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Banana (Musa acuminata) is the most important crop in the Canary Islands (38.9% of the total cultivated area). The main pathogen affecting this crop is the soil fungal Fusarium oxysporum f. sp. cubense subtropical race 4 (Foc-STR4), for which there is no effective control method under field conditions. Therefore, the use of native biological control agents may be an effective and sustainable alternative. This study aims to: (i) investigate the diversity and distribution of Trichoderma species in the rhizosphere of different banana agroecosystems affected by Foc-STR4 in Tenerife (the island with the greatest bioclimatic diversity and cultivated area), (ii) develop and preserve a culture collection of native Trichoderma species, and (iii) evaluate the influence of soil chemical properties on the Trichoderma community. A total of 131 Trichoderma isolates were obtained from 84 soil samples collected from 14 farms located in different agroecosystems on the northern (cooler and wetter) and southern (warmer and drier) slopes of Tenerife. Ten Trichoderma species, including T. afroharzianum, T. asperellum, T. atrobrunneum, T. gamsii, T. guizhouense, T. hamatum, T. harzianum, T. hirsutum, T. longibrachiatum, and T. virens, and two putative novel species, named T. aff. harzianum and T. aff. hortense, were identified based on the tef1-α sequences. Trichoderma virens (35.89% relative abundance) and T. aff. harzianum (27.48%) were the most abundant and dominant species on both slopes, while other species were observed only on one slope (north or south). Biodiversity indices (Margalef, Shannon, Simpson, and Pielou) showed that species diversity and evenness were highest in the healthy soils of the northern slope. The Spearman analysis showed significant correlations between Trichoderma species and soil chemistry parameters (mainly with phosphorus and soil pH). To the best of our knowledge, six species are reported for the first time in the Canary Islands (T. afroharzianum, T. asperellum, T. atrobrunneum, T. guizhouense, T. hamatum, T. hirsutum) and in the rhizosphere of banana soils (T. afroharzianum, T. atrobrunneum, T. gamsii, T. guizhouense, T. hirsutum, T. virens). This study provides essential information on the diversity/distribution of native Trichoderma species for the benefit of future applications in the control of Foc-STR4.

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The genus Pseudogymnoascus represents a diverse group of fungi widely distributed in different cold regions on Earth. Our current knowledge of the species of Pseudogymnoascus is still very limited. Currently, there are only 15 accepted species of Pseudogymnoascus that have been isolated from different environments in the Northern Hemisphere. In contrast, species of Pseudogymnoascus from the Southern Hemisphere have not yet been described. In this work, we characterized four fungal strains obtained from Antarctic marine sponges. Based on multilocus phylogenetic analyses and morphological characterizations we determined that these strains are new species, for which the names Pseudogymnoascus antarcticus sp. nov., Pseudogymnoascus australis sp. nov., Pseudogymnoascus griseus sp. nov., and Pseudogymnoascus lanuginosus sp. nov. are proposed. Phylogenetic analyses indicate that the new species form distinct lineages separated from other species of Pseudogymnoascus with strong support. The new species do not form sexual structures and differ from the currently known species mainly in the shape and size of their conidia, the presence of chains of arthroconidia, and the appearance of their colonies. This is the first report of new species of Pseudogymnoascus not only from Antarctica but also from the Southern Hemisphere.

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The preservation of mould-ripened salami was investigated during 48 days at 19-20 °C under different packaging conditions: (i) high barrier film filled with air, 100% N2 or under vacuum, (ii) biaxially oriented polypropylene film, (iii) microperforated polyethylene film and (iv) unpackaged. Sensory, texture profile, physicochemical and microbiological analyses were performed. Fungal quantification revealed two data groups. In group 1 (consisting of salami in microperforated polyethylene film, biaxially oriented polypropylene film and unpackaged) the conidium viability was relatively constant. In group 2 (salami preserved in high barrier film filled with air, 100% N2 or under vacuum) the conidium viability decreased due to the absence of oxygen and the high carbon dioxide volume fraction. SEM micrographs showed micromorphological changes in fungal structure; microperforated polyethylene film, biaxially oriented polypropylene film and unpackaged conditions preserved the conidial morphology, while high barrier film filled with air, 100% N2 or vacuum conditions collapsed the hyphae and most of the conidia. Salami packed in microperforated polyethylene film and biaxially oriented polypropylene film showed the most acceptable organoleptic characteristics and lower hardness and chewiness values after packaging.

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Novel Penicillium-like strains were isolated during the characterization of the mycobiota community dynamics associated with shrimp waste composting. Phylogenetic analysis of the partial ß-tubulin (BenA) gene and the ribosomal DNA internal transcribed spacer region (ITS1-5.8S-ITS2) sequences revealed that the novel strains were members of section Lanata-Divaricata and were closely related to Penicillium infrabuccalum DAOMC 250537T. On the basis of morphological and physiological characterization, and phylogenetic analysis, a novel Penicillium species, Penicillium pedernalense sp. nov., is proposed. The type strain is F01-11T (=CBS 140770T=CECT 20949T), which was isolated from whiteleg shrimp (Litopenaeus vannamei) heads waste compost in the Pedernales region (Manabí province, Ecuador).

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A basidiomycetous yeast, strain E2A-C3-II, was isolated from a marine sponge (Hymeniacidon sp.) collected at a depth of 6 m in Fildes Bay, King George Island, Antarctica. The phylogenetic analysis revealed that the yeast isolated is related to Leucosporidium drummii, Leucosporidiella muscorum and to the Leucosporidium scottii group, including Leucosporidiella creatinivora and Leucosporidiella yakutica. The analysis of the nucleotide differences and the genetic distances of the D1/D2 domain of the LSU rDNA gene and 5.8S ITS regions support that strain E2A-C3-II represents a new species. The novel species can be distinguished from L. drummii by its ability to assimilate L-sorbose, L-rhamnose, lactose and ribitol. The maximum temperature for growth was 25 °C. On the basis of morphological, biochemical and physiological characterization, and phylogenetic and nucleotide analysis, a novel basidiomycetous yeast species, Leucosporidium escuderoi f.a., sp. nov., is proposed. The type strain is E2A-C3-II(T) (=CBS 12734(T) =CECT 13080(T)). The Mycobank ( http://www.mycobank.org ) accession number is MB 804654. The nucleotide sequences of D1/D2 domain of the LSU rDNA gene and 5.8S-ITS regions obtained in this work have been deposited in Genbank under the Accession numbers JN181009 and JN197600, respectively.

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During the characterization of the mycobiota associated with shallow-water marine environments from Antarctic sea, a novel pink yeast species was isolated. Sequence analysis of the D1/D2 domain of the LSU rDNA gene and 5.8S-ITS regions revealed that the isolated yeast was closely related to Rhodotorula pallida CBS 320(T) and Rhodotorula benthica CBS 9124(T). On the basis of morphological, biochemical and physiological characterization and phylogenetic analyses, a novel basidiomycetous yeast species, Rhodotorula portillonensis sp. nov., is proposed. The type strain is Pi2(T) ( = CBS 12733(T)  = CECT 13081(T)) which was isolated from shallow-water marine sediment in Fildes Bay, King George Island, Antarctica.

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During the characterization of the microbiota biodiversity associated with grapes and wineries in different bioclimatic conditions of the Canary Islands (Spain), a novel yeast species was isolated from Lanzarote, the driest wine-producing region of the archipelago. Seven strains isolated from grapes, microvinifications and wineries are described. Sequence analysis of the D1/D2 domain of the LSU rDNA gene and 5.8S-ITS regions revealed that the isolates were phylogenetically a member of the genus Lachancea and are closely related to Lachancea meyersii NRRL Y-27269(T) and Lachancea nothofagi NRRL Y-48670(T). On the basis of morphological, biochemical and physiological characterization and phylogenetic analysis, a novel ascosporogenous yeast species, Lachancea lanzarotensis sp. nov., is proposed. The type strain is L2C-15(T) ( = CBS 12615(T) = CECT 13066(T)) which was isolated from grape berries of Vitis vinifera L. cv. Listán Negro red grape variety in Tinajo, Lanzarote. The MycoBank no. is MB 801390.

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The identification of acetic acid bacteria (AAB) from sound grapes from the Canary Islands is reported in the present study. No direct recovery of bacteria was possible in the most commonly used medium, so microvinifications were performed on grapes from Tenerife, La Palma and Lanzarote islands. Up to 396 AAB were isolated from those microvinifications and identified by 16S rRNA gene sequencing and phylogenetic analysis. With this method, Acetobacter pasteurianus, Acetobacter tropicalis, Gluconobacter japonicus and Gluconacetobacter saccharivorans were identified. However, no discrimination between the closely related species Acetobacter malorum and Acetobacter cerevisiae was possible. As previously described, 16S-23S rRNA gene internal transcribed spacer (ITS) region phylogenetic analysis was required to classify isolates as one of those species. These two species were the most frequently occurring, accounting for more than 60% of the isolates. For typing the AAB isolates, both the Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR and (GTG)5-PCR techniques gave similar resolution. A total of 60 profiles were identified. Thirteen of these profiles were found in more than one vineyard, and only one profile was found on two different islands (Tenerife and La Palma).

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The diversity of the mycobiota community occurring on the surface of fermented dry sausages and in the environment of the processing plants was studied. The manufacturing plants (five in total) were located in three different areas of southern Buenos Aires province (Argentina). Samples were collected from two types of fermented sausages (short and long ripening time) at two times of the year (winter and summer) and at different types of plants (artisanal and industrial). A total of 342 samples were examined and 822 isolates belonging to six genera and 16 fungal species were identified. In most cases, Penicillium was the genus most frequently isolated. Penicillium nalgiovense, P. nordicum, P. solitum and P. chrysogenum were the most abundant species. The characteristics of the plants and the season were the influencing factors on the composition of the mycobiota. Under conditions of high humidity and in ripening rooms with reduced ventilation, Mucor racemosus was most prevalent. Eurotium amstelodami and Aspergillus spp were detected mainly during the summer when the temperature was higher. A high number of species isolated from the surface of the sausage was also isolated from air samples. Geotrichum candidum, Alternaria infectoria and P. glabrum were found only in air samples. P. chrysogenum, P. nalgiovense and P. nordicum showed different levels of antibacterial activity in sensitive bioassay with Micrococcus luteus.

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INTRODUCTION: Staphylococcus lugdunensis is a coagulase-negative staphylococcus associated with a variety of clinical infections. In this paper we present the results of a comparative study using 4 methods to determine antimicrobial susceptibility to oxacillin and penicillin in 60 S. lugdunensis isolates. MATERIAL AND METHODS: We studied 60 S. lugdunensis isolates obtained from clinical specimens sent to our laboratory over an 8-year period. All isolates were free coagulase-negative and DNase-negative, and biochemically identified by API ID 32 STAPH (bioMérieux). Presence of mecA and ss-lactamase production were studied in all cases. Antimicrobial susceptibility was determined by the Vitek 2 System (bioMérieux) and broth microdilution (Wider) (Soria Melguizo) for penicillin and oxacillin, and the E-test (AB Biodisk) and cefoxitin disk diffusion test (BD BBLTM) for oxacillin. RESULTS: All isolates lacked the mecA gene and were susceptible to oxacillin by broth microdilution, E-test, and cefoxitin disk diffusion test. Only two isolates were oxacillin-resistant by the Vitek 2 System. Twenty-four isolates (40%) were ss-lactamase-positive, 4 after induction. Susceptibility testing to penicillin determined that 48 isolates showed concordance between the results obtained by broth microdilution and Vitek 2, but 12 isolates (20%), showed divergent results. CONCLUSIONS: We detected no resistance to oxacillin in S. lugdunensis. All the methods evaluated were adequate for determining oxacillin resistance. The Vitek 2 System is useful for detecting penicillin resistance, but the ss-lactamase test should be applied to isolates with a MIC=0.25microg/ml to avoid the interpretation of false resistance to this antibiotic.

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Introducción: Staphylococcus lugdunensis es un estafilococo coagulasa negativo relacionado con diversos tipos de infección. En este trabajo se presentan los resultados de un estudio comparativo mediante cuatro métodos para determinar la sensibilidad a oxacilina y penicilina. Material y métodos: se estudiaron 60 aislamientos de S. lugdunensis procedentes de muestras clínicas enviadas a nuestro laboratorio durante 8 años. Todos los aislados fueron coagulasa y DNasa negativos. La identificación se realizó bioquímicamente mediante API ID 32 STAPH (bioMérieux). En todos los casos se analizó la presencia de betalactamasa y la detección del gen mecA. La susceptibilidad antimicrobiana se determinó mediante: Vitek 2 System (bioMérieux) y microdilución en caldo (Wider) (Soria Melguizo) para oxacilina y penicilina; E-test (AB Biodisk) y método de difusión con disco de cefoxitina (BD BBLTM), para ensayar la sensibilidad a oxacilina. Resultados: todos los aislamientos fueron mecA negativos y sensibles a oxacilina en microdilución en caldo, E-test y en el método de difusión con cefoxitina, mientras que en Vitek 2 solamente dos aislamientos fueron resistentes a oxacilina; 24 (40%) fueron betalactamasa positivos, 4 tras inducción. Los resultados de susceptibilidad a penicilina mostraron que 48 aislamientos presentaban concordancia entre los obtenidos por microdilución en caldo y Vitek 2, pero 12 (20%) mostraron resultados discrepantes Conclusiones: en nuestro estudio no hemos hallado ningún aislamiento de S. lugdunensis resistente a oxacilina; los métodos de microdilución en caldo (Wider), E-test de oxacilina y difusión con disco de cefoxitina son adecuados para el estudio de sensibilidad a este antibiótico. El empleo del sistema Vitek 2 es útil para el estudio de la sensibilidad a penicilina si se aplica la prueba de betalactamasa a los aislamientos con concentración mínima inhibitoria (CMI) de 0,25μg/ml para evitar la interpretación de una falsa resistencia a dicho antibiótico (AU)

Introduction: Staphylococcus lugdunensis is a coagulase-negative staphylococcus associated with a variety of clinical infections. In this paper we present the results of a comparative study using 4 methods to determine antimicrobial susceptibility to oxacillin and penicillin in 60 S. lugdunensis isolates. Material and methods: We studied 60 S. lugdunensis isolates obtained from clinical specimens sent to our laboratory over an 8-year period. All isolates were free coagulase-negative and DNase-negative, and biochemically identified by API ID 32 STAPH (bioMérieux). Presence of mecA and ß-lactamase production were studied in all cases. Antimicrobial susceptibility was determined by the Vitek 2 System (bioMérieux) and broth microdilution (Wider) (Soria Melguizo) for penicillin and oxacillin, and the E-test (AB Biodisk) and cefoxitin disk diffusion test (BD BBLTM) for oxacillin. Results: All isolates lacked the mecA gene and were susceptible to oxacillin by broth microdilution, E-test, and cefoxitin disk diffusion test. Only two isolates were oxacillin-resistant by the Vitek 2 System. Twenty-four isolates (40%) were ß-lactamase-positive, 4 after induction. Susceptibility testing to penicillin determined that 48 isolates showed concordance between the results obtained by broth microdilution and Vitek 2, but 12 isolates (20%), showed divergent results. Conclusions: We detected no resistance to oxacillin in S. lugdunensis. All the methods evaluated were adequate for determining oxacillin resistance. The Vitek 2 System is useful for detecting penicillin resistance, but the ß-lactamase test should be applied to isolates with a MIC=0.25μg/ml to avoid the interpretation of false resistance to this antibiotic (AU)

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A case of fungal keratitis is presented in which corneal scrapings were obtained for microbiological studies, including morphological identification and molecular characterization of the etiologic agent. Comparative sequence analyses of the Internal Transcribed Spacer domain of 5.8S and 26S regions of nuclear rDNA showed 100% identity with different species of Alternaria and PCR-RFLP analysis of Intergenic Spacer regions revealed intraspecific variation. The combination of morphological and molecular characters resulted in the unambiguous identification of the causal agent as Alternaria alternata. Treatment with antifungals contributed to the improvement in the patient's lesions.

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Penicillium nalgiovense is a filamentous fungus that is acquiring increasing biotechnological importance in the food industry due to its widespread use as starter culture for cured and fermented meat products. Strains of P. nalgiovense can be improved by genetic modification to remove the production of penicillin and other potentially hazardous secondary metabolites, to improve its capacity to control the growth of undesirable fungi and bacteria on the meat product, and other factors that contribute to the ripening of the product in order to get safer and better quality foods. Genetic manipulation of P. nalgiovense has been limited by the lack of molecular genetics tools that were available for this fungus, particularly for "self-cloning" avoiding the use of exogenous DNAs. In this article we describe a series of vectors, selectable markers and transformation methods that can be used for efficient transformation of P. nalgiovense, gene cloning and expression. A uridine auxotrophic P. nalgiovense mutant with an inactive pyrG gene has been isolated. The P. nalgiovense wild-type pyrG gene was cloned and sequenced, and vectors carrying the gene were shown to complement the pyrG mutant. Autonomously replicating plasmids carrying the AMA1 region from Aspergillus nidulans transformed P. nalgiovense very efficiently; these plasmids were shown to be maintained as stable extrachromosomal elements in P. nalgiovense and could be rescued in Escherichia coli. The mitotic stability of self-replicative AMA1 plasmids in P. nalgiovense was higher than that reported for Penicillium chrysogenum.

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The presence of some fungi on a variety of food products, like cheeses or cured meat products, is beneficial for the ripening of the product and for the development of specific flavour features. The utilization of these fungi as starters, which are inoculated normally as asexual spores on the food products at the beginning of the ripening process, is becoming a usual procedure in the food industry. The starter culture also prevents undesirable fungi or bacteria from growing on the product. Penicillium nalgiovense is the most frequently used starter for cured and fermented meat products, but the fact that this fungus can secrete penicillin to the meat product makes it important to get strains unable to synthesize this antibiotic. In this work we report that P. nalgiovense strains impaired in penicillin production can be obtained by disruption of the pcbAB gene (the first gene of the penicillin biosynthetic pathway). When applied as starter on cecina (a salted, smoke-cured beef meat product from the region of León, Spain), the pcbAB-disrupted strain showed no differences with respect to the parental penicillin-producing strain in its ability to colonize the meat pieces and to control their normal mycoflora. Both strains exerted a similar control on the presence of bacteria in cecina. A similar proportion of penicillin-sensitive and penicillin-resistant bacteria were isolated from pieces inoculated with the penicillin-producing or the non-producing P. nalgiovense strains. The decrease of the bacterial population on the surface of cecina seems to be due to the higher competition for nutrients as a consequence of the inoculation and development of the P. nalgiovense mycelium and not due to the production of penicillin by this fungus. Penicillin production was less affected than growth in a solid medium with high NaCl concentrations; this suggests that the high salt concentration present in cecina is not a limiting factor for penicillin production by P. nalgiovense.

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Mycobiota growing on food is often beneficial for the ripening and development of the specific flavor characteristics of the product, but it can also be harmful due to the production of undesirable compounds such as mycotoxins or antibiotics. Some of the fungi most frequently isolated from fermented and cured meat products such as Penicillium chrysogenum and Penicillium nalgiovense are known penicillin producers; the latter has been shown to be able to produce penicillin when growing on the surface of meat products and secrete it to the medium. The presence of penicillin in food must be avoided, since it can lead to allergic reactions and the arising of penicillin resistance in human-pathogenic bacteria. In this article we describe a study of the penicillin production ability among fungi of the genus Penicillium that are used as starters for cheese and meat products or that are frequently isolated from food products. Penicillium griseofulvum was found to be a new penicillin producer and to have a penicillin gene cluster similar to that of Penicillium chrysogenum. No other species among the studied fungi were found to produce penicillin or to possess the penicillin biosynthetic genes, except P. verrucosum, which contains the pcbAB gene (as shown by hybridization and PCR cloning of fragments of the gene) but lacks pcbC and penDE. Antibacterial activities due to the production of secondary metabolites other than penicillin were observed in some fungi.

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