Ascospore Germination Research Articles

Survival, Germination, and Growth of Epichloë typhina and Significance of Leaf Wounds and Insects in Infection of Orchardgrass.

Epichloë typhina is an important stroma-producing endophytic ascomycete that is responsible for significant yield loss in orchardgrass (Dactylis glomerata) seed production fields. Infection is presumed to occur through leaves or stems, although details of the infection process and conditions that favor leaf infection are not well understood. The primary objectives of this study were to investigate the early stages of infection, including the effect of temperature or water potential on ascospore germination and subsequent growth of E. typhina, the tolerance of ascospores to desiccation, the requirement of leaf wounds for infection of orchardgrass by E. typhina, and the potential for insects to facilitate infection. Ascospores tolerated dry conditions, with at least 40% surviving 12 days under desiccation. Germination and growth of E. typhina was greatest at 25°C, with little to no growth at 5 and 35°C. Mycelial growth decreased with decreasing water potential from -0.3 to -10 MPa. Ascospore germination on leaves was predominantly hyphal at wound sites and iterative (conidiogenous) at sites without wounds. E. typhina typically entered leaves through wounds. Direct penetration was rarely observed and appeared to be associated with ascospore clusters. Germ tubes were significantly longer at sites with honeydew deposits from the bird cherry-oat aphid than at sites without honeydew. Growth of E. typhina was also observed at feeding sites of eriophyid mites, suggesting that leaf-wounding or sap-excreting insects support epiphyllous growth of E. typhina on leaves.

Twenty-five cultures of lichenizing fungi available for experimental studies on symbiotic systems

In this study we describe the techniques used to culture 25 mycobionts spanning three classes and five orders of the leotiomyceta (Ascomycota). We find that five media, including potato-carrot, malt extract-yeast extract (MY), Bold’s basal medium with nitrogen (NMBBM), oatmeal, and yeast extract with supplements (YES), are sufficient to induce ascospore germination of many lichenizing fungi and are also suitable for maintaining growth of the culture over the long term. Regular physical disruption of the cultures in liquid media is recommended to stimulate continued growth. Genomes of five of these lichen-forming fungal strains have been sequenced. The identity of each culture was confirmed by sequencing the nuclear ribosomal internal transcribed spacer (ITS) or the mitochondrial small subunit (mitSSU) from each strain. Additionally, the level of sequencing in terms of total number of genes sequenced for each taxon is provided. All fungal cultures have been deposited in public culture collections and, therefore, are available to the scientific community for conducting in vitro experiments.

The NADPH Oxidase Complexes in Botrytis cinerea: Evidence for a Close Association with the ER and the Tetraspanin Pls1

NADPH oxidases (Nox) are major enzymatic systems that generate reactive-oxygen species (ROS) in multicellular eukaryotes. In several fungi they have been shown to be involved in sexual differentiation and pathogenicity. However, in contrast to the well characterized mammalian systems, basic information on the composition, recruitment, and localization of fungal Nox complexes and on the molecular mechanisms of their cellular effects are still lacking. Here we give a detailed analysis of components of the Nox complexes in the gray mold fungus Botrytis cinerea. It had previously been shown that the two catalytic transmembrane subunits BcNoxA and B are important for development of sclerotia and for full virulence, with BcNoxA being involved in spreading of lesions and BcNoxB in penetration; BcNoxR functions as a regulator of both subunits. Here we present evidence (using for the first time a functional GFP fusion able to complement the ΔbcnoxA mutant) that BcNoxA localizes mainly to the ER and at the plasma membrane; BcNoxB shows a similar localization pattern, while the regulator BcNoxR is found in vesicles throughout the hyphae and at the hyphal tip. To identify possible interaction partners, which could be involved in the localization or recruitment of the Nox complexes, we functionally characterized the tetraspanin Pls1, a transmembrane protein, which had been suggested to be a NoxB-interacting partner in the saprophyte Podospora anserina. Knock-out experiments and GFP fusions substantiate a link between BcNoxB and BcPls1 because both deletion mutants have overlapping phenotypes (especially a defect in penetration), and the proteins show a similar localization pattern (ER). However, in contrast to the corresponding protein in P. anserina BcPls1 is important for female fertility, but not for ascospore germination.

Eisosome distribution and localization in the meiotic progeny of Aspergillus nidulans

In the model filamentous fungus Aspergillus nidulans, PilA and PilB, two homologues of the Saccharomyces cerevisiae eisosome proteins Pil1/Lsp1, and SurG, a strict orthologue of Sur7, are assembled and form tightly packed structures in conidiospores. As A. nidulans differs in its reproduction pattern from the Saccharomycotina in that it has the ability to reproduce through two different types of spores, conidiospores and ascospores, the products of the asexual and the sexual cycle respectively, we investigated the eisosome distribution and localization during the sexual cycle. Our results show that core eisosome proteins PilA, PilB and SurG are not expressed in hülle cells or early ascospores, but are expressed in mature ascospores. All eisosomal proteins form punctate structures at the membrane of late ascospores. In mature but quiescent ascospores, PilA forms static punctate structures at the plasma membrane. PilB also was observed at the ascospore membrane as well, with higher concentration at the areas where the two halves of ascospores are joined together. Finally, SurG was localized both at the membrane of ascospores and perinuclearly. In germlings originating from ascospores the punctate structures were shown to be composed only of PilA. PilB is diffused in the cytoplasm and SurG was located in vacuoles and endosomes. This altered localization is identical to that found in germlings originated from conidiospores. In germinated ascospores PilA foci did not colocalise with the highly mobile and transient peripheral punctate structures of AbpA, a marker for sites of clathrin-mediated endocytosis. Deletions of each one or all the three core eisosomal genes do not affect viability or germination of ascospores. In the presence of myriocin – a specific inhibitor of sphingolipid biosynthesis – PilA-GFP foci of ascospore germlings were less numerous and their distribution was significantly altered, suggesting a correlation between PilA foci and sphingolipid biosynthesis.

Autophagy genes Smatg8 and Smatg4 are required for fruiting-body development, vegetative growth and ascospore germination in the filamentous ascomycete Sordaria macrospora

Autophagy is a tightly controlled degradation process involved in various developmental aspects of eukaryotes. However, its involvement in developmental processes of multicellular filamentous ascomycetes is largely unknown. Here, we analyzed the impact of the autophagic proteins SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. A Saccharomyces cerevisiae complementation assay demonstrated that the S. macrospora Smatg8 and Smatg4 genes can functionally replace the yeast homologs. By generating homokaryotic deletion mutants, we showed that the S. macrospora SmATG8 and SmATG4 orthologs were associated with autophagy-dependent processes. Smatg8 and Smatg4 deletions abolished fruiting-body formation and impaired vegetative growth and ascospore germination, but not hyphal fusion. We demonstrated that SmATG4 was capable of processing the SmATG8 precursor. SmATG8 was localized to autophagosomes, whereas SmATG4 was distributed throughout the cytoplasm of S. macrospora. Furthermore, we could show that Smatg8 and Smatg4 are not only required for nonselective macroautophagy, but for selective macropexophagy as well. Taken together, our results suggest that in S. macrospora, autophagy seems to be an essential and constitutively active process to sustain high energy levels for filamentous growth and multicellular development even under nonstarvation conditions.

Isolates of Didymella bryoniae from South Carolina Remain Sensitive to DMI Fungicides Despite Multiyear Exposure

AbstractTriazole fungicides, which are sterol demethylation inhibitors, have become the primary systemic fungicides applied to cucurbits to control gummy stem blight caused by Didymella bryoniae. Isolates of D. bryoniae from South Carolina that were never exposed to tebuconazole or exposed for several years were tested for sensitivity to tebuconazole and difenoconazole. Colony diameters, percentage germination of ascospores and conidia, and germ tube lengths were measured when isolates were grown on agar amended with 0.10–10.0 mg/l tebuconazole and 0.01–1.0 mg/l difenoconazole. All 147 isolates tested were sensitive to tebuconazole and difenoconazole with mean EC50 values of 0.41 and 0.054 mg/l, respectively. Ascospore germination was greater than conidia germination on fungicide‐amended agar. Although the length of germ tubes arising from both spore types was reduced by both fungicides, the reduction was greater for ascospore germ tubes than for conidia germ tubes. Because many watermelon growers rotate crops among fields every two years, local populations of D. bryoniae have not been exposed repeatedly to tebuconazole. In addition, growers often apply a rotation of systemic and contact fungicides. Thus, despite exposure to tebuconazole for up to nine years, isolates of D. bryoniae from South Carolina remain sensitive to triazole fungicides.

Extratos de plantas de cobertura no desenvolvimento de Sclerotinia sclerotiorum

Extratos vegetais podem interferir no ciclo de vida dos fitopatógenos por promover ou inibir o desenvolvimento. Com base nessa propriedade, este trabalho foi realizado com o objetivo de estudar o efeito dos extratos aquosos das plantas de cobertura crotalária (Crotalaria juncea), braquiária (Brachiaria ruziziensis), capim-mombaça (Panicum maximum cv. mombaça), milheto (Pennisetum glaucum), feijão-guandu-anão (Cajanus cajan) e estilosantes (Stylosanthes capitata x Stylosanthes macrocephala) sobre o crescimento micelial, germinação carpogênica e micelial dos escleródios e germinação dos ascósporos do fungo Sclerotinia sclerotiorum. As etapas do experimento foram desenvolvidas em casa de vegetação, câmaras de crescimento e laboratório. As concentrações dos extratos utilizadas foram de 1%, 5%, 10% e 25%. Para verificar a influência dos extratos sobre a germinação micelial e carpogênica dos escleródios, foi utilizada somente a concentração de 25%. Os resultados sobre o crescimento micelial mostraram que o extrato da planta de Stylosanthes sp. inibiu o desenvolvimento do patógeno apenas na concentração de 25%. Para a germinação micelial dos escleródios, verificou-se que os extratos induziram a germinação 12 horas após a instalação do experimento, exceto o Stylosanthes sp. Na germinação dos ascósporos, apenas o extrato de Stylosanthes sp. impediu a germinação.

Biological Roles of the Podospora anserina Mitochondrial Lon Protease and the Importance of Its N-Domain

Mitochondria have their own ATP-dependent proteases that maintain the functional state of the organelle. All multicellular eukaryotes, including filamentous fungi, possess the same set of mitochondrial proteases, unlike in unicellular yeasts, where ClpXP, one of the two matricial proteases, is absent. Despite the presence of ClpXP in the filamentous fungus Podospora anserina, deletion of the gene encoding the other matricial protease, PaLon1, leads to lethality at high and low temperatures, indicating that PaLON1 plays a main role in protein quality control. Under normal physiological conditions, the PaLon1 deletion is viable but decreases life span. PaLon1 deletion also leads to defects in two steps during development, ascospore germination and sexual reproduction, which suggests that PaLON1 ensures important regulatory functions during fungal development. Mitochondrial Lon proteases are composed of a central ATPase domain flanked by a large non-catalytic N-domain and a C-terminal protease domain. We found that three mutations in the N-domain of PaLON1 affected fungal life cycle, PaLON1 protein expression and mitochondrial proteolytic activity, which reveals the functional importance of the N-domain of the mitochondrial Lon protease. All PaLon1 mutations affected the C-terminal part of the N-domain. Considering that the C-terminal part is predicted to have an α helical arrangement in which the number, length and position of the helices are conserved with the solved structure of its bacterial homologs, we propose that this all-helical structure participates in Lon substrate interaction.

Differential Sensitivity to Boscalid in Conidia and Ascospores of Didymella bryoniae and Frequency of Boscalid-Insensitive Isolates in South Carolina.

Since 2003, a 2:1 mixture of the fungicides boscalid and pyraclostrobin (Pristine) has been used widely on watermelon and other cucurbits, primarily to control gummy stem blight caused by Didymella bryoniae. Several isolates of D. bryoniae that were insensitive to boscalid at 10 mg/liter were found in a watermelon research plot in South Carolina in 2008. In total, 201 isolates collected between 1998 and 2009 were tested for sensitivity to boscalid by determining percentage germination of ascospores and conidia on media amended with boscalid at 0.01 to 10.0 mg/liter. All 31 isolates collected in 1998, 2002, or 2005 were sensitive to boscalid. Of the 170 isolates collected in or after 2006, 84.7% were insensitive to boscalid, including 19 of 30 isolates recovered from greenhouse-grown seedlings. The oldest insensitive isolates were obtained in 2006 from a greenhouse and in 2008 from a commercial field. Ascospores were less sensitive to boscalid than conidia. With boscalid at 1.0 mg/liter, 22.4% of ascospores but only 4.1% of conidia of 31 sensitive isolates germinated. Similarly, a mean of 68.6% of the ascospores and 54.1% of the conidia of 120 insensitive isolates germinated at 1.0 and 10.0 mg/liter. The 50% effective concentration (EC50) values based on ascospore germination were two to three times higher than values based on conidia germination. The significance of miscalculating EC50 values by considering only conidia was demonstrated in a greenhouse experiment. Twelve isolates that were sensitive, moderately insensitive, or highly insensitive based on conidia germination did not differ in relative virulence on boscalid-treated muskmelon seedlings when inoculum suspensions comprised ascospores alone or ascospores and conidia. This is the first report of differential sensitivity to a fungicide between conidia and ascospores in D. bryoniae. Because D. bryoniae produces conidia and ascospores on diseased hosts, both spore types should be used when calculating EC50 values for boscalid.

Ascospore discharge, germination and culture of fungal partners of tropical lichens, including the use of a novel culture technique

A total of 292 lichen samples, representing over 200 species and at least 65 genera and 26 families, were collected, mainly in Thailand; 170 of the specimens discharged ascospores in the laboratory. Generally, crustose lichens exhibited the highest discharge rates and percentage germination. In contrast, foliose lichen samples, although having a high discharge rate, had a lower percentage germination than crustose species tested. A correlation with season was indicated for a number of species. Continued development of germinated ascospores into recognizable colonies in pure culture was followed for a selection of species. The most successful medium tried was 2 % Malt-Yeast extract agar (MYA), and under static conditions using a liquid culture medium, a sponge proved to be the best of several physical carriers tested; this novel method has considerable potential for experimental work with lichen mycobionts.

Olpidium bornovanus-Mediated Germination of Ascospores of Monosporascus cannonballus: A Host-Specific Rhizosphere Interaction

Monosporascus cannonballus, a host-specific root-infecting ascomycete, is the causal agent of a destructive disease of melon (Cucumis melo L.) known as vine decline. Ascospores germinate only in the rhizosphere of melon plants growing in field soil. However, no germination occurs in the rhizosphere of melon plants if the field soil is heated to temperatures >50°C prior to infestation with ascospores. This observation suggested that germination is mediated by one or more heat-sensitive members of the soil microflora. Although bacteria or actinomycetes were heretofore suspected as the germination-inducing microbes, our data demonstrate that Olpidium bornovanus, an obligate, host-specific, root-infecting zoosporic fungus, is responsible. In four experiments conducted in autoclaved field soil amended with various population densities of culturally produced ascospores, significant ascospore germination was recorded only in the rhizosphere of cantaloupe seedlings colonized by O. bornovanus.

In vitro evaluation of Colombian plant extracts against Black Sigatoka (Mycosphaerella fijiensis Morelet)

In this work, 90 dichloromethane and methanol extracts obtained from 45 plants collected at the Natural Reserve Bremen – La Popa (Colombia) and at the Natural Regional Park Ucumarí (NRPU, Colombia) belonging to five botanical families were evaluated at 1000 mg/l, for their in vitro fungicide activity through the ascospore germ tube elongation and the measurement of the mycelial radial growth of Mycosphaerella fijiensis assays. The methanol extracts from the species Lycianthes acutifolia (Solanaceae) and Piper pesaresanum (Piperaceae); as well as, the dichloromethane extracts from P. pesaresanum and those from the Lauraceae family named Nectandra acutifolia and Ocoteca paulii, all inhibited M. fijiensis ascospore germination in 100% in the germinative tube elongation assay. With regards to the effects of the plant extracts on mycelial radial growth, the methanol extracts from P. pesaresanum and the dichloromethane one from N. acutifolia both showed 100% inhibition in this bioassay. Additionally, from the phytochemical screening on the dichloromethane and methanol extracts it was found that compounds such as alkaloids, phenols and terpenes were present in most of the extracts evaluated and they might be the cause of the antifungal activities reported.

Carrefour Mme. Gras: A wild-isolated Neurospora crassa strain that suppresses meiotic silencing by unpaired DNA and uncovers a novel ascospore stability defect

Ordinarily, RIP-induced erg-3 mutant Neurospora crassa ascospores and their erg+ siblings do not differ in stability during long-term storage. Consequently, the frequency of RIP-induced erg-3 mutants remains about constant regardless of the time that has elapsed between ascospore harvest and germination. We found, however, that RIP-induced erg-3 mutants were apparently selectively lost with time from among the ascospores stored from a cross with the wild-isolated Carrefour Mme. Gras strain from Haiti. The Haitian strain was also found to exert a dominant suppression of meiotic silencing by unpaired DNA. Similar loss of RIP-induced erg-3 mutant ascospores was seen among the stored ascospores from a subset of crosses heterozygous for the semi-dominant Sad-1 or Sad-2 suppressors of meiotic silencing. Our results suggest that crosses suppressed in meiotic silencing can compromise the stability during storage of ascospores that inherit RIP-induced mutations.

Molecular data reveal high host specificity in the phylogenetically isolated genus Massaria (Ascomycota, Massariaceae)

Molecular phylogenetic analyses of a four-gene sequence matrix (LSU, SSU, rpb2, tef1) demonstrate monophyly of the genus Massaria, which is placed as most basal lineage within Pleosporales. Data from microscopic morphology, pure cultures, and phylogenetic analyses of partial SSU-ITS-LSU rDNA and tef1 sequences revealed 17 taxa of Massaria, seven of which are described as new (M. ariae, M. aucupariae, M. campestris, M. mediterranea, M. parva, M. platanoidea, M. vindobonensis). Massarina macra is formally combined into Massaria. Synonymy of the genus Aglaospora with Massaria is confirmed, and the generic type Massaria inquinans is lecto- and epitypified. Massaria vomitoria, M. gigaspora and M. pyri, commonly considered as conspecific with M. inquinans, are shown to be distinct species. Due to homonymy, the new name M. gigantispora is introduced for M. gigaspora Fuckel. Several species are lecto- and/or epitypified. A key to all treated species is provided. Most Massaria species revealed by molecular phylogenetic analyses can be well characterised by a suite of morphological traits like ascospore shape, ascospore length and width, ascospore colour in the intact ascus vs. after ejection, size of pseudothecia, presence or absence of a black stromatic zone delimiting the pseudostroma, and staining of the substrate. Two different modes of ascospore germination were observed in pure culture, i.e. hyphal or by ejection of a naked protoplast, the former developing into hyphal colonies and the latter into meristematically growing colonies. Modes of ascospore germination and colony growth were found to be characteristic for the respective species. All species were found to be highly host-specific, disproving the wide host range given for Massaria inquinans in the literature. Biodiversity of Massaria was found to be centred on the genus Acer (seven species), where up to four species can occur on the same host species, and on Rosaceae (four species). Evidence for a hemibiotrophic life style and weak parasitism of Massaria is provided and discussed. Geographic distribution of species is reconsidered, concluding that Europe may be the centre of Massaria biodiversity.

Ume6p is required for germination and early colony development of yeast ascospores

Ume6p is a nonessential transcription factor that represses meiotic gene expression during vegetative growth in budding yeast. To relieve this repression, Ume6p is destroyed as cells enter meiosis and is not resynthesized until spore wall assembly. The present study reveals that spores derived from a ume6 null homozygous diploid fail to germinate. In addition, mutant spores from a UME6/ume6 heterozygote exhibited reduced germination efficiency compared with their wild-type sister spores. Analysis of ume6 spore colonies that did germinate revealed that the majority of cells in microcolonies following the first few cell divisions were inviable. As the colony developed, the viability percentage increased and achieved wild-type levels within approximately six cell divisions, indicating that the requirement for Ume6p in cell viability is transient. This function is specific for germinating spores as Ume6p has no or only a modest impact on the return to the growth ability of cells arrested at other points in the cell cycle. These results define a new role for Ume6p in spore germination and the first few subsequent mitotic cell divisions.

耐熱性カビの子嚢胞子発芽測定による新規 D 値試験法の開発

耐熱性カビの子嚢胞子発芽測定による新規 D 値試験法の開発

Germination of Monosporascus cannonballus ascospores in the rhizosphere: a host-specific response

The specificity of ascospore germination of Monosporascus cannonballus in the rhizosphere of 26 species/cultivars of plants belonging to eight families and 14 genera was examined. Results showed that ascospore germination occurred only in the rhizosphere of genera, species and cultivars belonging to the Cucurbitaceae family. Differences between the number of ascospore germlings attached to roots of the various cucurbit species/cultivars suggests qualitative and/or quantitative differences in a specific root exudate(s) which are presumably associated with the induction of ascospore germination.

The small serine-threonine protein SIP2 interacts with STE12 and is involved in ascospore germination in Sordaria macrospora

In fungi, the homoeodomain protein STE12 controls diverse developmental processes, and derives its regulatory specificity from different protein interactions. We recently showed that in the homothallic ascomycete Sordaria macrospora, STE12 is essential for ascospore development, and is able to interact with the alpha-domain mating-type protein SMTA-1 and the MADS box protein MCM1. To further evaluate the functional roles of STE12, we used the yeast two-hybrid approach to identify new STE12-interacting partners. Using STE12 as bait, a small, serine-threonine-rich protein (designated STE12- interacting protein 2, SIP2) was identified. SIP2 is conserved among members of the fungal class Sordariomycetes. In vivo localization studies revealed that SIP2 was targeted to the nucleus and cytoplasm. The STE12/SIP2 interaction was further confirmed in vivo by bimolecular fluorescence complementation. Nuclear localization of SIP2 was apparently mediated by STE12. Unlike deletion of ste12, deletion of sip2 in S. macrospora led to only a slight decrease in ascospore germination, and no other obvious morphological phenotype. In comparison to the Δste12 single knockout strain, ascospore germination was significantly increased in a Δsip2/ste12 double knockout strain. Our data provide evidence for a regulatory role of the novel fungal protein SIP2 in ascospore germination.

Effects of fungicides onin vitrospore germination and mycelial growth of the phytopathogensLeptosphaeria maculansandL. biglobosa(phoma stem canker of oilseed rape)

Phoma stem canker, caused by the coexisting related fungal pathogens Leptosphaeria maculans (Des.) Ces. & de Not and L. biglobosa Shoemaker & H Brun, is a major disease of winter oilseed rape in the UK. Annually, over 90% of UK crops receive at least one foliar application of fungicide, but little is known about the sensitivity of the more damaging L. maculans and the less damaging L. biglobosa to these fungicides. The effects of flusilazole, tebuconazole and Methyl Benzimidazole Carbamate (MBC) fungicides (benomyl and carbendazim) on the germination of ascospores, conidia and germ tube growth of both species were examined. Isolates collected from different oilseed rape crops in England and Wales were assessed for their mycelial growth on fungicide-amended medium, and ED(50) values were calculated. Leptosphaeria maculans and L. biglobosa differed in their sensitivity to fungicides. Conidial germination of L. maculans was more sensitive to these fungicides than that of L. biglobosa. Isolates of L. maculans had smaller ED(50) values for mycelial growth for all fungicides tested than isolates of L. biglobosa. These results suggest that fungicide applications might affect the structure of L. maculans/L. biglobosa populations in UK winter oilseed rape crops.

Behaviour ofNeonectria fuckelianacausing a pine canker disease in New Zealand

Neonectria fuckeliana (C. Booth) Castl. & Rossman is known to be associated with a stem canker disease of Pinus radiata D. Don in New Zealand plantation forests. Although N. fuckeliana has been previously recorded as a wound invader or weak pathogen of Picea and Abies species in the Northern Hemisphere, little is currently known about the basic biology of the fungus. This paper outlines early investigations into the spore production and dispersal of N. fuckeliana in New Zealand. Perithecia of N. fuckeliana occur frequently on pruned stubs and on the surface of cankers, and ascospores appear to be the primary means of dispersal for this fungus in New Zealand. Both field collections and spore trapping show that mature perithecia contain viable ascospores in all seasons, and spores are ejected and dispersed using moisture. The conidial phases are rarely found in the field. Optimum temperature for both growth of mycelium and ascospore germination was between 15 and 25 °C. Some spore germination occurred at temperatures as low as 5 °C, but above 25 °C spore germination was abnormal. Ascospores and perithecia favoured storage in lower temperatures: overall, ascospores from perithecia stored at room temperature gradually lost their viability, whereas those stored at 4 and –8 °C maintained their viability over an 18 month period.