Morphology and lipid body and vacuole dynamics during secondary conidia formation in Colletotrichum acutatum: laser scanning confocal analysis

Colletotrichum acutatum may develop one or more secondary conidia after conidial germination and before mycelial growth. Secondary conidia formation and germination were influenced by conidia concentration. Concentrations greater than 1x105 conidia/mL were associated with germination decrease and with secondary conidia emergence. Secondary conidia can form either alone or simultaneously with germ tubes and appressoria. Confocal analysis showed numerous lipid bodies stored inside ungerminated conidia, which diminished during germ tube and appressoria formation, with or without secondary conidia formation. They were also reduced during secondary conidia formation alone. While there was a decrease inside germinated conidia, lipid bodies appeared inside secondary conidia since the initial stages. Intense vacuolization inside primary germinated conidia occurred at the same time as the decrease in lipid bodies, which were internalized and digested by vacuoles. During these events, small acidic vesicles inside secondary conidia were formed. Considering that the conidia were maintained in distilled water, with no exogenous nutrients, it is clear that ungerminated conidia contain enough stored lipids to form germ tubes, appressoria, and the additional secondary conidia replete with lipid reserves. These results suggested a very complex and well-balanced regulation that makes possible the catabolic and anabolic pathways of these lipid bodies.     

Cell biology of conidial anastomosis tubes in Neurospora crassa

Although hyphal fusion has been well documented in mature colonies of filamentous fungi, it has been little studied during colony establishment. Here we show that specialized hyphae, called conidial anastomosis tubes (CATs), are produced by all types of conidia and by conidial germ tubes of Neurospora crassa. The CAT is shown to be a cellular element that is morphologically and physiologically distinct from a germ tube and under separate genetic control. In contrast to germ tubes, CATs are thinner, shorter, lack branches, exhibit determinate growth, and home toward each other. Evidence for an extracellular CAT inducer derived from conidia was obtained because CAT formation was reduced at low conidial concentrations. A cr-1 mutant lacking cyclic AMP (cAMP) produced CATs, indicating that the inducer is not cAMP. Evidence that the transduction of the CAT inducer signal involves a putative transmembrane protein (HAM-2) and the MAK-2 and NRC-1 proteins of a mitogen-activated protein kinase signaling pathway was obtained because ham-2, mak-2, and nrc-1 mutants lacked CATs. Optical tweezers were used in a novel experimental assay to micromanipulate whole conidia and germlings to analyze chemoattraction between CATs during homing. Strains of the same and opposite mating type were shown to home toward each other. The cr-1 mutant also underwent normal homing, indicating that cAMP is not the chemoattractant. ham-2, mak-2, and nrc-1 macroconidia did not attract CATs of the wild type. Fusion between CATs of opposite mating types was partially inhibited, providing evidence of non-self-recognition prior to fusion. Microtubules and nuclei passed through fused CATs.     

Heterokaryon incompatibility is suppressed following conidial anastomosis tube fusion in a fungal plant pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.     

Successful induction and recognition of conidiation,conidial germination and chlamydospore formation in pure culture of Morchella

Morels, fungi from the genus Morchella, are popular edible mushrooms. However, little knowledge of their asexual reproduction and inaccessible pure mitospores hamper illumination of their life cycle. Herein, we successfully induced conidiation, conidial germination and chlamydospore formation in pure culture of Morchella sextelata. Conidiation proceeded via four morphologically distinct stages: development of the conidiophore stalk, stalk branching, phialide differentiation, and conidium production. Terminal and intercalary chlamydospores were formed on conidial hyphae. The development of conidiophores occurred earlier, with more conidia produced, in cross-mating cultures than in single-spore cultures. Mature conidia were spherical and 2.5–8 μm in diameter, with a vast majority (nearly 99%) 2.5–5 μm in diameter. Each conidium contained one to three nuclei (80.2% conidia contained one nucleus, 19.1% contained two nuclei, and 0.7% contained three nuclei). The conidial nucleus diameter was 1–2 μm. The nuclear mitosis in detached conidia that was observed may benefit their colony initiation. Additionally, morel conidia formed conidial anastomosis tubes. Conidia (mitospores) likely not only function as spermatia, but also as reproductive propagules in Morchella. Further research is imperative to elucidate the relationship between the conidia and chlamydospores, and their unique function in the morel life cycle.     

球孢白僵菌在棉铃虫表皮上萌发的研究

在扫描电子显微镜下,观察了球孢白僵菌三株不同毒力菌株的分生孢子在棉铃虫幼虫表皮表面的萌发,发现高、低毒力菌株的分生孢子都能在棉铃虫幼虫表皮上萌发和生长,但高毒力菌株的萌芽管伸长至分生孢子长径的1.5～2.5倍时能发生定向生长,垂直向里穿透表皮,低毒力菌株的萌芽管呈蔓延生长,极少有穿透表皮的发生,偶见极度伸长的菌丝穿透表皮。此外,还发现球孢白僵菌穿透菌丝穿透表皮的过程不是垂直向里一步完成的。     

Targeted destruction of fungal structures of Erysiphe trifoliorum on flat leaf surfaces of Marchantia polymorpha

In this study, we observed the germination behaviour of airborne conidia from powdery mildews that settle on thalloid surfaces. We inoculated thalli (flat, sheet‐like leaf tissues) and gemmae (small, flat, sheet‐like leaf tissues that propagate asexually via bud‐like structures) of the common liverwort (Marchantia polymorpha) with conidia from tomato powdery mildew (Oidium neolycopersici; KTP‐02) and red clover powdery mildew (Erysiphe trifoliorum; KRCP‐4N) and examined their germination and subsequent appressorium formation under a high‐fidelity digital microscope. Conidial bodies and germ tubes of the inoculated KRCP‐4N conidia were destroyed on both the thalli and gemmae. The destruction of these fungal structures was observed only for KRCP‐4N conidia inoculated onto M. polymorpha on both leaf surfaces. No differences in destruction of the KRCP‐4N fungal structures between thalli and gemmae were observed. At 4 h post‐inoculation, destruction of the germ tube tip was observed when it reached the gemmae leaf surface. At 6 h post‐inoculation, the conidial bodies and germ tubes were destroyed. In contrast, KTP‐02 conidia were not destroyed and formed normal, well‐lobed appressoria on the surface of M. polymorpha gemmae.     

Dectin-1 and TLRs permit macrophages to distinguish between different Aspergillus fumigatus cellular states

Aspergillus fumigatus is a common cause of invasive and allergic pulmonary disease. Resting conidia of the filamentous fungus are constantly inhaled, but cause infection only after initiating hyphal growth. In this study, we have explored whether macrophages can distinguish between resting spores and the maturing, potentially invasive form of the fungus. Although macrophages bind and ingest A. fumigatus resting conidia efficiently, there is little inflammatory response; NF-kappabeta is not activated, inflammatory cytokines are not induced, and reactive oxygen species are not produced. However, maturing A. fumigatus conidia and germ tubes stimulate NF-kappabeta, secretion of proinflammatory cytokines and production of reactive oxygen by human monocyte-derived macrophages and murine macrophages from multiple anatomical sites. These responses are in part mediated by dectin-1, which binds cell wall beta-glucan that is not present on the surface of dormant conidia, but is present after cellular swelling and loss of the hydrophobic proteinaceous cell wall. Dectin-1 binding to germ tubes augments, but is not required for, TLR2-mediated inflammatory cytokine secretion. Dectin-1 recognition of germ tubes also stimulates TNF-alpha production in the absence of both TLR2 and MyD88 signaling. These data demonstrate one mechanism by which the pulmonary inflammatory response is tailored toward metabolically active cells, thereby avoiding unnecessary tissue damage with frequent inhalation of ubiquitous spores.     

Symptoms and Prepenetration Events Associated with the Infection of Common Bean by the Anamorph and Teleomorph of Glomerella cingulata f.sp. phaseoli

Glomerella cingulata f.sp. phaseoli and Colletotrichum lindemuthianum are the teleomorph and anamorph, respectively, of the pathogen causing anthracnose in common bean. The mechanisms relating to the sexual reproduction of this plant pathogen are still unclear, as are the infection structures involved and the symptoms produced. In the present study, bean plants were inoculated with ascospores and conidia, and the events taking place within the following 120 h were investigated using light microscopy and scanning electron microscopy. The symptoms exhibited by plants inoculated with the ascospores were milder than in those inoculated with conidia. Microscopy revealed that most of ascospores produced germ tubes and appressoria at an early stage (24 h after inoculation). From 48 h onwards, the formation of hyphae and the production of germ tubes and appressoria were great. In contrast, infections originating from conidia developed more slowly, and at 24 and 48 h, many non‐germinated conidia were present, whereas only few conidia developed germ tubes and appressoria. Ascospore germination and appressorium formation were similar on both resistant and susceptible cultivars. Hence, the symptoms and the temporal sequence of events associated with the infection of bean plants by the two fungal forms differed, although the structures produced were similar. This is the fist report comparing symptoms and prepenetration events between anamorph and teleomorph of G. cingulata f.sp. phaseoli in common bean.     

Conidia of the tomato powdery mildew <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> initiate germ tubes at a predetermined site

The emergence of germ tubes from the conidia of powdery mildew fungi is the first morphological event of the infection process, preceding appressoria formation, peg penetration and primary haustoria formation. Germination patterns of the conidia are specific in powdery mildew fungi and therefore considered useful for identification. In the present study, we examined conidial germination of the tomato powdery mildew Oidium neolycopersici KTP-01 in order to clarify whether germ tube emergence site in KTP-01 conidia is determined by the first contact of the conidia to leaves (as found for the conidia of barley powdery mildew), or alternatively is predetermined and is unrelated to contact stimulus. Highly germinative conidia of KTP-01 were collected from conidial pseudochains on conidiophores in colonies on tomato leaves using two methods involving an electrostatic spore attractor and a blower. In the electrostatic spore attraction method, the conidia were attracted to the electrified insulator probe of the spore collector—this being the first contact stimulus for the conidia. In addition, the blowing method was used as a model of natural infection; pseudochain conidia were transferred to detached leaves by air (1 m/s) from a blower. Thus, landing on the leaves was the first contact for the conidia. Furthermore, conidia were also blown onto an artificial membrane (Parafilm-coated glass slides forming a hydrophobic surface) or solidified agar plates in Petri dishes (hydrophilic surface). Eventually, almost all conidia on the probe and on tomato leaves or artificial hydrophobic and hydrophilic surfaces synchronously germinated within 6 h of incubation, indicating that the first contact of the conidia with any of the aforementioned substrata was an effective germination induction signal. Germ tube emergence sites were exclusively subterminal on the conidia. Moreover, the germ tubes emerged without any relation to the sites touched first on the conidia. Thus, the present study strongly indicates that conidia of O. neolycopersici produce germ tubes at a predetermined site.     

Attachment and germination of Entomophaga maimaiga conidia on host and non-host larval cuticle

The lepidopteran-specific fungal pathogen Entomophaga maimaiga is highly virulent against Lymantria dispar (gypsy moth) larvae, and other members of the family Lymantriidae. Numerous species in the subfamily Cuculliinae (Family Noctuidae) are not susceptible to E. maimaiga due to the inability of this fungus to penetrate the larval cuticle. Conidial attachment and germination were compared among five cuculliine species and L. dispar using bioassays and scanning electron microscopy. Although conidia were showered evenly across larvae during bioassays, on L. dispar conidia were most abundant on segments, where they adhered well to the cuticle and germinated at high percentages. Conidia on cuculliine cuticles were predominantly found in large, loose aggregations in intersegmental areas. Few conidia on cuculliine cuticle germinated and scanning electron microscopy revealed a thick film of mucous enveloping conidia. We hypothesize that the conidia on cuculliines become coated by this film and were only loosely attached to the larval cuticle. No such film was seen on L. dispar larvae where individual conidia appeared well attached. On L. dispar larvae many conidia also adhered to setae. To determine if hydrophobicity affected the ability of E. maimaiga conidia to attach and germinate on a substrate, a goniometer was used to determine relative hydrophobicity of larval cuticles. L. dispar cuticle was more hydrophobic than cuculliine cuticle, suggesting that a high level of hydrophobicity could be a required characteristic for hosts. Cuticles from four cuculliine species and L. dispar were sequentially extracted using hexane, chloroform, and methanol. Conidia were showered onto glass slides coated with the different extracts and germination was quantified. Methanol extracts of cuculliine cuticle consistently decreased germination, compared to all extracts of L. dispar cuticle. For all L. dispar extracts, the majority of conidia produced germ tubes, which is a normal prerequisite for cuticular penetration. For the cuculliines, conidia exposed to hexane and chloroform extracts produced secondary conidia as did all controls, but the conidia exposed to cuculliine methanol extracts that germinated produced germ tubes. These studies demonstrated that a range of factors act in concert to prevent E. maimaiga infection of the cuculliine species investigated.     

Conidia of <Emphasis Type="Italic">Erysiphe trifoliorum</Emphasis> attempt penetration twice during a two-step germination process on non-host barley leaves and an artificial hydrophobic surface

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.     

Relationship between conidial enzymes and germination of the apple blue mold,Penicillium expansum

The relationship between conidial enzymes of Penicillium expansum and spore germination was examined. The activities of xylanase and pectinase, but not of cellulase and amylase, were detected in the conidia. The levels of xylanase and pectinase were greatly enhanced by xylan and pectin as respective carbon sources in the basal medium. No conidia germinated in the basal medium without a carbon source. The type of carbon source and the enzyme levels of the conidia did not affect the rate of germination. However, a relationship was found between the enzyme levels and the elongation of the germ tubes.     

Live-cell imaging of conidial fusion in the bean pathogen, Colletotrichum lindemuthianum

Fusion of conidia and conidial germlings by means of conidial anastomosis tubes (CATs) is a common phenomenon in filamentous fungi, including many plant pathogens. It has a number of different roles, and has been speculated to facilitate parasexual recombination and horizontal gene transfer between species. The bean pathogen Colletotrichum lindemuthianum naturally undergoes CAT fusion on the host surface and within asexual fruiting bodies in anthracnose lesions on its host. It has not been previously possible to analyze the whole process of CAT fusion in this or any other pathogen using live-cell imaging techniques. Here we report the development of a robust protocol for doing this with C. lindemuthianum in vitro. The percentage of conidial germination and CAT fusion was found to be dependent on culture age, media and the fungal strain used. Increased CAT fusion was correlated with reduced germ tube formation. We show time-lapse imaging of the whole process of CAT fusion in C. lindemuthianum for the first time and monitored nuclear migration through fused CATs using nuclei labelled with GFP. CAT fusion in this pathogen was found to exhibit significant differences to that in the model system Neurospora crassa. In contrast to N. crassa, CAT fusion in C. lindemuthianum is inhibited by nutrients (it only occurs in water) and the process takes considerably longer.     

影响飞虱虫疠霉孢子萌发类型和侵染性芽管形成的生化因子

孢子萌发类型是决定飞虱虫疠霉能否入侵的重要因子之一 ,而不同营养基质及昆虫体壁理化特性直接影响到孢子的萌发类型。对氨基酸、脂类、糖类、矿物质、维生素及桃蚜(Myzuspersicae)和褐稻虱 (Nilaparvatalugen)粗提物研究发现 ,它们均在不同程度上对芽管形成有刺激作用。其中 ,L 半胱氨酸、L 天冬酰氨、山俞酸 (C2 2 :0 )、海藻糖、果糖、麦芽糖、甘油、抗坏血酸、叶酸、维生素B1、ZnSO4、FeSO4以及两种寄主粗提物对芽管形成刺激作用显著。而这些基质如L 半胱氨酸、抗坏血酸、叶酸、维生素B1、FeSO4、ZnSO4及豆蔻酸 (C1 4 :0 )、软脂酸 (C1 6 :0 )、硬脂酸 (C1 0 :0 )、花生酸 (C2 0 :0 )、山俞酸 (C2 2 :0 )、掬焦油酸 (C2 4 :0 )、核黄素、烟酸能显著抑制二级分生孢子的产生。相反对刺激芽管形成不明显的物质如丙氨酸、酪氨酸、葡萄糖、半胞糖、蔗糖、肌醇、糖原、维生素B6、NaCl和KCl等能显著促进二级分生孢子的形成。此外 ,有些物质在高浓度下刺激芽管形成 ,而在低浓度下能促进二级分生孢子的形成 ,如L 天冬酰氨、海藻糖、…     

Adhesion of Cochliobolus heterostrophus Conidia and Germlings to Leaves and Artificial Surfaces

Braun, E. J., and Howard, R. J. 1994. Adhesion of Cochliobolus heterostrophus conidia and germlings to leaves and artificial surfaces. Experimental Mycology 18, 211-220. We have examined the nonspecific attachment of Cochliobolus heterostrophus germlings to a variety of surfaces (glass, cellophane, Mylar, polystyrene, Teflon, maize leaves) in an effort to more fully characterize this important stage of pathogenesis. Washing experiments showed that conidia began adhering to glass just prior to germ tube emergence, about 20 min after hydration and inoculation. By 50-60 min after inoculation, over 90% of the germinating conidia resisted washing and remained firmly attached. Similar results were obtained with the other surfaces. Both sodium azide and cycloheximide prevented attachment, indicating that metabolic activity was required for adhesion. Light microscopy and cryo scanning electron microscopy were used to document a temporal and spatial relationship between attachment, appearance of extracellular matrix materials, and germ tube emergence. Attachment of conidia to the substratum was correlated with the appearance of extracellular material exuded from the tips of conidia just prior to germination. The two-layered sheath of matrix materials associated with germ tubes also surrounded appressoria and appeared to aid in attachment of these structures to leaves and artificial surfaces. We conclude that extracellular matrix is produced and/or secreted within 20 min of hydration and serves in the nonspecific attachment of germlings to the substrate.     

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.     

Effects of caffeine,cyclic 3′, 5′ adenosine monophosphate and cyclic 3′, 5′ guanosine monophosphate in the development of the mycelial form of Sporothrix schenckii

The kinetics of the development of the mycelial form of Sporothrix schenckii from yeast cells and conidia in a minimal basal medium with glucose at pH 4.0 and 25 °C were established. Germ tube formation was used as the index of germination for both yeast cells and conidia. Yeast cells were first observed to develop germ tubes after 3 h of incubation, reaching 92±5%, after 12 h of incubation. Germ tubes were first detected in conidia after 9 h of incubation, and 12 h after inoculation 92±6% of the conidia had germ tubes. After 24 h of incubation, fully developed, sporulating mycelia were observed from both yeast cells and conidia. A delay in germ tube formation from yeast cells was observed when But₂cAMP(10 mM) and But₂cGMP (10 mM) were added to the medium. Also the addition of caffeine, a cyclic nucleotide phosphodiesterase inhibitor, inhibited the yeast to mycelial transition. Conidial germination into the mycelial form was also inhibited when cAMP, But₂cAMP and caffeine were added to the medium. These results suggest the possible involvement of cyclic nucleotides in the control of dimorphism in S. schenckii.     

BcNoxD,a putative ER protein,is a new component of the NADPH oxidase complex in Botrytis cinerea

NADPH oxidases (Nox) are major enzymatic producer of reactive oxygen species (ROS). In fungi these multi‐enzyme complexes are involved in sexual differentiation and pathogenicity. However, in contrast to mammalian systems, the composition and recruitment of the fungal Nox complexes are unresolved. Here we introduce a new Nox component, the membrane protein NoxD in the grey mold fungus Botrytis cinerea. It has high homology to the ER protein Pro41 from Sordaria macrospora, similar functions to the catalytic Nox subunit BcNoxA in differentiation and pathogenicity, and shows similarities to phagocytic p22phox. BcNoxA and BcNoxD interact with each other. Both proteins are involved in pathogenicity, fusion of conidial anastomosis tubes (CAT) and formation of sclerotia and conidia. These data support our earlier view based on localization studies, for an ER‐related function of the Nox complex. We present the first evidence that some functions of the BcNoxA complex are indeed linked to the ER, while others clearly require export from the ER.     

The morphology of the imperfect states of powdery mildews (Erysiphaceae)

In contrast to popular belief, a rich variety of morphological characteristics exists in the imperfect states of powdery mildews. Because it has been generally assumed that species cannot be distinguished by their appressoria, haustoria, conidiophores, conidia, fibrosin bodies, and conidial germ tubes, their morphology has received little attention and several older publications have even been forgotten. As with the perfect states, few species can be recognized by one characteristic of the imperfect state alone but many species can be identified when a combination of several characteristics is used. Important characteristics are the location of the mycelium, the production of conidia singly or in chains, the presence or absence of conspicuous fibrosin bodies, the appressoria, the size and shape of the conidia, and the position and type of their germ tubes. Many species are associated with particular families or genera of plants and therefore these are included in a key to identify 131 species of powdery mildew. This key shows how much and especially how little is known about many species. It is hoped that this review will stimulate study of the morphology of the imperfect states of numerous species. Consideration of both the perfect and the imperfect state should result in a more natural classification of several genera, for exampleUncinula andErysiphe which at present include both species which produce conidia in long chains and those which produce conidia singly. It appears that there are two lines of development of the imperfect states. One is characterized by lobed appressoria and conidiophores which produce conidia singly. The other is characterized by more or less rounded, unlobed appressoria and conidiophores which produce conidia in chains. A better knowledge of all the different imperfect states may provide more information regarding the evolution of powdery mildews.