Mode of Infection of Phyllosticta maculata on Banana as Revealed by Scanning Electron Microscopy

The initial infection stages of Phyllosticta maculata on banana were studied using scanning electron microscopy. Conidial germination on the banana leaf surface commenced within 3 h postinoculation to produce a long and slender germ tube. The hyphae developed secondary branches and mostly grew randomly across the leaf surface. Appressoria were formed at the apex of the germ tubes within 18 h postinoculation and were variable in shape. A layer of an extracellular matrix surrounded the appressoria at the pathogen–host interface. On the fruit surface, conidia germinated to produce predominantly swollen germ tubes which functioned as lateral appressoria together with some slender ones. These germ tubes were formed within 3 h postinoculation. There was no stomatal penetration apparent on the leaf; instead, direct penetration through the cuticle with and without the formation of appressoria was observed. Cuticular degradation on the leaf surface was evident with a circular, darkened area around the point of penetration by hyphae or appressoria. The significant role of pycnidia and conidia in the epidemiology of the disease was further demonstrated in naturally infected leaf samples.     

Lipid mobilization and acid phosphatase activity in lytic compartments during conidium dormancy and appressorium formation of Colletotrichum graminicola

Colletotrichum graminicola, a pathogen of sorghum and corn, was investigated prior and during germination as to certain aspects of acid phosphatase activity and lipid mobilization. Ungerminated conidia cytoplasm was filled with lipid deposits, which were mobilized during the germination process. Cytochemical ultrastructural examination showed that conidia vacuoles exhibit acid phosphatase activity, which is suggestive of lytic activity. Lipid bodies, stored in the ungerminated conidia cytoplasm, were internalized by vacuoles in a process analogous to microautophagy and were apparently digested inside them. The lipid bodies disappeared and vacuoles became enlarged in conidial cells during germination. Appressoria also showed acid phosphatase activity in multiple heterogeneous vesicles which were, in most cases, juxtaposed with lipid bodies. These results suggest that the vacuolar system plays an important role during C. graminicola germination and that the initial stages of lipid metabolization are taking place inside the vacuoles.     

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.     

Microtubule dynamics during infection-related morphogenesis of Colletotrichum lagenarium.

Using a green fluorescent protein (GFP)-tubulin fusion protein, we have investigated the dynamic rearrangement of microtubules during appressorium formation of Colletotrichum lagenarium. Two alpha-tubulin genes of C. lagenarium were isolated, and GFP-alpha-tubulin protein was expressed in this fungus. The strain expressing the fusion protein formed fluorescent filaments that were disrupted by a microtubule-depolymerizing drug, benomyl, demonstrating successful visualization of microtubules. In preincubated conidia, GFP-labeled interphase microtubules, showing random orientation, were observed. At conidial germination, microtubules oriented toward a germination site. At nuclear division, when germ tubes had formed appressoria, mitotic spindles appeared inside conidia followed by disassembly of interphase microtubules. Remarkably, time-lapse views showed that interphase microtubules contact a microtubule-associated center at the cell cortex of conidia that is different from a nuclear spindle pole body (SPB) before their disassembly. Duplicated nuclear SPBs separately moved toward conidium and appressorium accompanied by astral microtubule formation. Benomyl treatment caused movement of both daughter nuclei into 70% of appressoria and affected appressorium morphogenesis. In conidia elongating hyphae without appressoria, microtubules showed polar elongation which is distinct from their random orientation inside appressoria.     

Mycosporine-alanine: A self-inhibitor of germination from the conidial mucilage ofColletotrichum graminicola

On soft substrata conidia ofColletotrichum graminicola germinated by the formation of germ tubes, whereas on hard, hydrophobic substrata they germinated by the formation of appressoria. Regardless of the substratum, conidia did not germinate at high conidium concentrations, suggesting the presence of an endogenous self-inhibitor. Bioassays to test for the inhibition of germination demonstrated the presence of a low-molecular-weight self-inhibitor in the mucilage which surrounds conidia as they are produced in acervuli. The inhibitory material exhibited an absorbance maximum of 310 nm, indicating that it is a member of the mycosporine family of compounds. The inhibitor was purified to homogeneity and shown by mass spectrometric analysis to have a molecular mass consistent with the structure corresponding to mycosporine-alanine.     

Percutaneous infection of Hylobius pales by Metarrhizium anisopliae

The manner in which Metarrhizium anisopliae infects larval and adult Hylobius pales was investigated histologically and by scanning electron microscopy. In the presence of fungal and bacterial contaminants on beetles, none or few conidia of M. anisopliae germinated. Antibiosis is suggested, since the inhibition could be eliminated by surface sterilization. On larvae, the contaminants were scarce, and spore germination was greater. With germination, the germ tubes typically grew extensively over the procuticle or sclerites before producing appressoria of various shapes and sizes. These appressoria consistently produced a light-transmissible mucoid substance. Conidia, germ tubes, and appressoria were frequently fused into infection cushions of random arrangement and size. Sclerites of larvae and adults were not penetrated by the fungus, whereas procuticle and metawings were.     

Electron microscopic observation of cell wall structure during appressorium formation in Colletotrichum lagenarium

Summary The formation of cell walls during the appressorium formation inColletotrichum lagenarium was observed by electron microscope on the materials prepared by replicas and sectioning. The outer layer of conidia cell walls ruptured at the time of germination and the inner layer bulged out to form a germ tube. The germ tubes and primordia of appressoria had smooth surface and were consisted of one-layered cell wall. However, as the appressorium matured, the electron dense materials appeared on the outer surface of the cell wall which grew into granules. These granules are believed to form the outer layer of appressoria. The under side of the appressorium in contact with the glass surface showed a round pore.Contribution No. 191.     

Development of appressoria on conidial germ tubes of <Emphasis Type="Italic">Erysiphe</Emphasis> species

Modes of branching of appressoria on conidial germ tubes of 36 Erysiphe spp. were studied. Only unlobed appressoria, termed alobatus pattern, were seen in E. lonicerae, E. magnifica and E. symphoricarpi. Viewed from above with light or scanning electron microscopes, other species had ± irregular lobing, but from below in the plane of contact with the substrate successive dichotomous branchings at 120° were seen to produce a five-lobed appressorium within 6 h. Each division produced a temporarily dormant outward-facing lobe and an inward limb that continued growth and division to form the axis of curved, hooked, single- or double-headed symmetrical or asymmetrical structures in a helicoid cyme-like pattern. Outlines of extracellular material after removal of germinated conidia confirmed this manner of branching. After 36 h some lobes re-divided forming botryose or jigsaw patterns even extending with extra appressoria to form candelabra-like structures. Conidia developed only one true germ tube; rarely secondary unswollen tubes emerged from spare shoulders or ends. The same true germ tubes developed initially on host surfaces, where secondary tubes and/or extensions from appressorial lobes grew into colony-forming hyphae. Lobed appressoria of Neoerysphe and Phyllactinia also branched at 120°. Podosphaera xanthii exhibited a simpler branching pattern.     

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.     

Differentiation inside multicelled macroconidia of Fusarium culmorum during early germination

Multicelled conidia are formed by many fungal species, but germination of these spores is scarcely studied. Here, the germination and the effects of antimicrobials on multicompartment macroconidia of Fusarium culmorum were investigated. Germ-tube formation was mostly from apical compartments. The intracellular pH (pH(in)) of the different individual cells of the macroconidia was monitored during germination. The pH(in) varied among different compartments and during different stages of germination. The internal pH was lowest in ungerminated cells and rose during germ-tube formation and was highest in new germ tubes. Antifungal compounds affect the pH(in) and differentiation of the conidia. The pH(in) inside the macroconidial compartments was lowered very fast in the presence of nystatin (1 and 4 microg/ml). At sublethal doses (0.3 microg/ml), the apical compartments were preferentially targeted showing lower pH(in) values. The reduced germination capacity of apical compartments under these conditions was compensated by an increased germination capacity of middle compartments.     

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.     

Secondary Sporangium Formation in Phytophthora Palmivora (Butl.) Butl

Very few sporangia of Phytophthora palmivora germinated directlyand produced secondary sporangia in distilled water and in solutionsof amino acids and carbohydrates at 30 °C. Although 1.0per cent (w / v) peptone and yeast-extract stimulated a highpercentage of germination by formation of germ tubes, less than1.0 per cent of the germinated sporangia produced secondarysporangia. Secondary sporangium formation was induced by transferringgerminated primary sporangia from a nutrient medium of sufficientlyhigh concentration to either distilled water or dilute solutionsof organic and inorganic compounds immediately after emergenceof the germ tubes. The percentage of germinated sporangia formingsecondary sporangia was influenced by both the nature and concentrationof the medium into which they were transferred. The secondarysporangia were significantly smaller than the primary sporangia. Phytophthora palmivora, germination, sporangium, Theobroma cacao L., cocoa     

cAMP-pKA signaling regulates multiple steps of fungal infection cooperatively with Cmk1 MAP kinase in Colletotrichum lagenarium

In Colletotrichum lagenarium, RPK1 encoding the regulatory subunit of PKA is required for pathogenicity. From the rpkl mutant that forms small colonies, we isolated three growth-suppressor mutants. All rpk1-suppressor mutants are nonpathogenic and contain amino acid changes in the PKA catalytic subunit Cpkl. To assess the roles of cyclic AMP (cAMP) signaling in detail, we generated knockout mutants of CPK1 and the adenylate cyclase gene CAC1. The cpk1 and cac1 mutants are nonpathogenic on cucumber. Interestingly, both of the mutants germinated poorly, suggesting involvement of cAMP signaling in germination. Germination defect in the cpk1 and cac1 mutants is partially rescued by incubation of the conidia at lower concentrations. Germinating conidia of the cpk1 and cac1 mutants can form appressoria, but the appressoria formed by them are nonfunctional, like those of the rpk1 mutant. Cytological analysis indicates that the appressoria of the cpk1 mutant contain larger numbers of lipid bodies compared with the wild type, whereas lipid levels in the rpk1 mutants are lower, suggesting cAMP-mediated regulation of lipid metabolism for appressorium functionality. Furthermore, the cpk1 and cacl mutants have a defect in infectious growth in plant. In C. lagenarium, Cmkl mitogen-activated protein kinase (MAPK) regulates germination, appressorium formation, and infectious growth. These results suggest that cAMP signaling controls multiple steps of fungal infection in cooperative regulation with Cmkl MAPK in C. lagenarium.     

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.     

Effects of exogenous nutrients on conidial germination and virulence against the silverleaf whitefly for two hyphomycetes

Exogenous protein and sugar sources were tested for their impact on conidial germination of two silverleaf whitefly (Bemisia argentifolii) pathogens: Beauveria bassiana and Paecilomyces fumosoroseus. In liquid culture, sugars stimulated only 5-27% germination of B. bassiana and < or =11% germination of P. fumosoroseus, whereas, yeast extract or peptone stimulated 95-100% germination. In the absence of additional nutrients, agar alone stimulated approximately 50% germination. Storing spores for different periods of time did not alter their general response to exogenous nutrients. When spores were germinated before being applied to third instar B. argentifolii, mortality was as much as 2.45 times greater and occurred more rapidly than that for fresh spores. For ungerminated conidia, the mean time to death from infection was 5.45 (SE = 0.16) and 4.74 (SE = 0.08) days for application rates of 37 and 144 conidia x mm(-2), respectively. When conidia were germinated before application, infection times dropped to 4.58 (SE = 0.16) and 4.45 (SE = 0.10) days, respectively. A likely explanation for the greater pathogenicity and virulence of germinated over ungerminated B. bassiana conidia is that only a fraction of the spores applied to whitefly nymphs actually germinate on the cuticle. For some specialized applications, such as greenhouse production systems, it may be beneficial to germinate spores immediately prior to application.     

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.     

The Effect of Orange Extract and Other Additives on Anthracnose of French Beans Caused by Colletotrichum lindemuthianum

Orange and carrot extract, pollen diffusate, peptone, and amixture of glucose and casamino acids greatly stimulated germinationof conidia of Colletotrichum lindemuthianum which was poor inwater. Urea and glucose and casamino acids separately had littleeffect. Orange extract and the glucose/casamino acids mixture alteredthe ways in which the pathogen grew after germination. Withincreasing concentration of these additives more spores germinatedwith appressoria at the ends of increasingly thicker germ tubesinstead of more directly with no or very thin germ tubes. Large-diametergerm tubes with no appressoria were also formed. These additives almost completely suppressed formation of lesionswhen added to spore suspensions placed on hypocotyls. Otheradditives that greatly stimulated spore germination did notaffect the development of lesions. Orange extract seems to control disease in the first place bychanging the form of growth of the parasite on the hypocotylsurface and not by altering the susceptibility of the host,though this may happen later. The implications of these results for the control of diseasescaused by this and similar fungi are discussed.     

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.     

Fine structure of germinatingPenicillium megasporum conidia

Summary Penicillium megasporum conidia have spore walls consisting of several layers. There is no visible change in the outer wall layers during spore germination, but the inner layers increases in thickness on only one side of the spore, resulting in a rupture of the outer wall layers and subsequently in germ tube formation. Invaginations in the plasma membrane disappear as the germ tube forms and emerges, and the nucleus migrates into the developing germ tube. Mitochondria gather at the base of the germ tube during its formation. During germination, the amount of lipid in the spore decreases and portions migrate into the germ tube. Membrane-bound, electron dense bodies are present in resting spores. These bodies decrease in size as germination proceeds, and the cytoplasm in the developing germ tube appears much more electron dense than the cytoplasm within the spore.