Fungal Germ Tubes Research Articles

In vivo studies on the nuclear behavior of the arbuscular mycorrhizal fungus Gigaspora rosea grown under axenic conditions

The distribution and fate of nuclei of the arbuscular-my-corrhizal fungusGigaspora rosea during late stages of axenic cultures were studied in fixed cultures by transmitted light, conventional and confocal laser scanning microscopy, and in live cultures with two-photon fluorescence microscopy. Mature specimens not yet showing apical septation displayed oval-shaped nuclei localized in lateral positions of the hypha all along the germ-tube length. Beside these, round-shaped nuclei were found to migrate along the central germ-tube core. Some (rare) germ-tube areas, delimited by septa and containing irregularly shaped, much brighter fluorescent nuclei were also found. Specimens that had just initiated the septation process after germ-tube growth arrest displayed round or oval-shaped nuclei in several portions of the germ tubes. These hyphal areas often alternated with other septa-delimited cytoplasmic clusters which contained distorted, brightly fluorescent nuclei. Completely septated specimens mostly lacked nuclei along their germ tubes. However, highly fluorescent chromatin masses appeared within remnants of cytoplasmic material, often compressed between close septa. Our results provide a first clear picture of the in vivo distribution of nuclei along arbuscular mycorrhizal fungal germ tubes issued from resting spores, and suggest that selective areas of their coenocytic hyphae are under specific, single nuclear control. They indicate as well that random autolytic processes occur along senescingG. rosea germ tubes, probably as a consequence of the absence of a host root signal for mycorrhizal formation. Finally, the data presented here allow us to envisage the fate of nuclei released by the germinating spore after nonsymbiotic fungal growth arrest.

Mechanisms Associated with Wheat Leaf Rust Resistance Derived fromTriticum monococcum

The infection of wheat (Triticum aestivum) line KS93U9 (Karl*3//PI 266844/PI 355520) with Puccinia recondita f. sp. tritici was studied to determine histological mechanisms of resistance. The development of fungal structures was studied at two host growth stages in KS93U9, its leaf rust-resistant T. monococcum parent lines PI 266844 and PI 355520, and its susceptible recurrent parent 'Karl'. T. monococcum accession Tm2126/5, previously reported to exhibit prehaustorial resistance to P recondita f. sp. tritici, was included in the study. Using fluorescence microscopy, infection sites on leaf sections infected with pathotype UVPrt9 and stained with Uvitex 2B were examined for, respectively, the percentage of prestomatal exclusion of the fungus (germ tubes not forming appressoria and appressoria not forming over stomata), aborted penetration (nonpenetrating appressoria and aborted substomatal vesicles), early abortion (less than six haustorium mother cells per infection site), and successfully established colonies. In general, the resistant lines responded similarly for prestomatal exclusion and aborted penetration, but differences in early abortion and colony formation were observed. In seedlings, prestomatal exclusion could be attributed to the inability of fungal germ tubes to produce appressoria, whereas the formation of nonstomatal appressoria occurred more commonly in adult plants. At both growth stages, most aborted penetration attempts were accounted for by arrested substomatal vesicles rather than nonpenetrating appressoria. All infection sites displaying early abortion in KS93U9 were associated with host cell necrosis, whereas no hypersensitivity was observed in primary leaves of Tm2126/5. In adult Tm2126/5 plants, however, a large proportion of the infection sites exhibited hypersensitivity. Similarly, infection sites in PI 266844 and PI 355520 were frequently accompanied by necrotic leaf tissue. Staining of the leaves with trypan blue and a saturated solution of picric acid in methyl salicylate, and viewing with phase contrast microscopy, showed that papillae commonly occurred at infection sites in the T. monococcum lines, but not in KS93U9. Expression of T. monococcum-derived resistance in KS93U9 was not different from hypersensitive reactions typically associated with existing sources of major gene resistance to wheat leaf rust. Furthermore, components of resistance indicated, that at a histological level, mechanisms of resistance in the hexaploid KS93U9 background were altered when compared with parent lines.

Development of leaf spot caused by Ramularia carthami and reaction of safflower cultivars

SUMMARYSafflower (Carthamus tinctorius L.) is an important oilseed crop in India. A severe leaf spot disease caused by Ramularia carthami Zaprom. was observed in 1983 and investigations were carried out in order to analyse the role of environmental factors in disease development. High temperature and normal relative humidity, with dry rainless days, favoured disease development. Studies on the nature of in vivo resistance and susceptibility to R. carthami showed that growth of the fungal germ-tube was slower on resistant than on susceptible safflower cultivars, where the germ-tube extended with or without branching and penetrated infected leaves through the stomata. Of the four safflower cultivars evaluated, none were entirely free from the disease, but on the basis of lesion number and size, cultivar IGFRI-116 was rated as highly resistant.

Noninhibitory binding of human interleukin-2-activated natural killer cells to the germ tube forms of Candida albicans

During incubation in vitro with yeast or germ tube forms of Candida albicans, only 2 to 6% of freshly isolated human natural killer (NK) cells (> 85% CD16+, CD56+, CD3-; < 15% CD3+; cytolytic for the NK-susceptible target K562 but not for the NK-resistant target DAUDI), were seen to interact with the fungal cells. As seen under the electron microscope, the contact area had a limited extent and was narrow, and neither the surface nor the intracytoplasmic organization of the NK cell was altered. In contrast, more than 30% of interleukin-2-activated NK (LAK) cells (> 96% CD16+, CD56+, CD3-; 1.5% CD3+; cytolytic for both K562 and DAUDI targets) interacted closely with the fungus. This interaction was particularly extensive with the surface of the fungal germ tube that was intimately enveloped by villous protrusions from the lymphocyte surface. The fungus-interacting LAK cell also showed a remarkable redistribution of surface microvilli and polarization of cytoplasmic organelles, such as the Golgi apparatus, centrioles, and granules, toward the area of fungal contact. Together with the elevated cytolytic potential against the K562 and DAUDI targets, all the morphological data suggested the presence of a potentially active lytic machinery in the fungus-interacting LAK cell. Nonetheless, two independent assays for anticandidal activity did not show consistent killing or fungal growth inhibition by either fresh NK or LAK cells. While offering direct evidence of the strong interaction between human LAK cells and the germ tubes, precursors of tissue-invasive hyphal forms of C. albicans, our observations also suggest that this interaction may not be sufficient to kill the fungus or arrest its growth.

Inhibition of phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase increases quantitative susceptibility of barley to powdery mildew ( Erysiphe graminis D.C.)

Seedling leaves of two barley lines, Algerian/4 * (f14) Man (S) and RISO 5678-S, were excised and supplied either with water (controls), the phenylalanine ammonia lyase inhibitors AOA ( α -aminooxy acetic acid) or AOPP ( α -aminooxy-β-phenylpropionic acid), or the cinnamyl alcohol dehydrogenase inhibitor OH-PAS ([[(2-hydroxyphenyl)amino]sulphinyl]acetic acid, 1,1-di-methylethyl ester) by immersing their cut ends in solution. After 24 h leaves were inoculated with compatible Erysiphe graminis f.sp. hordei conidia and returned to the test solution for 36 h incubation. Treatment with AOA had no effect on the percentage of conidial germlings which penetrated the barleys successfully (formed haustoria), nor on the occurrence of localized autofluorescent host cell responses associated with fungal primary germ-tubes or appressoria. However, both AOPP and OH-PAS treatment led to increased quantitative susceptibility of both barley lines as the percentage of appressoria penetrating host cells to form haustoria was approximately doubled. This was associated with reduction in both the frequency and intensity of autoflu orescent responses associated with fungal germ-tubes; in this respect, the effects of AOPP were slightly more pronounced than those of OH-PAS. The results support the view that in barley, as in oats, autofluorogenic compounds accumulating at sites of germ-tube contact with host cells are phenolic compound(s) synthesized de novo following pathogen attack, that these compound(s) are products synthesized as part of the lignin biosynthetic pathway, and that the compound(s) are involved with resistance to attempted penetration from appressoria.

Investigations of Entomophthora muscae (Cohn) (Entomophthorales: Entomophthoraceae) conidial infection on housefly Musca domestica (Linn.) by scanning electron microscopy

An entomogenous fungus, Entomophthora muscae, was studied morphologically with respect to the formation of primary and secondary conidia and germ tubes. The fungus penetrated the insect cuticle by germ tubes that were produced at the base of each conidium that penetrated directly through the cuticle. Fungal germ-tube formation and penetration of host integument were observed. The tough germ-tube penetration point seemed to provide abundant energy for the penetration of the host integument. Conidia not directly on the integument formed secondary conidia but were never observed to form germ tubes. Neither appressorium nor infection cushion was observed on the germ tubes.Key words: Entomophthora muscae, entomogenous fungus, insect mycosis, insect pathology, conidia.

Effects of PAL-specific inhibition on suppression of activated defence and quantitative susceptibility of oats to Erysiphe graminis

Conidia of Erysiphe graminis DC f.sp. avenae ex. Merat were applied at various densities (low = approximately 30 mm −2, medium = approximately 100 mm −2, high = approximately 300–400 mm −2) to first leaves of spring oat ( Avena sativa) cvs Selma and Maldwyn. As previously published, high inoculum density results in activated (enhanced) host cell defence to fungal penetration, and this correlates with elevation in the frequency and intensity of host cell autofluorescent responses. In the current study, leaves were infused with either water (control treatment) or the phenylalanine ammonia lyase (PAL) inhibitors α-aminooxy-β-phenylpropionic acid (AOPP) or α-aminooxy acetic acid (AOA) at concentrations known to be inhibitory to PAL from oats. Results confirm and extend previous observations showing that these PAL inhibitors suppress localized autofluorescent host cell responses to fungal germ tube contact and increase susceptibility to penetration (haustorium formation) from appressoria. The PAL inhibitors, particularly AOPP, suppressed activated defence due to inoculum density and decreased the frequency and intensity of host cell autofluorescence responses. The data support the hypotheses that the activated defence is related to autofluorogenic host responses, and that the autofluorogens are phenolic compounds. Involvement of phenolic compound metabolism in activated defence was shown by assays of the specific activity of PAL from control cv. Selma leaves inoculated with low and high densities of conidia. Compared to uninoculated controls, the specific activity of PAL was enhanced at 6 h after inoculation (when responses to the primary germ tubes of E. graminis were evident), but there was little difference between low and high density inoculations at this time. At 18 h after inoculation (when responses to fungal appressoria were evident), the specific activity of PAL was substantially greater from high than from low inoculum density inoculations. In a coincidental observation, brown colouration (suggesting phenol oxidation) visible in many epidermal cell papillae associated with appressorial attack in control leaves, was not present in most papillae from AOPP-treated leaves.

Accurate and Rapid Measurement of Lengths of Fungal Germ Tubes by Image Analysis

Accurate and Rapid Measurement of Lengths of Fungal Germ Tubes by Image Analysis

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1-2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.

Specimen Orientation for Transmission Electron Microscopic Studies of Fungal Germ Tubes and Appressoria on Artificial Membranes and Leaf Surfaces

(1988). Specimen Orientation for Transmission Electron Microscopic Studies of Fungal Germ Tubes and Appressoria on Artificial Membranes and Leaf Surfaces. Mycologia: Vol. 80, No. 4, pp. 586-590.

Specimen Orientation for Transmission Electron Microscopic Studies of Fungal Germ Tubes and Appressoria on Artificial Membranes and Leaf Surfaces

Specimen Orientation for Transmission Electron Microscopic Studies of Fungal Germ Tubes and Appressoria on Artificial Membranes and Leaf Surfaces

Fungitoxic effects of nonprotein imino acids on growth of saprophytic fungi isolated from the leaf surface of Calliandra haematocephala.

Four saprophytic and pathogenic fungi were isolated from the leaf surface of Calliandra haematocephala, a tropical legume known to contain large amounts of rare nonprotein imino acids in its leaves and seeds. The fungi Aspergillus niger, Aspergillus sp., Curvularia sp., and Penicillium sp. were cultured in the laboratory and tested for susceptibility to leaf extracts of the host plant and to proline, pipecolic acid, cis-5-hydroxypipecolic acid, and 2,4-trans-4,5-cis-4,5-dihydroxypipecolic acid. Fungal spore germination and germ tube growth were measured. Aspergillus sp. was inhibited by plant extracts and by pipecolic acid and cis-5-hydroxypipecolic acid. Curvularia sp. growth was stimulated by plant extracts and by pipecolic acid. The other two fungi were unaffected by any of the treatments. The data indicate that imino acids may play a role in the specific resistance of Calliandra spp. to Aspergillus sp.

Biochemical characteristics of sporelings of Puccinia graminis f. sp. tritici and protoplasts from wheat isolines of potential significance in specificity

The affinity of various lectins for protoplasts from isolines of wheat differing in their reaction to Puccinia graminis Pers. f. sp. tritici, Ericks and E. Henn. race 21 and for infection structures of the fungus was studied. Protoplasts of both isolines were agglutinated by concanavalin A and soybean lectin and lysed by wheat germ agglutinin. Pokeweed mitogen and wheat germ agglutinin bound to fungal germ tubes and appressoria but not to uredospore walls, substomatal vesicles or infection hyphae.Viability of protoplasts from either isoline was not affected when they were incubated with uredospores, germlings with and without infection structures or with an extract of the susceptible line, heavily infected by the fungus. No differences between polypeptide fractions prepared from protoplasts of the isolines were detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis.

The fine structure of Entomophthora apiculata and its penetration of Trichoplusia ni.

The fine structure of conidia and immature resting spores of the entomogenous fungus Entomophthora apiculata is described. The two cell types contain comparable organelles and quantities of lipid, and possess a similar cell wall structure. The large quantity of stored lipid and numerous mitochondria in each cell type indicate a capacity for great metabolic activity. Numerous dense, microbody-like organelles were observed in conidia and resting spores, and their possible functions are discussed. Third instar larvae of the cabbage looper Trichopulsia ni were exposed to conidia of E. apiculata. Fungal germ tube formation and penetration of the host integument were observed. The germ tubes did not form appressoria and appeared to penetrate the epicuticle by enzymatic and physical means. After penetration of the epicuticle the germ tube often bifurcates, and expands as two or more digit-like projections parallel to the lamellae of the exocuticle. The hyphae gradually extend inward, cleaving the lamellae of cuticle, and eventually enter the host's body cavity.

Regulation of Lignin Formation in Reed Canarygrass in Relation to Disease Resistance

Lignin content and enzymes involved in lignification were measured in leaf discs of reed canarygrass (Phalaris arundinacea L.) inoculated with Helminthosporium avenae and floated on water or solutions of cycloheximide (25 mug/ml). Fungal germ tubes did not penetrate localized lignified swellings, which formed beneath penetration sites, in the outer epidermal wall of discs floated on water. Within 18 hours, inoculated discs on water had higher lignin content and higher activity of the enzymes phenylalanine ammonia lyase, tyrosine ammonia lyase, hydroxycinnamate-CoA ligase and peroxidase than noninoculated discs on water. When inoculated tissues were floated on cycloheximide solutions, increases in lignin content and enzyme activities associated with lignin biosynthesis were inhibited, and the tissue was susceptible to fungal penetration. Lignin biosynthesis at the site of attempted fungal penetration may play an important role in the resistant response of reed canarygrass to leaf-infecting fungi.