Germ Tube Tip Research Articles

Development of special hyphal branches of Phyllactinia corylea on host and non-host surfaces

Hyphae of Phyllactinia corylea produce two kinds of special branches on the host surface: adhesion bodies which serve as fungal attachment and stomatopodia which enter the leaf through stomata. Conidial germination on host and non-host surfaces was examined with a scanning electron microscope to explain the stimuli responsible for development of the special branches, and the involvement of host recognition in the process. Conidia germinated within 4 h on host and non-host surfaces, but on non-host surfaces the emergence of the germ tube was not always directed towards the substratum. Adhesion bodies were formed from the tips of germ tubes at the first contact point on host and non-host surfaces. Development of stomatopodia was more specific and they were formed precisely over stomata on the host surface. Stomatopodia-like structures were occasionally formed over finely ridged leaf veins on the host surface and over some fine scratches on synthetic surfaces. The experiments showed that while conidial germination and development of adhesion bodies are in response to contact stimuli, the development of stomatopodia is a response to precise topographical signals, and the directional emergence and attached growth of germ tubes involve host recognition.

Influence of phototropic response of spore germ tubes on infection process in Colletotrichum lagenarium and Bipolaris oryzae

The germ tubes ofColletotrichum lagenarium showed negative phototropism to UV-blue (300–520 nm) and far-red (>700 nm) regions with maximum in the near ultraviolet (NUV) region, while monochromatic radiations of 575–700 nm (yellow-red region) induced positive phototropism with maximum in the red region. Green light (520–575 nm) was ineffective. Negative phototropism-inducing wavelength regions inhibited germ tube growth and positive phototropism-inducing wavelength regions promoted it significantly.Bipolaris oryzae did not show any phototropic response and light did not affect the germ tube growth. These results indicate that the lens effect, in combination with the light growth reaction and light growth inhibition, is the mechanism of the phototropism of germ tubes ofC. lagenarium. NUV radiation, which induced negative phototropism ofC. lagenarium, promoted appressorium formation, while red light, which induced positive phototropism, suppressed it significantly. In the case ofB. oryzae, light did not affect the infection structure formation. These results indicate that negative phototropism of germ tubes ofC. lagenarium favors the infection process by facilitating the contact of the tips of germ tubes with the host surface, while positive phototropism has the opposite effect.

The arrangement of F-actin and microtubules during germination of Mucor rouxii sporangiospores.

The distribution of F-actin microfilaments and microtubules was analyzed in germinating sporangiospores of Mucor rouxii by labeling with rhodamine-tagged phalloidin and by immunofluorescence microscopy. The transition from isodiametrical to apical growth was accompanied by a switch from uniform distribution of F-actin patches to a polarized accumulation of F-actin material at the germ tube tips. Immunoblotting of cell-free extracts of M. rouxii with a monoclonal anti-porcine alpha-tubulin antibody (TU-01) disclosed two discrete bands of alpha-tubulin suggesting the existence of two alpha-tubulin genes in this fungus. Immunofluorescence microscopy of germinating cells stained with the same antibody revealed an elaborate network of cytoplasmic microtubules that persisted during the entire germination process and extended into the apex of the germ tube. Although their precise roles remain undetermined, the observed arrangement of cytoskeletal elements during germination is consistent with their presumed involvement in cell wall morphogenesis: the long axial microtubules serving as long-distance conveyors of wall-building vesicles to the apical region while the concentrated F-actin patches mark the participation of microfilaments in the zone of intense vesicle exocytosis at the hyphal apex.

COMPARATIVE DEVELOPMENT OF THE RED ALGAL PARASITES BOSTRYCHIOCOLAX AUSTRALIS GEN. ET SP. NOV. AND DAWSONIOCOLAX BOSTRYCHIAE (CHOREOCOLACACEAE, RHODOPHYTA)1

ABSTRACTThe development of two red algal parasites was examined in laboratory culture. The red algal parasite Bostrychiocolax australis gen. et sp. nov., from Australia, originally misidentified as Dawsoniocolax bostrychiae (Joly et Yamaguishi‐Tomita) Joly et Yamaguishi‐Tomita, completes its life history in 6 weeks on its host Bostrychia radicans (Montagne) Montagne. Initially the spores divide to form a small lenticular cell, and then a germ tube grows from the opposite pole. Upon contact with the host cuticle, the germ tube penetrates the host cell wall. The tip of the germ tube expands, and the spore cytoplasm moves into this expanded tip. The expanded germ tube tip becomes the first endophytic cell from which a parasite cell is cut off that fuses with a host tier cell. The nuclei of this infected host cell enlarge. As parasite development continues, other host‐parasite cell fusions are formed, transferring more parasite nuclei into host cells. The erumpent colorless multicellular parasite develops externally on the host, and reproductive structures are visible within 2 weeks. Tetrasporangia are superficial and cruciately or tetra‐hedrally divided. Spermatia are formed in clusters. The carpogonial branches are four‐celled, and the carpogonium fuses directly with the auxiliary (support) cell. The mature carposporophyte has a large central fusion cell and sympodially branched gonimoblast filaments.Early stages of development differ markedly in Dawsoniocolax bostrychiae from Brazil. Upon contact with the host, the spore undergoes a nearly equal division, and a germ tube elongates from the more basal of the two spore cells, penetrates the host cell wall, and fuses with a host tier cell. Subsequent development involves enlargement of the original spore body and division to form a multicellular cushion, from which descending rhizoidal filaments form that fuse with underlying host cells. This radically different development is in marked contrast to the final reproductive morphology, which is similar to B. australis and has lead to taxonomic confusion between these two entities. The different spore germination patterns and early germ‐ling development of B. australis and D. bostrychiae warrant the formation of a new genus for the Australian parasite.

Hydrophobicity of contact surface induces appressorium formation inMagnaporthe grisea

Infection by Magnaporthe grisea, the causal agent of rice blast, requires the formation of a melanized, dome-shaped infection cell, called an appressorium. Little is known about the signals and mechanisms regulating this important developmental process. We have previously observed a correlation between hydrophobicity of the contact surface and appressorium formation. To evaluate this thigmotropic response more precisely, we measured appressorium formation on the surfaces of silicon wafers modified to create various degrees of hydrophobicity. We also examined the effects of artificial ridges created on polystyrene surfaces. Hydrophobic surfaces induced a high level of appressorium formation, whereas hydrophilic surfaces did not. Tips of germ-tubes did not respond to ridges of any particular height, but formed appressoria in a random manner. These results indicate that hydrophobicity of the substratum is a primary determinant and is sufficient to induce appressorium formation in M. grisea.

Characteristics of Extracellular Adhesions Produced DuringSaprolegnia FeraxSecondary Zoospore Encystment and Cystospore Germination

Fluorescence microscopy of lectin binding and electron microscopy of surface features were used in an investigation of adhesion characteristics of germinating secondary cystospores of Saprolegniaferax. Adhesive material at two locations bound S. ferax germlings to glass coverslips or to boiled onion epidermis. As secondary cystospores germinated, they released along the germ tube extracellular ad? hesive material to which fluorescently labeled wheat germ agglutinin bound. Similarly, this lectin also bound to portions of the adhesion pad between the secondary cystospore and its substrate. Scanning electron microscopy revealed that although material of the adhesion pad extended a short distance along the germ tube, the material at the germ tube tip was not continuous with this previously released adhesive material. Changes in the structure of the adhesion pad material indicated that it had fluidity when first released, but later its fluidity became constrained, possibly by progressive polymerization. Evidence suggests that adhesives found along the germ tube were released at the tip during elongation. The site of germ tube initiation appeared dependent on more cues than mere prealignment of the cystospore when a secondary zoospore adhered to a substrate along its ventral surface. It is concluded that S. ferax is well adapted to its aquatic environment because both secondary cystospores and colonizing germ tubes attached to substrates with adhesives.

Appressorium formation by Hemileia vastatrix

At germination, germ tubes extrude through germ pores of the urediniospore wall of Hemileia vastatrix . As many as five germ tubes can be formed from a single urediniospore, but only one elongates and extends randomly over the epidermis. Exploratory branches form along its length. Upon reaching the raised lips of the subsidiary cells of coffee leaves, or the guard cells of trench bean, a non-host, the germ tube tip, of either a side branch or a main axis of the germ tube, swells and a near-spherical appressorium is formed. A wedge-shaped structure, the appressorial wedge, develops from the base of the appressorium into the vestibule of the stoma. Upon reaching the stomatal pore, the appressorial wedge tapers to form an infection wedge. A protuberance is often found on the appressorium opposite the collapsed germ tube. Remnants of adhesive material are absent from the lower surfaces of the urediniospore, germ tube and appressorium, thus indicating that the germ tube must utilize another mechanism of attachment to the leaf surface.

CAMP Regulates Infection Structure Formation in the Plant Pathogenic Fungus Magnaporthe grisea.

Magnaporthe grisea, the causal agent of rice blast, is one of the most destructive fungal pathogens of rice throughout the world. Infection of rice by M. grisea requires the formation of an appressorium, a darkly pigmented, dome-shaped structure. The germ tube tip differentiates into an appressorium following germination of conidia on a leaf surface. When conidia germinate on growth medium or other noninductive surfaces, the emerging germ tube does not differentiate and continues to grow vegetatively. Little is known about the endogenous or exogenous signals controlling the developmental process of infection structure formation. We show here that a hydrophobic surface was sufficient for the induction of the appressorium. Furthermore, we demonstrate that the addition of cAMP, its analogs (8-bromo cAMP and N6-monobutyryl cAMP), or 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) to germinating conidia or to vegetative hyphae induced appressorium formation on noninductive surfaces. The identification of cAMP as a mediator of infection structure formation provides a clue to the regulation of this developmental process. Elucidation of the mechanism involved is not only of biological interest but may also provide the basis for new disease control strategies.

Differential effects of anticytoskeletal compounds on the localization and chemical patterns of actin in germinating conidia of Neurospora crassa.

Anti-actin drugs, cytochalasins A and B, inhibited both normal single, and benomyl-induced multiple, germ tube outgrowth from conidia of Neurospora crassa. Actin was cytochemically found to be concentrated in each of the benomyl-induced germ tube tips. No significant quantitative changes either in total actin or its isoforms were measured in the inhibitor-treated germlings. While intact microtubules are required for normal, monopolar axiation of the germ tube, they appear not to be necessary for benomyl-induced multipolar outgrowth which, in contrast, still requires intact actin microfilaments. Microfilaments and microtubules thus play complementary roles in the normal germination of conidia.

Differential effects of anticytoskeletal compounds on the localization and chemical patterns of actin in germinating conidia of Neurospora crassa

Anti-actin drugs, cytochalasins A and B, inhibited both normal single, and benomyl-induced multiple, germ tube outgrowth from conidia of Neurospora crassa. Actin was cytochemically found to be concentrated in each of the benomyl-induced germ tube tips. No significant quantitative changes either in total actin or its isoforms were measured in the inhibitor-treated germlings. While intact microtubules are required for normal, monopolar axiation of the germ tube, they appear not to be necessary for benomyl-induced multipolar outgrowth which, in contrast, still requires intact actin microfilaments. Microfilaments and microtubules thus play complementary roles in the normal germination of conidia.

Histopathology of the initial stages of the interaction between rose flowers and Botrytis cinerea

The early stages of the interaction between flowers of the cut rose cv. Sonia andBotrytis cinerea was investigated by scanning electron microscopy and light microscopy. Infection of petals by conidial germ tubes evoked a susceptible reaction. In contrast to general findings nutrient addition to the inoculum was not a prerequisite for this phenomenon. At the lower side of germ tube tips the cuticle was penetrated by infection pegs. Already at this early stage of the infection process, the infection sites were macroscopically visible as scattered white spots. After penetration, pegs enlarged to form infection hyphae, which invaded the periclinal wall of outer epidermal cells. At those sites, the petals formed outgrowths of variable appearance at their abaxial side. Thee outgrowths consisted of remanants of collapsed epidermal cells and of infection hyphae. Subsequent intra- and intercellular growth of hyphae led to a collapse of epidermal and mesophyll cells. The symptoms described generally developed within 24 h. After subsequent incubation the lesions became necrotic. Eventually, the necrosis would spread leading to the death of whole petals.

Ultrastructure of freeze-substituted appressoria produced by aeciospore germlings of the rust fungus Arthuriomyces peckianus

Aeciospores of Arthuriomyces peckianus germinated readily on moist strips of dialysis membrane and developing appressoria were observed within 3 to 6 h after spores were deposited on membranes. A single germ tube typically emerged from each binucleate spore and grew until its tip contacted the dialysis membrane. The germ tube tip was then transformed into a swollen appressorium that adhered tightly to the membrane, apparently as a result of an extracellular material that surrounded the appressorium base. Virtually all the spore cytoplasm and both nuclei moved into the germ tube and developing appressorium. Following a synchronous mitotic division of the two nuclei, a septum formed to delimit the now tetranucleate appressorium from the germ tube. As the appressorium matured, an apparently wall-less region developed in the central portion of the appressorium appressed against the dialysis membrane. In this region the fungus plasma membrane appeared to make direct contact with the underlying dialysis membrane. A funnel-like or cone-like structure referred to as the appressorial cone then developed in the wall-less region. The appressorial cone extended up into the cytoplasm of the appressorium and was lined by the fungal plasma membrane. Numerous branched elaborations of the plasma membrane were associated with the inner portion of the cone. Key words: rust fungus, infection structures, electron microscopy.

Host responses to fungal penetration in Evysiphe graminis f.sp. hordei infections in barley

Resistance mechanisms restricting penetration and establishment were investigated in an incompatible interaction using an avirulent race CC/3 (BMV 1 + 4) of Erysiphe graminis f.sp hordei on barley cv. Athos (BMR 2 + 5) and a compatible interaction using the universal susceptible cv. Golden Promise (BMR 0). In both interactions: (i) auto‐fluorescence of the host cell wall occurred adjacent to the primary germ tube tip after 4 h, and by 10 h near to the appressorial germ tube; (ii) strongly fluorescing papillae developed after 12 h, being frequently associated with failed penetrations; and (iii) only 30% of the attempted penetrations from appressorial lobes resulted in an incipient haustorium at 16 h. Hypersensitive reactions occurred in cells with an incipient haustorium in 30% of appressorial penetrations on Athos, but only in 4% of those on Golden Promise. Twenty per cent of hypersensitively reacting cells in Athos appeared dead by 14 h using trypan blue (membrane exclusion test), compared with 5% for neutral red, suggesting that plasma membrane damage is an early event in the hypersensitive response. Haustorial death was associated with host cell death, but did not precede it; appressorial death occurred 2‐4 h after that of the host cell.

Effect of Light on Uredospore Germination and Germ Tube Growth of Soybean Rust (Phakopsora pachyrhizi Syd.)

AbstractThe effect of light on uredospore germination and germ tube growth of Phakopsora pachyrhizi was studied. Frequency of uredospore germination was only partially reduced by high light intensity (> 1,9 * 104 mW * m−2). In uredospores unilaterally irradiated with polychromatic light germ tubes always emerged from the shadowed side. Already developed germ tubes showed a negative phototropic response. Both effects were inducible by low light intensities. Negative phototropism of germ tubes was a blue light effect. Light of 441 nm was more effective than that of 422 nm or 372 nm. Red light (> 600 nm) was ineffective, green light (513 nm) induced medium responses. In half‐side illumination studies longitudinal halves of germ tube tips and spores were irradiated under a microscope. The tips of the germ tubes bent into the illuminating beam. In half‐side illumination studies germ tubes always emerged from the illuminated spore halves. Under unilateral illumination liquid paraffin reversed this light “polarization” of spores and the negative phototropism of germ tubes. These results suggest that during unilateral illumination spores and germ tube tips act as a lens focussing the light on the wall farthest away from the light source., There, in uredospores emergence of germ tubes is stimulated and in germ tubes growth is inhibited. As a consequence, under unilateral illumination germ tubes emerge at the shadowed side of the spores and grow away from the light.

Microcycle conidiation induced by partial nitrogen deprivation in Neurospora crassa

Premature macroconidiation occurs at the tips of germ tubes elongated as presumptive conidiophores from macroconidia of Neurospora crassa incubated at 37 °C in nitrogen-limited shaken cultures. The use of different nitrogen sources indicated that this differentiation process could be induced by ammonium deprivation. This microcycle conidiation was also promoted by carbon deprivation and found not to be strain specific. At 25 °C, microconidia were produced in nutrient-limited shaken cultures. The results present evidence that microcycle conidiation in N. crassa can be obtained by simple manipulation of the conditions of culture.

Ultrastructure of Dormant, Converted, and Germinating Oospores of Pythium ultimum

The ultrastructural aspects of the conversion of constitutively dormant oospores of Pythium ultimum to thin-walled germinable oospores and their subsequent germination were studied by electron microscopy. During conversion, the thick inner oospore wall dissolves leaving the outer oospore wall as a loose but intact layer surrounding the spore. A thin germination wall, which eventually extends as the germ tube wall, becomes apparent around the protoplast. The protoplasm of the dormant oospore is predominantly reserve cytoplasm and includes a cortical layer of lipid bodies and a dense central ooplast. Converted oospores contain a more functional protoplasm including nuclei, mitochondria, endoplasmic reticulum, dictyosomes, and unattached ribosomes. The cortical lipids disperse within the cytoplasm while the ooplast breaks into fragments which are enveloped in membranes to form the densebody or “fingerprint” vacuoles characteristic of the oomycetous fungi. After sucrose is added, germinable oospores produce single germ tubes which rupture the outer oospore and oogonial walls. Germ tube tips are laden with dictyosome-derived vesicles.

Ultrastructure of conidium germination of Cochliobolus carbonus

Multiseptate conidia of Cochliobolus carbonus germinate in water by one or two polar germ tubes. The septa between cells within a conidium contain a central pore, which is plugged before germination but open during germination, so that all cells are interconnected. During germination, vacuoles enlarge, endoplasmic reticulum profiles increase in number, and mitochondria change from a spherical form with loosely arranged cristae to an elongated form with closely packed cristae. The conidium wall consists of an inner electron-translucent layer and an outer electron-opaque layer. The germ tube wall is continuous with the innermost layer of the conidium wall. Shortly after germination, a septum with a central pore forms in the germ tube by invagination where it emerges from the conidium. The germ tube wall is surrounded by a fibrillar sheath. The presence of apical vesicles in the germ tube tip is confirmed. The associations of Woronin bodies with septal pores and microbodies, of lipids with vacuoles, and of microbodies with lipids and vacuoles are discussed.

Ultrastructural aspects of encystation and cyst-germination in Phytophthora parasitica.

ABSTRACT Encystation in Phytophthora parasitica can be divided into 3 stages. In the first, the zoospores line their peripheries with flattened vesicles and nbrillar vacuoles in preparation for encystation. In the second stage, as the zoospores round up and shed their flagella, an initial wall is produced which takes the form of the mature cyst wall in thickness, but not in density. The participation of the flattened vesicles and fibrillar vacuoles in the formation of this initial wall is suggested by the disappearance of these organelles concomitant with wall formation. The third stage involves the maturation of the cyst wall and occurs only after dictyosomes produce vesicles which move to the cyst periphery and fuse to the plasmalemma. Germ tubes are formed in direct and indirect germination and involve the evagination of the plasmalemma and cyst wall proximal to an accumulation of dictyosome-derived vesicles. These vesicles remain at the germ-tube tip as it extends. In indirect germination the germ tube stops after having attained an average length of 6 μm and the vesicles appear to fuse at the hyphal apex, thus forming a cap. Lomasomes do not appear to be cell organelles with a specific function such as wall synthesis, but rather seem to represent aggregations of excess membranous material that have formed as a result of the discharge of vesicles at the cell periphery during wall formation. When dictyosome vesicles are inhibited from forming and moving toward the cell periphery, lomasomes are not formed.

STREPTOTINIA CAULOPHYLLI SP. NOV. PRODUCED IN CULTURE

Streptotinia caulophylli sp. nov. is described as a new species. Apothecia were produced in culture from conidia borne on streptoform conidiophores isolated from an overwintered petiole of Caulophyllum thalictroides. The connection between the perfect state and the imperfect state has been established. Microconidia were produced on the tips of spermatiophores and also on the tips of conidial germ tubes.

AN INVESTIGATION OF NUCLEAR NUMBER IN ALTERNARIA SOLANI

VARIATIONS IN isolates of Alternaria solani (Ell. and Mart.) Jones and Grout have been reported by many workers. Variations in pathogenicity of isolates of this fungus have been a serious handicap in screening for early blight resistances in existing lines of tomatoes, as well as in the development of new early blight resistant varieties. More intelligent work could be done in breeding for resistance if the pathogenic variation of the organism were understood. Heterocaryosis has been suggested as a mechanism of variation of A. solani. Neergaard (1943) reported that one isolate segregated into conidial, mycelial, and intermediate types. Brock (1950) and Henning and Alexander (unpublished) separated pathogenically different strains from isolates derived from single conidia. Stall (1954) reported that pathogenic differences existed among isolates from different germ tube tips from the same conidium. A cytological investigation of A. solani was undertaken to obtain additional information regarding the variation which occurs in this pathogen. Such an investigation aids in appraising the genetic composition of cultures derived from any particular vegetative structure, and could aid in finding techniques to minimize variations in culture. MATERIALS AND METHODS.-In this study, a sporulating isolate of A. solani was used. This isolate resembled wild type isolates in all apparent respects except that it sporulated without special cultural treatments. A modification of the cellophane-square method described by Hrushovetz (1956) was employed to stain conidia at various stages of development. Cellophane was cut itno 1 cm. X 2 cm. sections and sterilized in an approximate 3 per cent chlorine solution for 15 min. The sterilized cellophane sections were placed aseptically on Difco PDA in Petri dishes. Mycelia from mass transfers grew over the cellophane sections. When sporulation occurred on the cellophane, the sections were fixed and stained. Since nuclei could not be observed in pigmented cells in preliminary cytological work, a method was devised to bleach the pigment in cell walls. In developing a satisfactory method, water solutions of many chemicals were screened for pigment-bleach-