Influence of plant species on disease suppression by Pseudomonas fluorescens strain CHA0 with enhanced antibiotic production

Introduction of the recombinant cosmid pME3090 into Pseudomonas fluorescens strain CHAO, a good biocontrol agent of various diseases caused by soilborne pathogens, increased three- to five-fold the production of the antibiotic metabolites pyoluteorin (Pit) and 2,4-diacetylphlorogIucinol (Phi) in vitro. Strain CHAO/pME3090 also overproduced Pit and Phi in the rhizosphere of wheat infected or not infected with Pythium ultimum. The biocontrol activity of the wild-type and recombinant Straitis was compared using various plant pathogen-host combinations in a gnotobiotic system. Antibiotic overproduction affected neither the protection of wheat against P. ultimum and Gaeumannomyces graminis var. tritici nor the growth of wheat plants. In contrast, strain CHA0/pME3090 showed an increased capacity to protect cucumber against Fusarium oxysporum f. sp. cucumerinum and Phomopsis sclerotioides, compared with the wild-type strain CHAO, The antibiotic overproducing strain protected tobacco roots significantly better against Thielaviopsis basicola than the wild-type strain but drastically reduced the growth of tobacco plants and was also toxic to the growth of sweet com. On King's B agar and on malt agar, the recombinant strain CHA0/pME3090 inhibited all pathogens more than did the parental strain CHAO. Synthetic Pit and Phi were toxic to all fungi tested. Tobacco and sweet com were more sensitive to synthetic Pit and Phi than were cucumber and wheat. There was no correlation between the sensitivity of the pathogens to the synthetic antibiotics and the degree of disease suppression by strain CHAO pME3090. However, there was a correlation between the sensitivity of the plants and the toxicity of the recombinant strain. We conclude that the plant species rather than the pathogen determines whether cosmid pME3090 in P. fluorescens strain CHAO leads to improved disease suppression.

Multi-well fungal co-culture for de novo metabolite-induction in time-series studies based on untargeted metabolomics.

The induction of fungal metabolites by fungal co-cultures grown on solid media was explored using multi-well co-cultures in 2 cm diameter Petri dishes. Fungi were grown in 12-well plates to easily and rapidly obtain the large number of replicates necessary for employing metabolomic approaches. Fungal culture using such a format accelerated the production of metabolites by several weeks compared with using the large-format 9 cm Petri dishes. This strategy was applied to a co-culture of a Fusarium and an Aspergillus strain. The metabolite composition of the cultures was assessed using ultra-high pressure liquid chromatography coupled to electrospray ionisation and time-of-flight mass spectrometry, followed by automated data mining. The de novo production of metabolites was dramatically increased by nutriment reduction. A time-series study of the induction of the fungal metabolites of interest over nine days revealed that they exhibited various induction patterns. The concentrations of most of the de novo induced metabolites increased over time. However, interesting patterns were observed, such as with the presence of some compounds only at certain time points. This result indicates the complexity and dynamic nature of fungal metabolism. The large-scale production of the compounds of interest was verified by co-culture in 15 cm Petri dishes; most of the induced metabolites of interest (16/18) were found to be produced as effectively as on a small scale, although not in the same time frames. Large-scale production is a practical solution for the future production, identification and biological evaluation of these metabolites.

Efficacious treatment of non-dermatophyte mould onychomycosis with topical amphotericin B.

BACKGROUND: Dermatophytes are the main cause of onychomycosis, but various non-dermatophyte moulds (NDMs) are often the infectious agents in abnormal nails. In particular, Fusarium spp. and other NDMs are mostly insensitive to standard onychomycosis treatment with topical agents as well as with oral terbinafine and itraconazole.¦OBJECTIVE: The aim of this work is to report the efficacy of a topical amphotericin B solution on NDM onychomycosis in a series of 8 patients resistant to multiple conventional topical and systemic treatments.¦METHODS: Treatment consisted in the application of an optimized amphotericin B solution once daily to the affected nails and surrounding tissue. No mechanical debridement or medications were allowed except for trimming excessively long nails or in some cases occasionally applying urea-based cream to soften thickened nail plates.¦RESULTS: Onychomycosis was clinically cured in all patients after a 12-month treatment. Mycological cure was obtained in all but 1 patient.¦CONCLUSIONS: Topical amphotericin B is an efficacious, safe, cheap and easy-to-apply treatment which should be considered as first-line therapy for NDM onychomycosis.

Comportamento de fungos e de sementes de feijoeiro durante o teste de envelhecimento artificial

O teste de envelhecimento artificial, recomendado para avaliar o vigor de lotes de sementes, apresenta variabilidade em seus resultados; a ação dos fungos é considerada uma das causas dessa variabilidade. Este trabalho objetivou verificar os efeitos de diferentes períodos de envelhecimento artificial, no comportamento fisiológico de sementes do feijoeiro e dos fungos Aspergillus spp., Penicillium spp., Fusarium oxysporum e Colletotrichum lindemuthianum, inoculados artificialmente. Foram conduzidos testes de sanidade, germinação, tetrazólio, emergência, condutividade elétrica e lixiviação de potássio. As respostas obtidas, dependentes da duração do período de envelhecimento, indicaram efeitos da espécie fúngica presente. Concluiu-se que o teste de envelhecimento artificial associa a expressão de causas fisiológicas e sanitárias, o que prejudica a interpretação dos dados obtidos; a presença de fungos, principalmente de Aspergillus spp., pode ser considerada como capaz de interferir de modo negativo no desempenho das sementes envelhecidas artificialmente.

Contaminação endógena por Aspergillus spp. em milho pós-colheita no Estado do Paraná

Sessenta amostras de milho pós-colheita foram avaliadas quanto à contaminação fúngica endógena e o potencial toxígeno de espécies do gênero Aspergillus e seus teleomorfos. Quarenta grãos aparentemente sadios de cada amostra foram desinfestados em NaClO e incubados em câmara úmida a 25±1ºC para exteriorização dos fungos, que posteriormente foram isolados em ágar Czapek-Dox. Foram identificadas as espécies Aspergillus flavus, A. parasiticus, Eurotium amstelodami e E. chevalieri. O potencial toxígeno dos fungos A. flavus e A. parasiticus foi avaliado quanto à síntese de aflatoxinas em meio ágar-coco. Espécies do gênero Eurotium foram avaliadas quanto à síntese de esterigmatocistina, nos meios ágar-amendoim e trigo triturado. A porcentagem de grãos contaminados variou entre 0 e 100%, prevalecendo os gêneros Aspergillus, Penicillium e Fusarium. A espécie predominante foi a A. flavus (64%), seguida por E. amstelodami (19%), E. chevalieri (10%) e A. parasiticus (7%). A partir de 109 isolados de A. flavus, evidenciou-se que 73 isolados sintetizaram aflatoxinas B1 e B2, 20 sintetizaram B1, sete sintetizaram B1 e G1, três sintetizaram B1, B2 e G1 e em seis não foi detectada a síntese de aflatoxina. A síntese de esterigmatocistina pelas espécies E. amstelodami e E. chevalieri não foi detectada.

Controle da fusariose em plantas de pimenta-do-reino com bactérias endofíticas: sobrevivência e respostas morfofisiológicas

Oito bactérias endofíticas de plântulas de pimenta-do-reino foram testadas em casa de vegetação, objetivando controlar Fusarium solani f. sp. piperis e avaliar respostas morfofisiológicas das plantas tratadas com os agentes de controle. Plantas com quatro meses de idade tiveram o sistema radicular tratado com as suspensões bacterianas (10(9) ufc mL-1) por 10 minutos, e foram plantadas em vasos com solo natural infestado artificialmente com o patógeno (0,25%). O delineamento experimental foi inteiramente casualizado, com três repetições para avaliar o comportamento fotossintético, a produção e a alocação de biomassa, e cinco repetições para avaliar a mortalidade das plantas. As avaliações foram feitas 120 dias após a instalação do ensaio. O isolado B0 (Methylobacterium radiotolerans) controlou F. solani f. sp. piperis, provocando redução significativa do número de plantas mortas. Na ausência do patógeno, M. radiotolerans funcionou ainda como promotor de crescimento. Os demais isolados testados não apresentaram efeito na diminuição da mortalidade das plantas. A aplicação das bactérias endofíticas não afetou a taxa fotossintética instantânea das plantas, à exceção de B6, que teve efeito negativo. As plantas tratadas com M. radiotolerans priorizaram a alocação de carbono para a parte aérea.

Alterações dos padrões de isoenzimas em sementes de milho infectadas por fungos

Este trabalho teve como objetivo estudar a interferência dos fungos Aspergillus flavus, Fusarium moniliforme e Penicillium spp. sobre padrões eletroforéticos das sementes de milho. Tais padrões são, normalmente, utilizados na identificação de cultivares e na certificação da pureza genética da espécie em estudo. Sementes da cultivar C-805 foram infectadas artificialmente com os referidos fungos; outra parte delas foi tratada com Benomil e Thiabendazol, e ainda outra parte (controle) não foi tratada. As amostras foram acondicionadas em câmara de crescimento (25°C, 95% de umidade relativa) por um período de 30 dias. Na análise eletroforética foi avaliada também uma amostra de sementes que não permaneceu em câmara de crescimento, visando detectar possíveis interferências das condições do ambiente de crescimento sobre os padrões eletroforéticos. Os resultados obtidos permitiram concluir que a infecção das sementes com os fungos Aspergillus flavus, Fusarium moniliforme e Penicillium spp. promove alterações nos padrões eletroforéticos das isoenzimas malato-desidrogenase, esterase, fosfatase ácida, peroxidase e glutamato-oxalacetato-transaminase. A infecção das sementes com Aspergillus flavus promove alterações tanto na intensidade como no número de bandas dos padrões isoenzimáticos da álcool-desidrogenase e malato-desidrogenase.

Diagnostic des onychomycose a moisissures et importance pour le traitement [Diagnosis of non dermatophyte onychomycosis ant its relevance for treatment].

PCR methods are reliable and suitable to in situ identify dermatophytes, yeasts and non dermatophyte moulds (NDM) in onychomycosis. Onychomycosis insensitive to standard treatment with topical agents as well as with oral terbinafine or itraconazole revealed Fusarium spp., Acremonium spp. and Aspergillus spp. as infectious agents. However, NDM onychomycosis could be efficiently cured using topical amphotericin B. In conclusion, correct fungal species identification is important in onychomycoses in order to prescribe adequate treatments since dermatophytes and moulds have different sensitivities to antifungal drugs.

Survival of pathogens on soybean debris under no-tillage and conventional tillage systems

A study was conducted in the subtropical area of Southern Brazil to determine the survival of pathogens in soybean residues under conventional and no-tillage cultivation systems from March to September of 1998 and 1999. The pathogens most frequently isolated were Colletotrichum truncatum, Phomopsis spp., Cercospora kikuchii, Fusarium spp., Macrophomina phaseolina, and Rhizoctonia solani. Other fungi isolated were Myrothecium roridum, Penicillium sp., Chaetomium sp., Epicoccum sp., Corynespora cassiicola and Trichoderma sp. The percent of survival of each pathogen varied according to the month and the year. Survival of C. truncatum, Phomopsis spp. and C. kikuchii were significantly reduced (p<0.05) from the first to the last evaluation either on buried debris or maintained on the soil surface. On the other hand, M. phaseolina and Fusarium spp. were either not affected or favored by burying the debris. The frequency of recovery of Fusarium spp. increased specially in debris kept under the soil. The loss of biomass, measured by debris weight along the period of this study, showed a reduction of 44.4% in the conventional system and 34.9% in the no-tillage system in 1998, when rain was better distributed. In 1999, the reduction was 48.2% and 39.0% for the conventional and no-tillage system, respectively.

Controle químico de fungos associados a sementes de sorgo e proteção contra fungos do solo

Este trabalho teve como objetivo avaliar a eficiência de alguns fungicidas no controle de fungos associados a sementes de sorgo cultivar CMS 182R e na proteção delas contra fungos do solo. A microbiota das sementes de sorgo, composta por Fusarium moniliforme, Penicillium spp., Alternaria tenuis, Cladosporium spp. e Phoma sorghina, foi patogênica a essas sementes, e os fungos Pythium aphanidermatum e Sclerotium rolfsii, infectantes do solo de monocultivo de sorgo, também se mostraram patogênicos. Os fungicidas captan, thiram, captan + thiabendazole, thiram + thiabendazole, iprodione + thiram e tolylfluanid foram os mais eficientes no controle não só de toda a microbiota das sementes, como também na proteção das sementes contra Pythium aphanidermatum e Sclerotium rolfsii.

Efeito fungistático de Bacillus thuringiensis e de outras bactérias sobre alguns fungos fitopatogênicos

Quatro isolados bacterianos da rizosfera de Drosera villosa var. villosa (B1, B2, B3, B4) e dois isolados de Bacillus thuringiensis (B5 e B6), sendo B6 produtor da toxina bioinseticida Cry1Ab, foram avaliados quanto à capacidade de inibir os fungos fitopatogênicos Fusarium solani f. sp. phaseoli, Fusarium solani f. sp. glycines, Fusarium oxysporum e Colletotrichum sp. A cepa mais efetiva foi B1 que inibiu o crescimento dos quatro fungos até o 26º dia. B. thuringiensis inibiu o crescimento de três destes, o que indica que possui atividade antifúngica e abre um novo campo de estudo para a utilização do B. thuringiensis.

Relação entre características morfológicas da cariopse e fusariose em milho

Fusarium moniliforme causa sérios prejuízos no rendimento do milho. Suspeita-se que a entrada do fungo na cariopse ocorra pela cicatriz do estilete, e que este fato e o nível de infestação por F. moniliforme estejam relacionados com características morfológicas da cariopse. O objetivo deste trabalho foi identificar características morfológicas que conferem resistência a F. moniliforme em cariopses de milho. Cariopses de seis linhagens de milho, com e sem inoculação do patógeno, foram avaliadas utilizando-se de estereomicroscópio e o microscópio eletrônico de varredura. Características morfológicas da cariopse, tais como: tegumento com saliências e reentrâncias acentuadas, pericarpo pouco espesso, amido menos compacto e presença do canal estilar favorecem a penetração de F. moniliforme.

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.

Characterization of the surface hydrophobicity of filamentous fungi.

A method for the quantitative analysis of the hydrophobicity of the mycelial mat of filamentous fungi based on contact angle measurements is presented. It was tested for a range of fungi belonging to the classes of basidiomycetes, ascomycetes and deuteromycetes. The measured contact angles of the mycelial mats ranged between hydrophilic (<30 degrees) for the deuteromycetes Fusarium oxysporum Fo47 GUS1 and Trichoderma harzianum P1[pZEGA1] and hydrophobic (>60 degrees) for the ascomycete Cladosporium sp. DSE48.1b and the basidiomycetes Paxillus involutus WSL 37.7, Hebeloma crustiliniforme WSL 6.2, Suillus bovinus WSL 48.1 and Laccaria bicolor WSL 73.1. For some fungi, variations in the hydrophobicity of the mycelium depending on the growth medium, the physiological state and the exposure to water were distinguished.

Fungos endofíticos isolados de ápices caulinares de pupunheira cultivada in vivo e in vitro

A perda de plantas micropropagadas ocorre, principalmente, pela presença de microrganismos, responsáveis pela morte das plantas no início da cultura ou em seu estabelecimento no campo. O trabalho teve como objetivo a identificação, por taxonomia clássica, e por meio de técnicas moleculares, de fungos presentes nos ápices caulinares de pupunheiras sadias, cultivadas no campo, e a comparação com os fungos isolados, em plantas micropropagadas há dois anos. Os isolados da microbiota fúngica endofítica, das plantas cultivadas in vitro, foram: Fusarium oxysporum, Neotyphodium sp. e Epicoccum nigrum; e das plantas in vivo, foram: Fusarium sp., F. proliferatum, F. oxysporum, Colletotrichum sp., Alternaria gaisen, Neotyphodium sp. e Epicoccum nigrum. As sete espécies de fungos foram reintroduzidas in vitro na planta hospedeira, demonstrando diferentes comportamentos. Neotyphodium sp. e E. nigrum estabeleceram uma interação endofítica com a planta, e as demais comportaram-se como patógenos, diminuindo o desenvolvimento das plântulas em relação às plantas sem inoculação. As espécies endofíticas apresentam potencial para o uso no controle biológico de patógenos de pupunha.