associated with stem blight and dieback of blueberry plants in Peru

The production of blueberry in Peru has been increasing over the last years. Dieback and stem blight of plants are the most prominent disease symptoms observed in blueberry orchards. This study evaluated the diversity and pathogenicity of Lasiodiplodia and Neopestalotiopsis species associated with dieback of blueberry plants in Peru. A collection of isolates was initially subjected to Microsatellite-primed PCR (MSP-PCR) fingerprinting and further characterised by sequence analyses of the rDNA internal transcribed spacer region (ITS), translation elongation factor 1-alpha (tef1-α) and β-tubulin (tub2). The phylogenetic analyses revealed the presence of Lasiodiplodia theobromae, L. laeliocattleyae and Neopestalotiopsis rosae. Lasiodiplodia laeliocattleyae and N. rosae are reported for the first time in blueberry plants associated with dieback and stem blight symptoms. Additionally, N. rosae is newly reported from Peru. Inoculation trials of blueberry plants confirmed the pathogenicity of Lasiodiplodia and Neopestalotiopsis species and revealed differences in aggressiveness among species and isolates.

Biscogniauxia mediterranea associated with cork oak (Quercus suber) in Tunisia: Relationships between phenotypic variation, genetic diversity and ecological factors

Biscogniauxia mediterranea is a xylariaceous ascomycete responsible for ‘charcoal disease’, the most frequent disease of cork oak in the Mediterranean basin. However, little is known regarding this fungus in North Africa. This is the first report on the morphological and genetic diversity of B. mediterranea populations in Tunisia. A total of forty-eight isolates from cork oak trees, corresponding to three different sites and three tree parts, were identified as B. mediterranea based on morphological and molecular diagnosis. Variability among the isolates was evaluated using morphological traits and microsatellite-primed PCR (MSP-PCR). B. mediterranea isolates showed high variability in culture, specifically in colony color. A high level of genetic diversity within populations and within tree parts was detected (H = 0.417, I = 0.605, and H = 0.415, I = 0.601 respectively). Significant genetic differentiation among populations (G ST = 0.116 and Φ ST = 0.137; p < 0.001) was determined. However, a low genetic differentiation among tree parts (G ST = 0.066 and Φ ST = 0.018; p = 0.07) was observed. Principal coordinate analysis showed that genetic divergence was partially related to the geographical origin. Cluster analysis based on morphological traits showed that the grouping of the isolates occurs independently of the geographic location and the tree part. The effective number of alleles (Ne), the Nei's gene diversity index (H), the Shannon's information index (I), the colony color and the presence of exudates were significantly correlated with the altitude and the rainfall. The number of alleles (Na), the percentage of polymorphic loci (PPL), the texture and the density of the mycelium were significantly correlated with the temperature. These results provide a deeper understanding of population genetics of B. mediterranea and its adaptation to environmental conditions which could help to develop control strategies against charcoal canker disease.

Preservation of diversity and oenological properties of wine yeasts during long-term laboratory maintenance: A study of strains of a century-old Tokaj wine yeast collection

The effect of long-term laboratory maintenance on the biological diversity and certain oenolological properties of wine yeasts was investigated by the analysis of 17 strains isolated from Tokaj (Hungary) wines more than a century ago and maintained since then in culture collections. The analysis of the D1/D2 domains of the large subunit ribosomal RNA genes and the ITS sequences assigned them all to S. cerevisiae but divided them into two groups. One group showed sequence identity with wine strains occurring mainly in countries of the region. The combined results of the interdelta, RAPD, microsatellite-primed PCR (MSP), mtDNA and karyotype analyses found both groups diverse and defined each strain as unique, demonstrating that the long period of maintenance under identical conditions has not resulted in convergent evolution in their genomes. High diversity was detected also in certain phenotypic traits of oenological relevance (osmotolerance, killer phenotype, production of glycerol, ethanol, H2S and volatile acids). In the microvinification experiments all strains performed better than the laboratory strain S288c and were comparable to the industrial strain used as a control or even surpassed it in certain parameters. Thus, the oenological abilities of wine yeasts can be preserved over long periods of maintenance under laboratory conditions.

Analysis of the genetic diversity and phylogenetic relationships of Biscogniauxia mediterranea isolates associated with cork oak

Biscogniauxia mediterranea is one of the most frequent fungal pathogens involved in cork oak decline in the Mediterranean Basin, causing charcoal canker. In Portugal, this disease is widespread on adult declining trees but nowadays it increasingly affects young trees and exhibits atypical symptoms, leading to the hypothesis that some change in the fungus may have occurred. In order to evaluate the genetic diversity and phylogenetic relationship of B. mediterranea associated with cork oak, 102 isolates were obtained from young and adult trees of Quercus suber and other hosts species with different disease expression, from several Mediterranean countries. The collection of isolates was analyzed by individual and multigene phylogenies using Maximum-Likelihood approach based on nucleotide sequences of the internal transcribed spacers of ribosomal DNA, translation elongation factor 1-α and β-tubulin genes, and by microsatellite-primed PCR profiles. Sequence analyses separated the Mediterranean isolates from those from other regions, while MSP-PCR analysis revealed relevant but unstructured diversity among the Mediterranean isolates under study, making this a monophyletic but diverse population. Considering the adaptive capacity of the fungus in the Mediterranean-climate ecosystems and the present climatic change scenario, all conditions are gathered to favor aggravation of the disease in cork oak stands.

Trichosporon heliocopridis sp. nov., a urease-negative basidiomycetous yeast associated with dung beetles (Heliocopris bucephalus Fabricius)

Ninety-six yeast isolates associated with dung beetles (Heliocopris bucephalus Fabricius) were examined based on a culture-dependent method. A comparison of the colony morphology and PCR-fingerprints obtained by (GTG)5 microsatellite-primed PCR indicated that 84 of these isolates belonged to one group. Five strains (DD1-1T, DD2-33, DD4-11, DD5-15 and DD6-1) were selected as the representatives of this main group, where each of the five selected strains had been derived from a different dung beetle collected in northern Thailand. A comparison of the D1/D2 domain sequence of the large subunit rRNA gene (LSU D1/D2) and the internal transcribed spacer (ITS) sequences revealed that these five strains were the same and were related to the genus Trichosporon. Phylogenetic analysis based on the LSU D1/D2 plus ITS sequences placed this group within the Trichosporon brassicae clade, but it was clearly separated from any known species. In addition, physiological tests showed that this group had the unusual property of the inability to hydrolyse urea, which was distinctly different from the related taxon. Therefore a novel yeast species named Trichosporon heliocopridis sp. nov. (ex-type strain DD1-1T = TISTR 5946T = JCM 30786T = CBS 14168T) is proposed. The MycoBank number is MB812098.

Morphological and genetic diversity of Biscogniauxia mediterranea associated to Quercus suber in the Mediterranean Basin

Biscogniauxia mediterranea is a widespread fungus that causes charcoal disease on cork oak and other hardwood hosts. It had been considered a secondary pathogen causing the disease only in stressed hosts. However, its frequency and severity have been increasing, inclusive in young trees without other decline signs and developing atypical symptoms. The present work aims to assess the fungus' variability in the Mediterranean basin, following cork oak geographical distribution. A collection of 36 isolates originated from cork oak in Portugal and other Mediterranean countries, from other hosts and from trees with different ages and disease expression were analyzed by cultural characteristics, conidial dimensions, and growth rates at different temperatures and by microsatellite-primed PCR profiles. Clustering UPGMA analyses combining different parameters were preformed. All the approaches revealed high level of intraspecific polymorphism among Mediterranean isolates, not allowing relating the disease development with any analyzed features. The results highlighted the variability of this fungus that induces its adaptation ability in the present worrying scenario of climatic change. All the conditions are gathered to favor the aggravation of the disease in cork oak stands.

Characterization of Iranian nonaflatoxigenic strains of Aspergillus flavus based on microsatellite-primed PCR.

Out of fifty-two Iranian nonaflatoxigenic strains of Aspergillus flavus,collected from various substrates (soil and kernel) and sources (peanut, corn and pistachio), fifteen representatives were selected according to their different geographical origins (six provinces: Guilan and Golestan, Ardebil, Fars, Kerman and Semnan) and vegetative compatibility groups (VCGs, IR1 to IR15) for microsatellite-primed PCR analysis. Two inter-simple sequence repeat (ISSR) primers AFMPP and AFM13 were used to determine polymorphism and the relationship among strain isolates. A. flavus isolates were identified by their morphologies and their identities were confirmed by PCR amplification using the specific primer pair ITS1 and ITS4. The results revealed variations in the percentages of polymorphisms. In the ISSR analysis, primers AFMPP and AFM13 generated a total of 18 and 23 amplicons among the fungal strains, out of which 12 (66.7%) and 22 (95.7%) were polymorphic, respectively. Cluster analysis of the ISSR data was carried out using 1 D DNA gel image analysis. The two dendrograms obtained through these markers showed six different clusterings of testing nonaflatoxigenic A. flavus L strains, but we noticed that some clusters were different in some cases. The microsatellite-primed PCR data revealed that the Iranian nonaflatoxigenic isolates of A. flavus were not clustered according to their origins and sources. This study is the first to characterize Iranian nonaflatoxigenic isolates of A. flavususing ISSR markers.

Effect of Denture-Related Stomatitis Fluconazole Treatment on Oral Candida albicans Susceptibility Profile and Genotypic Variability.

Denture-related stomatitis (DRS) is the most common condition affecting removable-denture wearers, and Candida albicans the most frequent pathogenic agent. Systemic antifungal treatment is indicated but recurrences are frequent. The aim of this study was to characterize the oral load, fluconazole susceptibility profile and genotypic variability of oral C. albicans isolates from patients with DRS before (T0), immediately after fluconazole treatment (Tat) and after 6-months follow-up (T6m). Eighteen patients presenting DRS and treated with fluconazole were followed at the Faculty of Dentistry of Oporto University. Seventy C. albicans isolates were obtained and identified using standard cultural and biochemical multi-testing. Fluconazole susceptibility was tested by E-test®. Microsatellite-primed PCR was performed to assess the genotypic variability of C. albicans isolates. The patients’ mean age was 58.0±3.2 years, and 55.6%/44.4% had total/partial dentures. Before treatment, 22.2%, 44.4% and 33.3% of the patients presented DRS type I, II or III, respectively. Fluconazole treatment healed or improved DRS in 77.8% of the patients, accompanied by an 83.5% reduction in oral C. albicans load. However, after 6-months, oral C. albicans load increased significantly and DRS severity was similar to the one observed before treatment. Moreover, the prevalence of patients presenting fluconazole resistant isolates of C. albicans increased significantly throughout the study: T0-5.6%, Tat-10.0% and T6m-42.9%. A change in the genotypic variability of C. albicans isolates was also verified, being mostly associated to fluconazole susceptibility profile change. In conclusion, fluconazole presents a good short-term DRS treatment efficiency, but may be associated to a long-term emergence of C. albicans fluconazole resistance.

Microsatellite-primed PCR for intra-species genetic relatedness in Trichophyton ajelloi strains isolated in poland from various soil samples.

Trichophyton ajelloi is a geophilic dermatophyte that specializes in the decomposition of native keratin. It exists in soil with a permanent influx of keratin matter. In the present study, two PCR-based methods were used for the identification and intra-species differentiation of T. ajelloi strains isolated from 3 types of soils with different physicochemical properties. The first method, employed for molecular identification, was PCR amplification of the 5.8S rRNA gene and its flanking regions encoding internal transcribed spacers (ITSs), followed by restriction enzyme digestion using endonuclease HinfI. The second method, employed for molecular differentiation, was microsatellite-primed PCR (MSP-PCR) using the repetitive oligonucleotide (GACA)4. All the T. ajelloi strains were also identified using a traditional culture method. Our results showed that molecular identification using the PCR-restriction fragment length polymorphism (PCR-RFLP) method agreed with the identification made using the traditional approach. On the other hand, PCR-RFLP results showed no strain differentiation, while MSP-PCR using the (GACA)4 primer identified different varieties among the T. ajelloi strains. The reasons for the intra-species differentiation of T. ajelloi have been discussed.

Genetic diversity of Valsa malicola isolates assessed by microsatellite-primed PCR (MP-PCR)

Genetic diversity of 40 isolates of Valsa malicola in Iran was investigated by MP-PCR. Isolates were obtained from different host plants in diverse regions during 2004–2010. Of the six microsatellite primers tested, only four primers; (ACTG)4, (GACA)4, (AC)8 and (CGA)5, were able to amplify DNA fragments. With these four primers, 120 loci were identified; of which eight were monomorphic (6.7%) and 112 were polymorphic (93.3%). Approximate size of amplified DNA fragments ranged from 0.2 to 3 kb. Observed high genetic diversity in isolates of V. malicola indicates the eligibility of the marker to investigate ranks below the species level. The results of cluster analysis indicated that isolates are related to each other with 45.63% similarity and four groups (1, 2, 3 and 4) were identified in the dendrogram. Group 3 included 37 isolates that were obtained from different hosts and geographical regions and each of groups 1, 2 and 4 only had one isolate. Some correlations between identified groups and host origins or geographic distributions of the isolates were found.

Molecular characterization and a multiplex allele-specific PCR method for detection of thiabendazole resistance in Penicillium expansum from apple

Thiabendazole (TBZ) is commonly used as a postharvest treatment for control of blue mold in apples caused by Penicillium expansum. Different point mutations in the β-tubulin gene conferring benzimidazole resistance have been reported in plant pathogens, but molecular mechanisms of TBZ resistance in P. expansum from apple in Washington State are unknown. Determination of TBZ resistance level showed that all 102 TBZ-resistant (TBZ-R) isolates were highly resistant. Sequencing of the majority of the β-tubulin gene showed that 76 TBZ-R isolates harboured the E198V mutation and 26 harboured the F167Y mutation, and all the sensitive isolates did not possess any of the mutations, indicating that these two point mutations in the β-tubulin gene were correlated with TBZ resistance in P. expansum from apple in Washington State. There was no association between levels of TBZ resistance and types of point mutations (E198V or F167Y) in the β-tubulin gene. A multiplex allele-specific PCR assay was developed to detect these two mutations simultaneously. Microsatellite-primed PCR derived presence-absence matrix used to assess the genetic relationship among 56 isolates suggested that the resistance mutations originated several times independently and that there was no correlation between the types of point mutation and the genetic background of the isolates.

Zygosaccharomyces sapae sp. nov., isolated from Italian traditional balsamic vinegar

Fourteen yeast isolates were recovered from two traditional balsamic vinegar (TBV) samples collected in the provinces of Modena and Reggio Emilia, Italy. Microsatellite-primed-PCR (MSP-PCR) was used to de-replicate the isolate collection into two representative strains, ABT301(T) and ABT601. Phylogenetic analysis based on the D1/D2 domains of the 26S rRNA gene indicated that these strains represented a distinct species of the genus Zygosaccharomyces, closely related to Zygosaccharomyces rouxii and Zygosaccharomyces mellis. Physiological and morphological tests supported the recognition of a novel taxon of halotolerant, osmotolerant, non-psychrotolerant and maltose-fermentation-negative yeasts showing a chain or star-shaped pattern of budding cells, which remained attached to each other. Morphological observations offered evidence of ascospore formation. A novel species, Zygosaccharomyces sapae sp. nov., is proposed to accommodate these strains, with strain ABT301(T) (= CBS 12607(T) = MUCL 54092(T)) as the type strain. Based on D1/D2 domain phylogenetic analysis, the novel strains shared the highest sequence similarity (100 %) with Zygosaccharomyces sp. strain NCYC 3042, previously isolated from sugar [James, S. A., Bond, C. J., Stratford, M. & Roberts, I. N. (2005). FEMS Yeast Res 5, 747-755]. However, based on phylogenetic (internal transcribed spacers, ITS), PCR fingerprinting and physiological analyses, marked differences were observed between the novel species and strain NCYC 3042, and these results are discussed in more detail.

Cryphonectria naterciae: A new species in the Cryphonectria–Endothia complex and diagnostic molecular markers based on microsatellite-primed PCR

In a recent study intended to assess the distribution of Cryphonectria parasitica in Portugal, 22 morphologically atypical orange isolates were collected in the Midwestern regions. Eleven isolates were recovered from Castanea sativa, in areas severely affected by chestnut blight and eleven isolates from Quercus suber in areas with cork oak decline. These isolates were compared with known C. parasitica and Cryphonectria radicalis isolates using an integrated approach comprising morphological and molecular methods. Morphologically the atypical isolates were more similar to C. radicalis than to C. parasitica. Phylogenetic analyses based on internal transcribed spacer (ITS) and β-tubulin sequence data grouped the isolates in a well-supported clade separate from C. radicalis. Combining morphological, cultural, and molecular data Cryphonectria naterciae is newly described in the Cryphonectria–Endothia complex. Microsatellite-primed PCR fingerprinting with (GACA) 4 primer discriminated between C. naterciae, C. radicalis, and C. parasitica.

Fungus Symbionts Colonizing the Galleries of the Ambrosia Beetle Platypus quercivorus

Isolations were made to determine the fungal symbionts colonizing Platypus quercivorus beetle galleries of dead or dying Quercus laurifolia, Castanopsis cuspidata, Quercus serrata, Quercus crispula, and Quercus robur. For these studies, logs from oak wilt-killed trees were collected from Kyoto Prefecture, Japan. Fungi were isolated from the: (1) entrances of beetle galleries, (2) vertical galleries, (3) lateral galleries, and (4) the larval cradle of P. quercivorus in each host tree. Among the fungus colonies which appeared on YM agar plates, 1,219 were isolated as the representative isolates for fungus species inhabiting in the galleries based on their cultural characteristics. The validity of the visual classification of the fungus colonies was checked and if necessary properly corrected using microsatellite-primed PCR fingerprints. The nucleotide sequence of the D1/D2 region of the large subunit nuclear rRNA gene detected 38 fungus species (104 strains) of which three species, i.e., Candida sp. 3, Candida kashinagacola (both yeasts), and the filamentous fungus Raffaelea quercivora were isolated from all the tree species. The two yeasts were most prevalent in the interior of galleries, regardless of host tree species, suggesting their close association with the beetle. A culture-independent method, terminal restriction fragment length polymorphism (T-RFLP) analysis was also used to characterize the fungus flora of beetle galleries. T-RFLP patterns showed that yeast species belonging to the genus Ambrosiozyma frequently occurred on the gallery walls along with the two Candida species. Ours is the first report showing the specific fungi inhabiting the galleries of a platypodid ambrosia beetle.

Characterization of Fusarium avenaceum from lupin in central Alberta: genetic diversity, mating type and aggressiveness

Root rot, caused by Fusarium avenaceum (teleomorph: Gibberella avenacea), is an important disease of lupin (Lupinus angustifolius) and other crops in western Canada. Fusarium isolates collected from infected roots of lupin in central Alberta were characterized using two RAPD primers, three microsatellite-primed PCR markers, diagnostic primers for putative mating type, sequence comparison of the translation elongation factor 1α (EF1α) and internal transcribed spacer (ITS) region, and assessments of aggressiveness on lupin seedlings. The teleomorph phase of F. avenaceum has been reported only twice in the world, but both mating types were found throughout the province. Isolates of mating type 2 were slightly more aggressive than mating type 1 on five lupin cultivars. All 42 isolates of F. avenaceum assessed in this study had a unique multilocus genotype, which indicates a high level of genotypic diversity in the pathogen population. Cluster analysis did not reveal an association between the geographic location of the isolates and genetic relatedness based on genetic markers. Instead, the F. avenaceum isolates divided into two distinct groups, with isolates from both groups occurring together at many sites. Phylogenetic analysis also revealed high diversity and the presence of some European-like isolates within the Canadian pathogen population. The high genotypic diversity of isolates and the presence of both mating types across sites provide an indication that sexual reproduction may occur within populations of F. avenaceum in Alberta.

Phylogeny, morphology and pathogenicity of Diaporthe and Phomopsis species on almond in Portugal

The ascomycete genus Diaporthe includes plant pathogens and endophytes on a wide range of hosts including economically important crops. Anamorphs are coelomycetous and reside in the genus Phomopsis. Phomopsis amygdali is the causal agent of twig canker and blight of almonds. In a recent survey of dieback of almonds in Portugal, the most frequent fungi detected were Diaporthe/Phomopsis species. Isolates from almond and other Prunus species were characterised and grouped according to their microsatellite-primed PCR (MSP-PCR) profiles and representatives of the different groups were selected for a phylogenetic study based on the ITS rDNA region (ITS1–5.8S–ITS2). Combining morphological, cultural, molecular and pathogenicity data, three species were distinguished. Phomopsis amygdali was shown to be the main pathogen on almond and is epitypified in the present work. Diaporthe neotheicola is reported for the first time on this host. A third species represented by a single isolate could not be unequivocally identified.