Germling fusion via conidial anastomosis tubes in the grey mould Botrytis cinerea requires NADPH oxidase activity

In many filamentous ascomycete species, the early steps of colony development include fusion between germinating vegetative spores (conidial germlings). Often these fusion events are mediated by specialized hyphal structures, so-called conidial anastomosis tubes (CATs). Here, we show that germling fusion in the grey mould Botrytis cinerea is mediated by hyphal structures possessing the typical features of CATs. Formation of these structures is delayed when spores are germinating on complex media compared to growth on poor substrates. Fusion frequency is also influenced by the growth conditions of the precultures from which spores were obtained. During germination on hydrophobic plant surfaces, which induce pathogenic development, CAT formation is significantly suppressed. Screening of existing B. cinerea gene knockout mutants identified strains lacking the NADPH oxidase BcNoxA or the potential Nox regulator BcNoxR as fusion deficient, suggesting a potential role of reactive oxygen species (ROS) signalling in CAT formation and fusion.     

The potential biocontrol agent Pseudomonas antimicrobica inhibits germination of conidia and outgrowth of Botrytis cinerea

AIMS: Antifungal metabolites of Pseudomonas antimicrobica have previously been shown to inhibit conidial germination of the grey mould pathogen Botrytis cinerea. In this study, metabolites of the bacterium have been tested at different stages of Botrytis germination to determine their effects on germ tube production and extension. METHODS AND RESULTS: Metabolites were added to conidia that had been pre-incubated for either 120 or 255 min. Pseudomonas antimicrobica inhibited B. cinerea conidial germination and caused a significant reduction in germ tube extension, irrespective of the stage of germination. Abnormal germination and a reduction in the frequency of lateral branching of the germ tubes in the presence of the metabolites were also reported, suggesting interference with normal hyphal development. CONCLUSION: The bacterium can inhibit germination of conidia and extension of germ tubes at different stages of Botrytis development. SIGNIFICANCE AND IMPACT OF THE STUDY: The antagonistic activity of the bacterium has promising implications for its use as a biocontrol agent.     

Live-cell imaging of endocytosis during conidial germination in the rice blast fungus,Magnaporthe grisea

Although there is growing evidence that endocytosis is important in hyphal tip growth, it has not previously been shown to occur during fungal spore germination. We have analysed and characterized endocytosis during the germination of living conidia of the rice blast fungus, Magnaporthe grisea. Conidia treated with the endocytic markers Lucifer Yellow carbohydrazide, FITC-dextran, and FM4-64 were imaged by confocal microscopy. Internalization of these fluorescent marker dyes by conidia was blocked by chemical and temperature treatments that inhibit endocytosis, and the sequential staining of organelles by the membrane-selective dye FM4-64 was consistent with dye internalization by endocytosis. FM4-64 uptake occurred within 2-3 min of conidial hydration, more than 40 min before the emergence of the germ tube. The times at which each of the three conidial cells initiated dye internalization were different as were the rates of dye uptake by each cell. Using these techniques we have demonstrated for the first time that ungerminated and germinated spores of filamentous fungi undergo endocytosis. Furthermore, internalization of FITC-dextran and Lucifer Yellow carbohydrazide by germinating conidia provides the first direct evidence for fluid-phase endocytosis in a filamentous fungus. FM4-64 was internalized by both ungerminated conidia and conidial germlings on the rice leaf suggesting that endocytosis might play a significant role in spore germination and germ tube growth during the pre-penetration phase of infection.     

Fine structure of resting and germinatingPenicillium chrysogenum conidiospores

Summary The fine structure of resting and germinating conidia ofPenicillium chrysogenum has been examined by electron microscopy. In addition to enlargement of the cells, a number of changes in ultrastructure become evident as morphogenesis proceeds. The newly synthesized germ tube is continuous with the corresponding layers of the conidial wall. Some conidial wall layers, however, do not extend into the hyphal wall. Several sections showing initial septum synthesis suggest that a septal pore is not a necessary structural entity. A characteristic orientation of the initial septum formed after germination is described. Aside from numerical considerations, no significant changes occur in nuclei, mitochondria, or ribosomes. The electron micrographs illustrate the presence of spherosomes, lomasomes, and nucleoli; the possible significance of these structures is discussed.     

Development of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> on Tolerant and Susceptible <Emphasis Type="Italic">Olea europaea</Emphasis> L. cultivars: A Microscopic Analysis

Colletotrichum acutatum is a cosmopolitan and damaging plant pathogen of temperate, subtropical, and tropical fruits and causes anthracnose on olive (Olea europaea L.). Three olive cultivars showing a variable response to infection by C. acutatum were selected to a preliminary study of pathogen development. Fruit samples, from susceptible and tolerant cultivars, were taken at 0, 24, 48, 72, and 192 h after inoculation for a microscopic and histological study of the infection and colonization process. The aim of this study was to compare the infection process: conidial germination, germ tube and appressorium formation, hyphal growth, and mesocarp colonization in susceptible and tolerant olive cultivars as a condition for further exploration of disease development, which is required to develop cultivars with improved resistance to anthracnose. The rate of mesocarp colonization differed between the susceptible and tolerant cultivars, and both intracellular hemibiotrophy and subcuticular intramural necrotrophy were observed. Hemibiotrophic infection predominated in the moderately tolerant cultivar.     

A novel Botrytis cinerea-specific gene BcHBF1 enhances virulence of the grey mould fungus via promoting host penetration and invasive hyphal development

Botrytis cinerea is the causative agent of grey mould on over 1000 plant species and annually causes enormous economic losses worldwide. However, the fungal factors that mediate pathogenesis of the pathogen remain largely unknown. Here, we demonstrate that a novel B. cinerea-specific pathogenicity-associated factor BcHBF1 (h yphal b ranching-related f actor 1), identified from virulence-attenuated mutant M8008 from a B. cinerea T-DNA insertion mutant library, plays an important role in hyphal branching, infection structure formation, sclerotial formation and full virulence of the pathogen. Deletion of BcHBF1 in B. cinerea did not impair radial growth of mycelia, conidiation, conidial germination, osmotic- and oxidative-stress adaptation, as well as cell wall integrity of the ∆Bchbf1 mutant strains. However, loss of BcHBF1 impaired the capability of hyphal branching, appressorium and infection cushion formation, appressorium host penetration and virulence of the pathogen. Moreover, disruption of BcHBF1 altered conidial morphology and dramatically impaired sclerotial formation of the mutant strains. Complementation of BcHBF1 completely rescued all the phenotypic defects of the ∆Bchbf1 mutants. During young hyphal branching, host penetration and early invasive growth of the pathogen, BcHBF1 expression was up-regulated, suggesting that BcHBF1 is required for these processes. Our findings provide novel insights into the fungal factor mediating pathogenesis of the grey mould fungus via regulation of its infection structure formation, host penetration and invasive hyphal branching and growth.     

Fine Structure of Germinating Botrytis fabae Sardina Conidia

The fine structure of germinating Botrytis fabae conidia wasstudied using both chemically stained sections and freeze-etchedreplicas. Germinating conidia have fewer organelles than restingconidia, glycogen is absent, and prevacuoles have disappeared.Endoplasmic reticulum which occurs as small strands close tothe cell wall of resting conidia becomes, on germination, multiplesheets surrounding the nuclei. A cross wall is formed at thebase of the germ tube soon after germination commences. Thenew wall material which appears to be continuous with this septalwall is produced, at least partly, from a new wall layer laiddown in the centre of the old conidial wall. An apical corpuscleis present at the apex of young germ tubes. Freeze-etched preparationsshow the formation of lomasomes by the passage of vesicles throughthe plasmalemma of conidia and germ tubes. In young hyphae lomasomescontain a complex arrangement of branching tubules. Some ofthe particles on the outer plasmalemma of young hyphae are arrangedin a geometrical pattern.     

Molecular and cellular events during the germination of conidia of Sporothrix schenckii

Hyaline, non pigmented microconidia of Sporothrix schenckii were harvested and allowed to form germ tubes in a basal medium with glucose at pH 4.0 and 25 °C. These conditions supported only the development of the mycelial form of Sporothrix schenckii in a reproducible, synchronized manner which allowed further analysis of the early cellular events ocurring during the germination of the conidia. The relationship between macromolecular synthesis (DNA, RNA and protein synthesis) and nuclear division, hyphal growth and septum formation were established. Following inoculation, protein synthesis was observed after 10 minutes followed by RNA synthesis, after 1 h and DNA synthesis after 2 h. The first nuclear division was observed during the 9 to 12 h interval after inoculation. Germ tube formation slightly preceeded nuclear division and was first evidenced 9 h after the induction of germination but was not completed until 12 h after inoculation. Septation was first observed in the germ tubes 0.25 m from the mother cell-germ tube function 9 h after induction of germination.     

cAMP regulation of "pathogenic" and "saprophytic" fungal spore germination

We report on the elucidation of two separate pathways of spore germination in a plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene. Conidia of the fungus can germinate either from one side or from both sides, depending on external conditions. In shake culture that includes an extract made up from fresh peas, the unicellular conidium divides and one of the two cells develops a germ tube. On a solid surface this germ tube differentiates an appressorium. In rich medium without pea extract, germination is highly similar to Aspergillus spore germination: the conidium swells, forms a single germ tube and then divides and forms a second germ tube. Conidia that germinate in a rich medium do not form appressoria even on a solid surface and are non-pathogenic. In rich medium, cAMP stimulates germination in rich liquid cultures and induces appressoria formation on a hard surface. In pea extract cAMP induces swelling and formation of irregular germ tubes and appressoria. Our results suggest that plant surface signals induce pathogenic-specific spore germination in a cAMP-independent manner. cAMP is required for saprophytic germination and for appressorium formation.     

A p21-activated kinase is required for conidial germination in Penicillium marneffei

Asexual spores (conidia) are the infectious propagules of many pathogenic fungi, and the capacity to sense the host environment and trigger conidial germination is a key pathogenicity determinant. Germination of conidia requires the de novo establishment of a polarised growth axis and consequent germ tube extension. The molecular mechanisms that control polarisation during germination are poorly understood. In the dimorphic human pathogenic fungus Penicillium marneffei, conidia germinate to produce one of two cell types that have very different fates in response to an environmental cue. At 25 degrees C, conidia germinate to produce the saprophytic cell type, septate, multinucleate hyphae that have the capacity to undergo asexual development. At 37 degrees C, conidia germinate to produce the pathogenic cell type, arthroconidiating hyphae that liberate uninucleate yeast cells. This study shows that the p21-activated kinase pakA is an essential component of the polarity establishment machinery during conidial germination and polarised growth of yeast cells at 37 degrees C but is not required for germination or polarised growth at 25 degrees C. Analysis shows that the heterotrimeric G protein alpha subunit GasC and the CDC42 orthologue CflA lie upstream of PakA for germination at both temperatures, while the Ras orthologue RasA only functions at 25 degrees C. These findings suggest that although some proteins that regulate the establishment of polarised growth in budding yeast are conserved in filamentous fungi, the circuitry and downstream effectors are differentially regulated to give rise to distinct cell types.     

甾醇生物合成抑制剂粉锈宁对苹果黑星病菌发育的影响

采用电子显微镜和细胞化学技术,研究了杀菌剂粉锈宁（甾醇生物合成抑制剂）对苹果黑星病菌在苹果叶片上发育的影响。观察结果表明,接种前24h施药对病菌入侵有明显的影响。表现为分生孢子的萌发受阻,推迟萌发及萌发率降低;并引起芽管畸形,不能形成附着胞。接种后6天（显症前）施药,可引起叶片角质层下菌丝细胞和子座细胞的原生质坏死、细胞壁不规则增厚及液泡增大, 从而使菌丝进一步发育受阻。接种后12天（显症后）施药,不仅导致菌丝、子座细胞发生上述变化, 而且引起分生孢子和分生孢子梗塌陷、畸形,阻止了病菌的进一步产孢和扩展。细胞化学定位分析结果表明,?-1,3-葡聚糖和几丁质这两种胞壁主要成分在对照菌丝和药剂处理后的菌丝细胞壁内含量有很大差异。在药剂处理的菌丝细胞壁中,这两种成分的标记密度明显高于对照菌丝,表明杀菌剂对病菌质膜透性的不利影响使?-1,3-葡聚糖和几丁质在菌丝细胞壁中过度累积。     

Spatial Uncoupling of Mitosis and Cytokinesis during Appressorium-Mediated Plant Infection by the Rice Blast Fungus Magnaporthe oryzae

To infect plants, many pathogenic fungi develop specialized infection structures called appressoria. Here, we report that appressorium development in the rice blast fungus Magnaporthe oryzae involves an unusual cell division, in which nuclear division is spatially uncoupled from the site of cytokinesis and septum formation. The position of the appressorium septum is defined prior to mitosis by formation of a heteromeric septin ring complex, which was visualized by spatial localization of Septin4:green fluorescent protein (GFP) and Septin5:GFP fusion proteins. Mitosis in the fungal germ tube is followed by long-distance nuclear migration and rapid formation of an actomyosin contractile ring in the neck of the developing appressorium, at a position previously marked by the septin complex. By contrast, mutants impaired in appressorium development, such as Δpmk1 and ΔcpkA regulatory mutants, undergo coupled mitosis and cytokinesis within the germ tube. Perturbation of the spatial control of septation, by conditional mutation of the SEPTATION-ASSOCIATED1 gene of M. oryzae, prevented the fungus from causing rice blast disease. Overexpression of SEP1 did not affect septation during appressorium formation, but instead led to decoupling of nuclear division and cytokinesis in nongerminated conidial cells. When considered together, these results indicate that SEP1 is essential for determining the position and frequency of cell division sites in M. oryzae and demonstrate that differentiation of appressoria requires a cytokinetic event that is distinct from cell divisions within hyphae.     

Distinct roles for intra- and extracellular siderophores during Aspergillus fumigatus infection

Siderophore biosynthesis by the highly lethal mould Aspergillus fumigatus is essential for virulence, but non-existent in humans, presenting a rare opportunity to strategize therapeutically against this pathogen. We have previously demonstrated that A. fumigatus excretes fusarinine C and triacetylfusarinine C to capture extracellular iron, and uses ferricrocin for hyphal iron storage. Here, we delineate pathways of intra- and extracellular siderophore biosynthesis and show that A. fumigatus synthesizes a developmentally regulated fourth siderophore, termed hydroxyferricrocin, employed for conidial iron storage. By inactivation of the nonribosomal peptide synthetase SidC, we demonstrate that the intracellular siderophores are required for germ tube formation, asexual sporulation, resistance to oxidative stress, catalase A activity, and virulence. Restoration of the conidial hydroxyferricrocin content partially rescues the virulence of the apathogenic siderophore null mutant Delta sidA, demonstrating an important role for the conidial siderophore during initiation of infection. Abrogation of extracellular siderophore biosynthesis following inactivation of the acyl transferase SidF or the nonribosomal peptide synthetase SidD leads to complete dependence upon reductive iron assimilation for growth under iron-limiting conditions, partial sensitivity to oxidative stress, and significantly reduced virulence, despite normal germ tube formation. Our findings reveal distinct cellular and disease-related roles for intra- and extracellular siderophores during mammalian Aspergillus infection.     

Factors influencing pathogenicity of Fusarium tumidum on gorse (Ulex europaeus)

Factors promoting pathogenicity of Fusarium tumidum on gorse (Ulex europaeus) were determined to develop a novel strategy for delivering this potential mycoherbicide using insects as vectors of inoculum. Fusarium tumidum sprayed as a suspension of 1×10⁶ conidia mL^?1 on at least 50% of a gorse plant reduced shoot dry weight by 45% (P<0.05). A minimum of 910 viable conidia were required to cause a lesion on leaves. The leaves and flowers were more susceptible to infection than stems, spines and pods. Generally, wounding of gorse leaves and stem increased F. tumidum infection, most likely through releasing nutrients that enhanced conidial germination and hyphal growth. We showed in a separate experiment that conidial germination (93%) and germ tube length (407 µm) were greater when incubated in 0.2% gorse extract solution for 24 h than in water (62% germination, germ tube length 42 µm). Inoculation of gorse with a F. tumidum conidial suspension supplemented with 0.2% gorse extract resulted in a shoot dry weight reduction (P=0.012) equivalent to that of plants that were wounded and inoculated. It is concluded that wounding of older tissues (which mimics insect damage) is required to facilitate F. tumidum infection of mature gorse plants.     

冠盘二胞Marssonina coronaria侵染不同抗性苹果叶片的组织病理学研究

利用光学和电子显微镜,从组织细胞学水平系统研究了冠盘二胞Marssonina coronaria在苹果抗、感病品种叶片上的侵染过程及侵染后寄主细胞的超微结构特征。结果表明：冠盘二胞的侵入和定殖过程可以分为6个阶段：孢子萌发与芽管形成、附着胞形成、侵入细胞角质层、在叶肉细胞内产生吸器、菌丝在叶肉细胞间和细胞内扩展、分生孢子盘形成。随着菌丝扩展,受侵寄主细胞出现细胞壁加厚,细胞壁降解,质壁分离,叶绿体内淀粉粒、嗜饿颗粒积累,叶绿体基粒片层瓦解,线粒体空泡化等现象。在不同抗性的苹果品种上,分生孢子萌发率差别不明     

Microcycle conidiation in Penicillium urticae: an ultrastructural investigation of conidial germination and outgrowth.

A cultivation system has been developed for Penicillium urticae (NRRL 2159A) which yields 'microcycle' conidiation in submerged culture. Spherical growth of conidia was initiated by incubation at 37 degrees C in a growth-favoring medium. Transfer of these enlarged conidia to a nitrogen-poor medium at 35 degrees C resulted in synchronous germination and limited outgrowth followed by roughly synchronous conidiogenesis. An ultrastructural study of the germination stage indicated nuclear migration into the emerging germ tube whose new cell wall was an extension of the parent conidium's innermost cell wall layer. Septal formation at the neck of the germ tube followed. The septal pore was filled with particulate material and the septal membranes possessed unusual linear elements in their median hydrophobic zones. The germ tube, which possessed a smooth-surfaced plasma membrane, continued to elongate with periodic septum formation. The parent conidium and later the proximal germ tube showed progressive vacuolation and the cytoplasm became largely occupied by electron-translucent material. In older cells the septal pore was blocked by Woronin bodies. Compared with normal conidial germination this microcycle' germination is far more synchronous and the resultant germling is morphologically simpler. In ultrastructural terms, however, germination appears to be identical with that obtained at 28 degrees C.     

Filipin is a reliable in situ marker of ergosterol in the plasma membrane of germinating conidia (spores) of Penicillium discolor and stains intensively at the site of germ tube formation

Filipin, a widely used fluorescent sterol marker is also a potent antibiotic. In this study we address the reliability of filipin as a monitor of ergosterol in fungal cells. A revised staining protocol was developed to minimize any biological effect of the compound. Germinating conidia of Penicillium discolor stained with filipin, displayed a fluorescent cap at the location of germ tube appearance and formation. During germ tube emergence, the fluorescent intensity of the cap increased. This was confirmed by HPLC as an increase of the net cellular ergosterol content. Filipin staining is absent during early germination, while FM dyes, similar molecules, stain the plasma membrane after 1 h. This indicates that the conidial cell wall is no barrier for filipin. To evaluate if filipin does bind ergosterol in situ, natamycin, more specific to ergosterol, was added before filipin staining. This resulted in a marked decrease in fluorescence indicating high ergosterol levels. This was characterized further in ergDelta-mutant cells of Saccharomyces cerevisiae containing altered sterols. Here ergosterol containing cells showed a high fluorescence decrease. Taken together, these data suggest that filipin monitors an ergosterol-enriched cap in germinating conidia at the site of germ tube formation. Furthermore, the sterol-rich cap decreases and reappears after a period of actin disruption. Myriocin that affects sphingolipid synthesis results in an increase of cellular ergosterol and overall filipin fluorescence, but not at the ergosterol cap, where fluorescence is significantly lowered. In conclusion, in this work we have demonstrated an effective revised method for ergosterol staining with filipin and demonstrated its specificity in both Penicillium and Saccharomyces.