Development of mutants of coniothyrium minitans with improved efficiency for control of sclerotinia sclerotiorum

Coniothyrium minitans (CM) is hyperparasitic to Sclerotinia sclerotiorum (SS), a pathogen of many economically important crops. In this paper, we describe the isolation of improved mutants of CM, using a UV - irradiation regime, with altered chitinase production and tolerance to high concentration of iprodione, which are effective against SS. Three out of the 59 mutants obtained inhibited the mycelial growth of CM. Infectivity of sclerotia by the new mutants was assayed by the plant-tissue-based system using carrot segments. More than 80% of sclerotia were colonized by the mutants and the wild-type CM. The mutant strains retained ability to produce significant amounts of chitinase. The mutants differed from their wild-type strain in appearance, morphology and sporulation. In conclusion, the results presented here provide evidence that the new biotypes of C. minitans are effective in controlling S. sclerotiorum.

Strains of Coniothyrium minitans reduce the emission of apothecia of Sclerotinia sclerotiorum

Sclerotia of Sclerotinia sclerotiorum (Ss) can survive for long time in soil and are the main inoculum source of the white mold disease. An alternative for reducing this inoculum is the use of parasites, such as Coniothyrium minitans (Cm). We evaluated the potential of Cm isolates for the biological control of Ss in beans. The effect of the temperature on the growth of 15 isolated of Cm was evaluated in vitro. The hyperparasitism ability of Cm was evaluated in soil infested with sclerotia and conditioned in pots. The infested soil was treated with conidia suspension of the antagonists, fluazinan or sterile distilled water. After seven days at 20°C, the sclerotia were removed from soil and placed inside Petri dishes over bean leaves previously disinfested. The germination and parasitism of sclerotia were evaluated after 7 to 10 days. To evaluate the apothecia emission, soil infested with sclerotia of Ss and treated as described was maintained at 18°C and the number of emerged apothecia was counted up to 84 days after inoculation. The emergence of bean plants in soil infested with sclerotia and mycelium of the pathogen and treated as described was evaluated in greenhouse. The ideal temperature for growth of Cm isolates varied from 18 to 19°C and at 30-35°C they were complete inhibited. The isolates of Cm promoted less than 10% of reduction in viability of the sclerotia, but they significantly reduced the emission of apothecia. Two isolates increased the emergence of plants in relation to the inoculated check, but was significantly lower than the non-inoculated check. Field tests will be conduct to confirm the potential of the selected isolates to reduce the inoculum source of the pathogen.

Determination of Coniothyrium minitans conidial and germling lectin avidity by flow cytometry and digital microscopy

The avidity of conidia and 48-h-old germlings of Coniothyrium minitans for FITC-conjugated lectins was characterised by flow cytometry and digital microscopy. Six isolates of C. minitans representing three morphological types were compared. Binding of Con A, SBA and WGA by conidial populations varied markedly in extent and pattern between isolates, however, with increasing culture age, conidia from all isolates demonstrated a significant reduction in lectin avidity. Germling isolates bound significantly different amounts of lectins and lectin binding differed significantly with locality. Spore walls of all germlings from all isolates bound more ConA compared with hyphal apices and mature hyphal walls. In contrast, hyphal apices of the majority of germling isolates, readily bound SBA and mature hyphal walls of germling isolates bound more WGA than other regions of the germlings. Such differential lectin binding by conidia and germlings may influence their specific surface interactions and adherence characteristics.

Hydrophobicity and surface electrostatic charge of conidia of the mycoparasite Coniothyrium minitans

The effect of increasing culture age on cell surface hydrophobicity (CSH) and cell surface electrostatic charge (measured as zeta potential) of conidia from five isolates of Coniothyrium minitans representing three different morphological types was examined. Conidial CSH of three isolates (A2 960/1, CH1 and CH2) decreased with culture age, whereas CSH of two others (B 1300/2 and IMI 134523) remained high for the whole 42 day experimental period. In contrast, cell surface electrostatic charge decreased uniformly in conidia of all five isolates for the first 34 d and then rose slightly at 42 d. The variation in cell surface electrostatic charge (spectrum width) of the sampled conidia decreased with age for all five isolates. In all five isolates cell surface electrostatic charge of conidia became increasingly negative as the pH of the buffer used to suspend conidia was increased from pH 3.0 to 9.0. No relationship between colony morphology of C. minitans and conidial CSH and cell surface electrostatic charge was found.

Controle biológico de Sclerotinia sclerotiorum por Coniothyrium minitans: ensaios preliminares.

O mofo-branco ou podridão de esclerotinia, causada por Sclerotinia sclerotiorum, é uma doença de grande importância agronômica, devido ao fato de ser de difícil controle e provocar grandes perdas à cultura atacada. O feijão sofre consideráveis estragos por ser altamente susceptível à doença. Em condições de safra outono?inverno e de alta umidade proporcionada por irrigação, o patógeno se espalha com extrema facilidade. O patógeno produz estruturas de resistência, denominados escleródios, que podem durar muitos anos no solo. Uma das alternativas propostas para o manejo da doença é o controle biológico, pela utilização de parasitas de escleródios e apotécios. Entre estes, o fungo Coniothyrium minitans é amplamente estudados em países do hemisfério norte. O presente trabalho teve como objetivo verificar preliminarmente a capacidade de isolados de C. minitans como agente de biocontrole de S. sclerotiorum em biotestes em laboratório. Nas condições dos testes, verificou-se que os isolados avaliados promoveram apenas ligeira redução na viabilidade dos escleródios e apotécios e foram altamente sensíveis ao principal fungicida utilizado no controle da doença. Novos testes deverão ser realizados para verificar o potencial de C. minitans contra o patógeno.

Seleção de isolados de Coniothyrium minitans para o controle de Sclerotinia sclerotiorum.

2009

Supressão de apotécios de Sclerotinia sclerotiorum em solo por Coniothyrium minitans.

O mofo-branco (Sclerotinia sclerotiorum, Ss) afeta a cultura do feijão (Phaseolus vulgaris L.) principalmente em cultivos de outono-inverno. O agente de controle biológico (ACB) Coniothyrium minitans (Cm) é utilizado em países de clima temperado como alternativa para controle da doença. O antagonista é especialista em parasitar escleródios e reduzir o inóculo primário do patógeno. Porém, não existem trabalhos com o ACB nas condições brasileiras. Objetivou-se avaliar a capacidade de isolados do ACB em suprimir a emissão de apotécios do patógeno. O ensaio foi conduzido em blocos ao acaso com cinco repetições em câmara de crescimento a 20±2˚C e fotoperíodo de 12h. Foram realizados 10 tratamentos: oito isolados de Cm, fungicida (fluazinam) e testemunha. Cada repetição consistiu de uma caixa gerbox (11x11x3,5 cm) contendo solo solarizado, tratado com esporos do ACB (107 con./ml, 300L/ha de calda), dose recomendada do fungicida ou ADE (testemunha). Em cada repetição depositaram-se 12 escleródios de Ss produzidos em meio cenoura-fubá. Verificou-se redução significativa (Tukey, 5%) na emissão de apotécios de Ss pelos isolados de Cm (56 a 93%), sendo alguns tratamentos superiores ao fungicida (76%). C. minitans tem grande potencial para controle do mofo-branco do feijoeiro.

Crescimento de isolados de coniothyrium minitans em diferentes temperaturas.

O mofo-branco causado por Sclerotinia slerotiorum é uma das doenças mais destrutivas do feijoeiro. Agentes de controle biológico (ACB) vêm sendo usados com sucesso para o controle do patógeno em diversos países. Entre estes, Coniothyrium minitans tem se destacado. O objetivo deste trabalho foi avaliar o efeito da temperatura sobre o crescimento de isolados do antagonista. O experimento foi composto de 15 isolados de C. minitans e cinco temperaturas (15, 20, 25, 30 e 35ºC) em câmaras de crescimento na ausência de luz. Foram feitas quatro repetições para cada isolado e temperatura, sendo cada repetição composta por uma placa de Petri. Os diâmetros da colônia no sentido transversal e longitudinal de crescimento foram medidos após 4, 7, 11, 14 e 19 dias, quando as primeiras repetições atingiram o crescimento máximo. O efeito da temperatura sobre o crescimento radial dos isolados de C. minitans foi semelhante. A temperatura ideal de crescimento variou de 18 a 19ºC para todos isolados e nas temperaturas de 30 e 35ºC não houve crescimento. Ainda não existem estudos acerca da utilização do antagonista em condições brasileiras. Essa espécie é resistente à decomposição por luz, porém é sensível a altas temperaturas, como verificado. Portanto, C. minitans tem potencial para ser usado nos cultivos de outono-inverno no Centro-sul do Brasil, onde as temperaturas são mais amenas. Os isolados de C. minitans serão avaliados quanto à capacidade parasítica de escleródios e apotécios do patógeno em condições de campo em cultivos de feijão de outono-inverno.

Arbuscular mycorrhizal fungi and collembola non-additively increase soil aggregation

Soil aggregation is a principal ecosystem process mediated by soil biota. Collembola and arbuscular mycorrhizal (AM) fungi are important groups in the soil, and can interact in various ways. Few studies have examined collembola effects on soil aggregation, while many have quantified AM effects. Here, we asked if collembola have any effect on soil aggregation, and if they alter AM fungi-mediated effects on soil aggregation.

We carried out a factorial greenhouse study, manipulating the presence of both collembola and AM fungi, using two different plant species, Sorghum vulgare and Daucus carota. We measured root length and biomass, AMF (and non-AMF) soil hyphal length, root colonization, and collembolan populations, and quantified water stable soil aggregates (WSA) in four size classes.

Soil exposed to growth of AMF hyphae and collembola individually had higher WSA than control treatments. Moreover, the interaction effects between AMF and collembola were significant, with nonadditive increases in the combined application compared to the single treatments.

Our findings show that collembola can play a crucial role in maintaining ecological sustainability through promoting soil aggregation, and point to the importance of considering organism interactions in understanding formation of soil structure. (C) 2011 Elsevier Ltd. All rights reserved.

Acanthamoeba polyphaga trophozoite binding of representative fungal single cell forms

Acanthamoeba polyphaga trophozoites bind yeast cells of Candida albicans isolates within a few hours, leaving few cells in suspension or still attached to trophozoite surfaces. The nature of yeast cell recognition, mediated by an acanthamoebal trophozoite mannose binding protein is confirmed by experiments utilizing concentration dependent mannose hapten blocking. Similarly, acapsulate cells of Cryptococcus neoformans are also bound within a relatively short timescale. However, even after protracted incubation many capsulate cells of Cryptococcus remain in suspension, suggesting that the capsulate cell form of this species is not predated by acanthamoebal trophozoites. Further aspects of the association of Acanthamoeba and fungi are apparent when studying their interaction with conidia of the biocontrol agent Coniothyrium minitans. Conidia which readily bind with increasing maturity of up to 42 days, were little endocytosed and even released. Cell and conidial surface mannose as determined by FITC-lectin binding, flow cytometry with associated ligand binding analysis and hapten blocking studies demonstrates the following phenomena. Candida isolates and acapsulate Cryptococcus expose most mannose, while capsulate Cryptococcus cells exhibit least exposure commensurate with yeast cellular binding or lack of trophozoites. Conidia of Coniothyrium, albeit in a localized fashion, also manifest surface mannose exposure but as shown by Bmax values, in decreasing amounts with increasing maturity. Contrastingly such conidia experience greater trophozoite binding with maturation, thereby questioning the primacy of a trophozoite mannose-binding-protein recognition model.

Eine neue Rosenkrankheit, verursacht durch den Pilz Coniothyrium Wernsdorffiae

Von Dr. R. Laubert

Etudes sur le rot livide de la vigne (coniothyrium diplodiella) /

Mode of access: Internet.

Untersuchungen mit Bodenpilzen aus der Rebstock-Rhizosphäre unterschiedlich bewirtschafteter Rebanlagen des Rheingaus unter besonderer Berücksichtigung ihrer Pathogenität

Ziel der Untersuchungen war, Pilze aus geschädigtem und ungeschädigtem Wurzelmaterial konventionell und ökologisch bewirtschafteter Weinbergsböden zu isolieren und diese auf ihre Durchsetzungsfähigkeit gegenüber den anderen Arten bzw. deren Pilzmetabolitsuspensionen unter unterschiedlichen Nahrungsbedingungen zu prüfen und eine eventuelle substratabhängige Verhaltensänderung bei den Spezies in Interaktion festzustellen. Zudem wurde in weiteren In-vitro- Versuchen das pathogene Potenzial der gefundenen Arten gegenüber Vitis spp. getestet. Hintergrund dieser Untersuchungen war die Hypothese, dass Absterbeerscheinungen in Rebanlagen nicht durch die Reblaus per se verursacht werden, sondern dass ein Zusammenhang zwischen der Bewirtschaftungsmethode und dem Schadbild in reblausbefallenen Rebanlagen besteht und dessen Entstehung auf pathogenkonduktive und –suppressive Eigenschaften des Bodens zurückgeführt werden kann. Aus rund 2400 Wurzelproben konnten insgesamt 49 Pilzarten isoliert und bestimmt und mehr als die Hälfte davon in Wurzeln beider Versuchsflächen nachgewiesen werden. Ein Großteil der Pilze wurde sowohl in geschädigten als auch in ungeschädigten Wurzelgeweben identifiziert. Darunter waren Arten, die in der Literatur als Parasiten und Saprobier beschrieben werden, aber auch Arten, die eine andere Lebensweise pflegen oder deren Lebensweise nicht bekannt ist. Mit Hilfe von Interaktionsversuchen auf unterschiedlichen Nährmedien (einem Voll- und einem Mangelmedium) konnte bei den untersuchten Arten teilweise starke substratabhängige Verhaltensänderung in Interaktion mit bestimmten Pilzkolonien festgestellt und auf die Verfügbarkeit von organischem Kohlenstoff zurückgeführt werden. Starke Konkurrenz um organischen Kohlenstoff und dadurch entstehende fungistatische und antibiotische Effekte können in diesem Zusammenhang pathogenkonduktive bzw. pathogensuppressive Bodeneigenschaften fördern oder hemmen. Weiterhin konnte gezeigt werden, dass alle 15 in vitro an Vitis spp. inokulierten Pilze (Absidia glauca, Acremonium kiliense, Aspergillus ustus, Cylindrocarpon magnusianum, Cylindrocarpon sp., Fusarium culmorum, F. detonianum, F. oxysporum, F. sacchari, F. semitectum, Gliocladium roseum, Leptosphaeria coniothyrium, Penicillium expansum, Trichoderma harzianum, T. pseudokoningii), unter denen sich auch als Saprobier bekannte Arten befanden (P. expansum, T. harzianum), selbst bei Verfügbarkeit organischer Kohlenstoffverbindungen im Substrat, gegenüber Vitis spp. ein fakultativ pathogenes Potenzial besitzen. Diese aus In-vitro-Interaktionsversuchen gewonnenen Erkenntnisse geben Hinweise darauf, welchen Einfluss die Bewirtschaftung, insbesondere die Versorgung der Weinbergsböden mit organischem Kohlenstoff, auf fakultativ pathogene Sekundärparasiten in Form von Bodenpilzen und folglich auf die Entwicklung von Schadbildern an durch die Reblaus prädispositionierten Rebpflanzen in vivo haben kann.