Entomopathogenic fungal (Hyphomycetes) collection: assessment of conidial viability

Twenty four strains of the entomopathogenic fungi (Hyphomycetes) Beauveria bassiana, Metarrhizium anisopliae, Nomuraea rileyi, Paecilomyces farinosus, P. fumosoroseus and P. lilacinus, maintained in the culture collection of Embrapa-Centro Nacional de Pesquisa de Recursos Genéticos e Biotecnologia (Cenargen) and preserved by lyophilization and in liquid nitrogen, had their conidial viability assessed. Germination rates of 16- to 84-month-old cultures stored in liquid nitrogen decreased, on average, less than 13.3%. For 29- to 49-month-old cultures preserved by lyophilization, the viability loss ranged, on average, from 28.6 to 94.5%. The results demonstrated the efficiency of the tested methods, especially liquid nitrogen, in preserving the viability of entomopathogenic fungi.

Changes in arbuscular mycorrhizal fungal phenotypes and genotypes in response to plant species identity and phosphorus concentration.

* Arbuscular mycorrhizal fungi (AMF) are plant symbionts that improve floristic diversity and ecosystem productivity. Many AMF species are generalists with wide host ranges. Arbuscular mycorrhizal fungi individuals are heterokaryotic, and AMF populations are genetically diverse. Populations of AMF harbor two levels of genetic diversity on which selection can act, namely among individuals and within individuals. Whether environmental factors alter genetic diversity within populations is still unknown. * Here, we measured genetic changes and changes in fitness-related traits of genetically distinct AMF individuals from one field, grown with different concentrations of available phosphate or different host species. * We found significant genotype-by-environment interactions for AMF fitness traits in response to these treatments. Host identity had a strong effect on the fitness of different AMF, unearthing a specificity of response within Glomus intraradices. Arbuscular mycorrhizal fungi individuals grown in novel environments consistently showed a reduced presence of polymorphic genetic markers, providing some evidence for host or phosphate-induced genetic change in AMF. * Given that AMF individuals can form extensive hyphal networks colonizing different hosts simultaneously, contrasting habitats or soil properties may lead to evolution in the population. Local selection may alter the structure of AMF populations and maintain genetic diversity, potentially even within the hyphal network of one fungus.

Plantes noves per la flora de Montserrat.

En aquestes notes indiquem la troballa de trenta-tres plantes noves per la flora de Montserrat, de les quais fem alguns comentaris ecologies i corológics

La cartografia de la flora del Montseny en reticle UTM d'1 km. de costat: plantejament i primers resultats

El present treball és un primer escrit sobre la cartografia corològica de les plantes vasculars en el massis del Montseny (Serralada Pre-litoral), en el qual exposem el plantejament del projecte i oferim uns primers résultats. Hi incloem també un mapa per espècie prenent com a unitat espacial el quadrat d'1 km de costat del reticle UTM. La presència de 1'espècie a cada quadrat és indicada en très graus d'abundància: espècie présent o rara, espècie fréquent i espècie abundant. L'àrea estudiada comprèn 513 quadrats d'1 km de costat, que pertanyen a 12 quadrats de 10 km de costat de la zona 31T del reticle UTM: DG 33, DG 43, DG 53, DG 63, DG 32, DG k2, DG 52, DG 62, DG 3 1 , DG kl, DG 51 i DG 6 l . Com a primera aportaciô presentem 10 mapes amb la distribució de Quercus ilex, Fagus sylvatica, Abies alba, Taxus baccata, Betula pendula, Cistus laurifolius, Cardamine heptaphylla, Ramonda myconii i Equisetum hyemale.

Arbuscular mycorrhiza: the challenge to understand the genetics of the fungal partner.

Arbuscular mycorrhizal symbioses occur between fungi and the majority of plant species. They are important for plant nutrition, plant growth, protection from pathogens, plant diversity, nutrient cycling, and ecosystem processes. A key goal in research is to understand the molecular basis of the establishment, regulation, and functioning of the symbiosis. However, lack of knowledge on the genetics of the fungal side of this association has hindered progress. Here, we show how several key, recently discovered processes concerning the genetics of arbuscular mycorrhizal fungi could be essential for ultimately understanding the molecular genetics of this important symbiosis with plants.

Contribució a la flora dels macromicets de l'illa de Mallorca

Com a resultat de les prospeccions micològiques realitzades a Mallorca entre els anys 1983 i 1990, donem a conèixer un catàleg de 218 taxons (8 Ascomycetes i 210 Basidiomycetes), dels quals creiem que 74 corresponen a citacions noves per a l'illa. En destaquem, entre d'altres: Cordyceps militaris (L.) Link, Daldinia vernicosa (Schw.) Ces. et de Not., Dichomitus campestris (Quél.) Domanski et Ori., Agaricus lanipes (Moell. et Schaeff.) Sing., Amanita boudieri Barla, Clitocybe lituus (Fr.) Metr., Hygrocybe reai Mre., Inocybe tenebrosa Quél., Leucopaxillus tricolor (Peck) Kühn, i Russula seperina Dupain.

Amostragem do banco de semente e flora emergente de plantas daninhas

O objetivo desta pesquisa foi determinar o número de amostras necessárias para estimar com precisão aceitável a quantidade de sementes no solo e a flora de plantas daninhas emergentes, em áreas experimentais e de lavoura, para auxiliar na tomada de decisão das estratégias de manejo das plantas daninhas. A amostragem do solo para quantificação do banco de sementes foi feita com trado tubular de 5,0 cm de diâmetro, na profundidade de 0 a 10 cm. A flora emergente foi contada por meio de um gabarito de ferro nas dimensões de 0,5x0,5 m, de forma aleatória na área. O número de amostras necessário foi estimado em razão da média de sementes da amostra, para uma determinada precisão (CV = 20% ou 40%). Foi estimado que, nas áreas experimentais, para médias de 10 a 20 sementes/amostra de solo (500 a 1.000 sementes/m²) e coeficiente de variação de 20%, são necessárias entre 40 e 90 amostras, respectivamente; com 40% (menor precisão), entre 10 e 20 amostras. Considerando o mesmo intervalo em áreas de lavoura, representativas de glebas homogêneas, o tamanho de amostragem necessária é cerca de três vezes maior. Levantamentos da flora daninha emergente apresentam menor associação dos dados (média e variância) entre si, portanto, são menos apropriados para decisões de manejo.

Identity and combinations of arbuscular mycorrhizal fungal isolates influence plant resistance and insect preference

1. Accumulating evidence indicates that plant resistance against above-ground herbivores can be affected by the presence of arbuscular mycorrhizal fungi (AMF) in association with the host plant. Little is known, however, about how AMF composition can influence herbivore choice to feed on a particular plant. 2. Unravelling the preference-performance hypothesis in a multitrophic context is needed to expand our knowledge of complex multitrophic interactions in natural systems. If given mycorrhizal fungal genotypes increase attractiveness for a herbivore (reduced plant resistance), then the benefits of increased unpalatability provided by the mycorrhizal fungi (increased plant resistance) might be outweighed by the increased herbivore recruitment. 3. This was addressed by designing three experiments to test the effects of different AMF genotypes, inoculated either alone or in combination, to measure intraspecific AMF effects on plant resistance and insect herbivore preference. Using strawberry (Fragaria vesca L.) plants that were colonised by eight different combinations of Rhizophagus irregularis isolates, we measured effects on plant growth, insect growth and survival, as well as feeding preferences of a generalist herbivore caterpillar (Spodoptera littoralis Boisduval). 4. Overall, it was found that: (i) AMF influenced plant resistance in an AMF genotype-specific manner; (ii) some AMF inoculations decreased insect performance; (iii) insects preferentially chose to feed more on leaves originating from non-mycorrhizal plants; but also that (iv) in a whole plant bioassay, insects preferentially chose the biggest plant, regardless of their mycorrhizal status. 5. Therefore, AMF-mediated trade-offs between growth and resistance against herbivores have been shown. Such trade-offs, particularly driven by plant attractiveness to herbivores, buffer the positive effects of the mycorrhizal symbiosis on enhanced plant growth.

Diagnostic and identification in situ of fungal pathogens in superficial mycosis

Fungi are divided in 3 groups in the field of medical mycology. The dermatophytes are filamentous fungi able to grow on keratinized tissues from human or animals. They are the main cause of superficial and cutaneous mycoses of the skin and its appendix (hair and nail). The yeasts, or dimorphic fungi, can be responsible of diverse types of infections (superficial to deep mycoses). The moulds include all Non-dermatophyte Filamentous Fungi (NDF). In medical mycology, the most representative moulds are Aspergillus spp., Fusarium spp. and Mucor spp. Diagnosis of mycosis is currently based on direct mycological examination of biological samples, as well as macroscopic and microscopic identification of the infectious fungus in culture assay. However, culture assays were found to remain sterile in roughly 40% of cases otherwise positive by direct mycological examinations. Additionally, results from culture assays are often difficult to interpret as various NDF are sometimes isolated. This thesis work is composed of three projects focusing on the development of new assays for direct in situ identification of fungi from dermatological samples. Part 1. A Polymerase Chain Reaction - Terminal Restriction Fragment Length Polymorphism assay (PCR-TRFLP) targeting the 28S rDNA was developed to identify dermatophytes and NDF in nails with suspected onychomycosis. This method is faster and more efficient than culture. It further enables the distinction of more than one agent in case of mixed infection. A fast and reliable assay for the identification of dermatophytes and NDF in onychomycosis was found to be highly relevant since onychomycosis with Fusarium spp. or other NDF are weakly responsive or unresponsive to standard onychomycosis treatments with oral terbinafine and itraconazole. Part 2. A nested PCR-sequencing assay targeting the 28S rDNA was developed to identify dermatophyte species in skin and hair samples. This method is especially suitable for tinea capitis where dermatophytes identification is critical for subsequently prescribing the adequate treatment. The challenge presented when performing direct PCR fungi identification in skin and hair differs from that seen in onychomycosis as small amount of material is generally collected, few fungal elements are present in the clinical sample and one dermatophyte among a dozen species must be identified. Part 3. Fusarium spp. is currently isolated from nails with a frequency of 15% of that of dermatophytes in the laboratory of Mycology of the CHUV (2005-2012). The aim of this work was to examine if the intensive use of terbinafine and itraconazole could be a cause of the high incidence of Fusarium nail infections. For that purpose, two different methods, specific PCR and TRFLP, were used to detect both Fusarium spp. and Trichophyton spp. in nails of previously treated or untreated patients. TRFLP assay was found to be less sensitive than classical PCR assays specifically detecting Fusarium spp. or Trichophyton spp. Independently of the detection method used, the prevalence of Fusarium spp. appears not to be higher in patients previously treated by oral standard treatment with terbinafine and azoles which are highly effective to fight Trichophyton spp. in nails. In many cases Fusarium sp. was detected in samples of patients not previously subjected to antifungal therapy. Therefore, these treatments do not appear to favor the establishment of Fusarium spp. after elimination of a dermatophyte in nail infection. - En mycologie médicale, les champignons sont classés en 3 groupes. Les dermatophytes sont des champignons filamenteux capables de se développer dans les tissus kératinisés des hommes et des animaux, ils représentent la principale cause des mycoses superficielles et cutanées de la peau et de ses appendices (ongles et cheveux). Les levures, ou champignons dimorphiques, peuvent être responsables de divers types d'infections (superficielles à profondes). Les moisissures incluent tous les champignons filamenteux non-dermatophytes (NDF), les Aspergillus spp., les Fusarium spp. et les Mucor spp. sont les principales espèces rencontrées. Le diagnostic d'une mycose est basé sur un examen mycologique direct des prélèvements biologiques ainsi que sur l'identification macroscopique et microscopique du champignon infectieux isolé en culture. Cependant, dans environ 40% des cas, l'identification de l'agent pathogène est impossible par cette méthode car la culture reste stérile, bien que l'examen direct soit positif. De plus, la croissance de moisissures et/ou autres contaminants peut rendre l'interprétation de l'examen difficile. Ce travail de thèse est composé de trois projets focalisés sur le développement de nouvelles méthodes d'identification des champignons directement à partir d'échantillons dermatologiques. Projet 1. Une méthode de Réaction en chaîne de polymérase couplée à du polymorphisme de longueur des fragments de restriction terminaux (PCR-TRFLP), en ciblant l'ADN ribosomal 28S, a été développée pour l'identification des dermatophytes et moisissures dans les ongles avec suspicion d'onychomycoses. Cette technique s'est avérée plus rapide et plus efficace que la culture, permettant l'identification de plusieurs champignons en même temps. Posséder une méthode d'identification rapide et fiable des dermatophytes et des NDF dans les onychomycoses a été jugée nécessaire du fait que les Fusarium et d'autres NDF sont peu ou pas sensibles aux traitements oraux standards à la terbinafine et à Γ itraconazole. Projet 2. Une PCR nichée couplée au séquençage d'un fragment de l'ADN ribosomal 28S a été développée afin de différencier les dermatophytes dans la peau et les cheveux. Cette méthode est particulièrement adaptée au cas de tinea capitis, où l'identification du dermatophyte est essentielle afin de prescrire le traitement adéquat. Le problème de l'identification du pathogène fongique dans les cheveux et la peau diffère des onychomycoses car de petites quantités sont prélevées chez les patients, peu d'éléments fongiques sont présents et il faut discriminer un dermatophyte parmi une douzaine d'espèces potentielles. Projet 3. Au laboratoire de Mycologie du CHUV, les Fusarium ont été isolé dans les ongles à une fréquence de 15% pour la période 2005-2012. Le but de ce travail était d'examiner si l'utilisation intensive de terbinafine et d'itraconazole pouvait être une des causes de la forte incidence des infections des ongles par Fusarium. A cet effet, deux méthodes ont été utilisées pour détecter à la fois Fusarium spp. et Trichophyton spp., la PCR spécifique et le TRFLP. Indépendamment de la méthode choisie, il en résulte que la prévalence des Fusarium η'apparaît pas liée à un traitement au préalable des patients avec de la terbinafine ou des azoles, thérapies très efficaces contre les Trichophyton spp. dans les ongles. De plus, il existe de nombreux cas où Fusarium était détecté chez des patients non traités.

ECIL-3 classical diagnostic procedures for the diagnosis of invasive fungal diseases in patients with leukaemia.

Invasive fungal diseases (IFDs) continue to cause considerable morbidity and mortality in patients with haematological malignancy. Diagnosis of IFD is difficult, with the sensitivity of the gold standard tests (culture and histopathology) often reported to be low, which may at least in part be due to sub-optimal sampling or subsequent handling in the routine microbiological laboratory. Therefore, a working group of the European Conference in Infections in Leukaemia was convened in 2009 with the task of reviewing the classical diagnostic procedures and providing recommendations for their optimal use. The recommendations were presented and approved at the ECIL-3 conference in September 2009. Although new serological and molecular tests are examined in separate papers, this review focuses on sample types, microscopy and culture procedures, antifungal susceptibility testing and imaging. The performance and limitations of these procedures are discussed and recommendations are provided on when and how to use them and how to interpret the results.

Nonmolecular methods for the diagnosis of respiratory fungal infections.

Diagnosis of invasive fungal pneumonias by conventional culture methods is difficult to assess and often delayed. Nonmolecular fungal markers have emerged as an important adjunctive tool to support their diagnosis in combination with other clinical, radiologic, and microbiological criteria of invasive fungal diseases. Concerns about the sensitivity and specificity of some tests in different patient populations should lead to warnings about their widespread use. None can identify the emerging and particularly deadly fungal pathogens responsible for mucormycosis. The role of nonmolecular fungal markers should be better defined in combination with other microbiological and radiologic tools in preemptive antifungal strategies.

Flora e estrutura da vegetação secundária após o uso de diferentes trituradores florestais

O objetivo deste trabalho foi caracterizar e comparar a estrutura de florestas secundárias, com quatro anos de idade, no nordeste do Pará, que foram formadas após o uso dos trituradores florestais Ahwi FM600 (T1) e Tritucap (T2). O estudo foi realizado em cinco áreas de pequenos produtores, cada uma dividida em parcelas que totalizavam 60 m² por área de estudo. Foi encontrada diversidade média (H') de 2,84 para as áreas T1 e 2,95 para as T2. A densidade média foi 530.167 indivíduos ha-1 nas áreas T1 e 459.556 indivíduos ha-1 nas T2; a média de área basal das T1 foi 1,25 m² ha-1 e a das T2, 4,55 m² ha-1; a biomassa seca estimada para as áreas T1 foi de 2,37 t ha-1 e 6,68 t ha-1 para as T2. Ocorrem diferenças estruturais nas florestas secundárias, formadas após a utilização dos trituradores.