Hyphal Growth Patterns Research Articles

Effects of UVC Irradiation on Growth and Apoptosis of Scedosporium apiospermum and Lomentospora prolificans.

Scedosporium apiospermum and Lomentospora prolificans are important fungal species isolated from immunocompromised patients. Previous studies have demonstrated that these filamentous fungi exist as saprophytes in the soil and showed the highest minimum inhibitory concentration to several drugs. We aimed to examine how UVC affects the S. apiospermum and L. prolificans by investigating the role of UVC on growth, induction of apoptosis by ethidium bromide (EB)/acridine orange (AO) staining, and transcriptomic study of caspase recruitment domain family, member 9 (CARD-9) gene. Our studies showed that 15 minutes of exposure to UVC light effectively increased reduction in both organisms and caused changes in colony morphology, color, and hyphal growth pattern. After 15 min of UVC irradiation, apoptotic cells were quantitated by EB/AO staining, and the percentage of apoptosis was 96.06% in S. apiospermum and 28.30% in L. prolificans. CARD-9 gene expression results confirmed that apoptosis was induced in S. apiospermum and L. prolificans after UVC treatment and that S. apiospermum showed a higher expression of apoptosis signaling than L. prolificans. Our study explored the effects of UVC in the inactivation of S. apiospermum and L. prolificans. We hope that our data is useful to other researchers in future studies.

Construction of new GFP-tagged fusants for Trichoderma harzianum with enhanced biocontrol activity

Abstract Trichoderma is one of the most exploited biocontrol agents for the management of plant diseases. In biocontrol ecology, the critical factors are detection, and the monitoring and recovery of specific biocontrol agents either naturally present or deliberately released into the environment. Protoplast fusion is an appropriate tool for the improvement of biocontrol Trichoderma strains. Protoplast isolation from Trichoderma harzianum was achieved using 24 h culture age, 6.6 mg/ml Novazym L 1412 at 30°C which resulted the maximum protoplast yield of 5 × 108/ml. The self-fused protoplasts were regenerated and 12 fusants were selected based on their growth rate on 2% colloidal chitin medium. Next, a comparison was done for chitinase and antagonistic activity. Transcriptomic analysis based on quantitative real-time RT-PCR, demonstrated that T8-05 fusant expressed 1.5 fold of chit42 transcript as compared with the parental line. This fusant with 7.02±0.15U chitinase activity showed a higher growth inhibition rate (100%) than the parent strain, against Rhizoctonia solani. To obtain a genetically marked fusant that can be used as a biomonitor, the fusant was cotransformed with the gfp and amdS genes. The morphology and viability of selected cotransformant (FT8-7MK-05-2) was similar to the parent. Green fluorescing conidia were observed within the first 2 days of incubation in the soil, and this was followed by the formation of chlamydopores after 60 days. The colonisation of the gfp-tagged fusant was also monitored visually on R. solani sclerotia by scanning electron microscopy. Production of gfp-tagged fusant of Trichoderma spp. provides a potentially useful tool for monitoring hyphal growth patterns and the population of biocontrol agent isolates introduced into environmental systems.

Rapid Response by the Ectomycorrhizal (ECM) Community of a Lodgepole Pine Forest to Diesel Application as Indicated by Laccase Gene Fragment Diversity

A combination of field baiting and molecular methods was used to identify basidiomycete fungi that are able to withstand conditions within diesel contamination in a pine forest soil. Diesel was applied to 3, 1 meter x 1 meter blocks, which penetrated to a depth of 4 cm. After two weeks fungi that exhibited positive growth responses were identified by amplifying laccase (phenol oxidase) gene fragments using primers specific to basidiomycete laccase genes. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) gene repeat was amplified as well to provide a broad community screen, to indicate the pool of basidiomycete fungi that could respond to diesel application. Results indicated that ectomycorrhizal (ECM) fungi in the genera Russula, Piloderma and the ECM species Tricholoma saponaceum (rather than wood-rotting basidiomycetes) responded rapidly to diesel application. These results support recent hypotheses regarding the ecological role of ECM fungi in carbon cycling via exploitation of short chain phenolic carbon compounds. Further the ECM fungi that responded correspond roughly to classification of ECM exploration type, a criterion that describes ECM fungi on the basis of their hyphal growth patterns and potential for enzymatic activity. Finally, because it is apparent that these ECM fungi can survive and express laccase genes under conditions created by diesel contamination, these data provide new models for phytoremediation and site restoration strategies that utilize ECM fungi and pines.

Cochliobolus heterostrophus G-protein alpha and beta subunit double mutant reveals shared and distinct roles in development and virulence

Heterotrimeric G proteins transduce extracellular signals to control development in eukaryotes, including filamentous fungi. Targeted disruption of their α- and β-subunit genes has shown that fungal G proteins play essential roles in sexual and asexual sporulation, hyphal growth pattern and virulence. The G-protein β-subunit gene CGB1 of Cochliobolus heterostrophus, the agent of Southern leaf blight, is essential for virulence and sporulation, while one of the α-subunit genes, CGA1, has roles in several developmental pathways. We constructed a strain with insertions/deletions at both CGA1 and CGB1. The double mutant, like the single mutants, was deficient in mating and appressorium formation and its hyphae followed a straight path rather than the typical meandering growth pattern on a hard surface. The two genes shared a combined role in determining pigmentation, surface hydrophobicity, and resistance to different stresses. However, in contrast to the single mutants, the double mutants developed white-gray to completely white colonies that are hydrophilic and form wet, autolytic-appearing patches. These phenotypes resemble some of those conferred by mutations in the MAPK gene CHK1, suggesting crosstalk between MAPK and G-protein pathways. Loss of signal-transduction functions, while reducing virulence, increases resistance to some stresses. A model is proposed for the interactions between the signaling pathways.

Loss of cAMP-Dependent Protein Kinase A Affects Multiple Traits Important for Root Pathogenesis by Fusarium oxysporum

The soilborne fungal pathogen Fusarium oxysporum causes vascular wilt and root rot diseases in many plant species. We investigated the role of cyclic AMP-dependent protein kinase A of F. oxysporum (FoCPKA) in growth, morphology, and root attachment, penetration, and pathogenesis in Arabidopsis thaliana. Affinity of spore attachment to root surfaces of A. thaliana, observed microscopically and measured by atomic force microscopy, was reduced by a loss-of-function mutation in the gene encoding the catalytic subunit of FoCPKA. The resulting mutants also failed to penetrate into the vascular system of A. thaliana roots and lost virulence. Even when the mutants managed to enter the vascular system via physically wounded roots, the degree of vascular colonization was significantly lower than that of the corresponding wild-type strain O-685 and no noticeable disease symptoms were observed. The mutants also had reduced vegetative growth and spore production, and their hyphal growth patterns were distinct from those of O-685. Coinoculation of O-685 with an focpkA mutant or a strain nonpathogenic to A. thaliana significantly reduced disease severity and the degree of root colonization by O-685. Several experimental tools useful for studying mechanisms of fungal root pathogenesis are also introduced.

Quantification of fungal colonization in modified wood: Quantitative real-time PCR as a tool for studies on Trametes versicolor

Abstract Traditional wood preservatives based on biocides are effective against wood-deteriorating organisms because of their toxicity. By contrast, modified woods are non-toxic by definition. To investigate the efficiency of various wood modifications, quantitative real-time polymerase chain reaction (qPCR) was used to profile the DNA amounts of the white-rot fungus Trametes versicolor (L.) [Lloyd strain CTB 863 A] during an 8-week-long growth period in treated Pinus sylvestris (L.) sapwood. The studied wood was modified by acetylation, furfurylation, and thermal treatment. The traditional wood preservatives bis-(N-cyclohexyldiazeniumdioxy)-copper (Cu-HDO) and chromated copper arsenate (CCA) were used as references, whereas untreated P. sylvestris (L.) sapwood served as a control. The maximum levels of fungal DNA in native wood occurred at the end of the experiment. For all wood treatments, the maximum fungal DNA level was recorded after an incubation period of 2 weeks, followed by a decline until the end of the trial. For the preservative-treated woods, Cu-HDO showed the lowest level of fungal DNA throughout the experiment, indicating that exploratory hyphal growth is limited owing to the phytotoxicity of the treatment. The other treatments did not inhibit the exploratory hyphal growth phase. We conclude that qPCR studies of hyphal growth patterns within wood should provide a powerful tool for evaluating and further optimizing new wood protection systems.

Spatial distribution of Raffaelea quercivora hyphae in transverse sections of seedlings of two Japanese oak species

Summary To compare the distributions of Raffaelea quercivora hyphae within seedlings of a susceptible species, Quercus crispula, and a resistant species, Q. glauca, we examined water conductance at transverse sections. Raffaelea quercivora was inoculated into the stems of seedlings in July (summer) and October (autumn) and observed by light and fluorescence microscopy at 1 and 2 weeks after inoculation. The hyphal distribution patterns expressed as the Iδ index differed between the species at each occasion. The hyphal growth was wider in Q. crispula than in Q. glauca in July inoculation, but did not differ in October inoculation. Non-conductive sapwood in Q. crispula in transverse section was wider than that in Q. glauca at each occasion. The differences of hyphal growth and hyphal distribution patterns between species as little as 1 week after inoculation reflect differences in host susceptibility to the fungus.

Hyphal Growth Patterns and Recurrence of Fungal Keratitis after Lamellar Keratoplasty

To evaluate growth patterns of fungal pathogens in corneas and recurrence of fungal keratitis after lamellar keratoplasty (LK). Retrospective noncomparative study. One hundred seventy-four patients (174 eyes) with fungal keratitis who underwent LK at Shandong Eye Institute from January 2000 through November 2006. Medical records of each patient were retrospectively reviewed. Hyphal growth patterns in corneas were evaluated by histopathological examination. Fungal recurrence after LK was observed during the follow-up. Pathogens, hyphal growth patterns, and postoperative fungal recurrence. The pathogens were Fusarium (85.1%), Aspergillus (6.3%), Alternaria (4.6%), Penicillium (2.3%), and Candida (1.7%). Most Fusarium hyphae (91.2%) lay parallel to the corneal stromal lamellae, whereas most Aspergillus (90.9%) grew vertically. Recurrence of fungal keratitis was found in 15 patients (8.6%) after LK, and the pathogens were F. oxysporum (33.3%), F. solani (26.7%), F. moniliforme (13.3%), Aspergillus flavus (13.3%), Aspergillus fumigatus (6.7%), and Aspergillus terreus (6.7%). In cases of fungal recurrence, the majority of hyphae (80%) grew vertically. There was a higher recurrence rate in patients with vertically growing hyphae (46.2%) than in those with horizontally growing hyphae (2%) (chi(2) = 54.664, P<0.001), as well as in those with Aspergillus keratitis (36.4%) versus those with Fusarium keratitis (7.4%) (chi(2) = 10.031, P = 0.002). Reproducibility of the fungal recurrence rate was moderate in the patients with different hyphal growth patterns (kappa = 0.534) but poor in those with different fungal pathogens (kappa = -0.044). Hyphal growth patterns in corneas differ not only in the same fungal genus but also in the same species. The fungal recurrence rate after LK in patients with hyphae growing horizontally is much lower than that in those with hyphae growing vertically. Growth patterns of fungal pathogens may be an important factor for fungal recurrence after LK.

Mutational Analysis of the Glycosylphosphatidylinositol (GPI) Anchor Pathway Demonstrates that GPI-Anchored Proteins Are Required for Cell Wall Biogenesis and Normal Hyphal Growth inNeurospora crassa

Using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (GPI) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus Neurospora crassa. Mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the N. crassa GPI anchor biosynthetic pathway, have been characterized. GPI anchor mutants exhibit colonial morphologies, significantly reduced rates of growth, altered hyphal growth patterns, considerable cellular lysis, and an abnormal "cell-within-a-cell" phenotype. The mutants are deficient in the production of GPI-anchored proteins, verifying the requirement of each altered gene for the process of GPI-anchoring. The mutant cell walls are abnormally weak, contain reduced amounts of protein, and have an altered carbohydrate composition. The mutant cell walls lack a number of GPI-anchored proteins, putatively involved in cell wall biogenesis and remodeling. From these studies, we conclude that the GPI anchor pathway is critical for proper cell wall structure and function in N. crassa.

Concerted regulation of all hyphal tips generates fungal fruit body structures: experiments with computer visualizations produced by a new mathematical model of hyphal growth

Filamentous hyphal growth is inherently suited to kinetic analysis, and in many respects the fungal mycelium can be viewed as a very mechanical biological system, which lends itself to mathematical modelling. The mathematics of hyphal tip extension growth are well-established. However, even though a hyphal growth equation can be written with confidence, and we have a good understanding of the effects of tropisms on growth, it is not easy to form a mental picture of the behaviour of large populations of hyphal tips. What is required, and what we believe we have produced, is a mathematical model that is sufficiently sophisticated to produce a realistic visualization of fungal hyphal growth. This provides us with a cyberfungus that can be used for experimentation on the theoretical rules that might govern hyphal patterning, hyphal interactions, and tissue formation and organ development by actually visualizing the virtual hyphal growth patterns that result from different regulatory scenarios. From a series of model experiments the most significant observation is that complex fungal fruit body shapes can be simulated by applying the same regulatory functions to all of the growth points active in a structure at any specific time. No global control of fruit body geometry is necessary. No localized regulation is necessary. The shape of the fruit body emerges from the concerted response of the entire population of hyphal tips, in the same way, to the same signals.

Breaking dormancy is spores of the arbuscular mycorrhizal fungus Glomus intraradices: a critical cold-storage period.

To elucidate the effect of cold storage on spore dormancy in the arbuscular mycorrhizal (AM) fungus Glomus intraradices, spores were cold stratified at 4 degrees C, for either 0, 3, 7, 14, 90 or 120 days, prior to germination tests at 25 degrees C. The results showed that cold stratification longer than 14 days significantly increased spore germination. Moreover, the longer cold storage periods clearly reduced spore mortality from 90% to 50% and considerably altered the hyphal growth pattern. Long polarized hyphae were only observed after cold stratification periods longer than 14 days, involving consequences for root infectivity. The results clearly show that environmental factors, e.g., coldness, can affect the physiology of AM fungal spores.

Growth of Epichloë / Neotyphodium and p-endophytes in leaves of Lolium and Festuca grasses

Epichloe spp. (Clavicipitaceae) and their close asexual relatives, Neotyphodium spp., form systemic endophytic associations with Pooideae grasses. Interactions between Lolium and Festuca host grasses and fungal endophytes were examined in studies focusing on leaves of natural associations and also of plants into which the endophyte was introduced by seedling inoculation. Light microscopy as well as fungal isolation was used to locate the position of hyphae, while transmission electron microscopy was used to examine host/fungus interactions at the cellular level. These studies provide support for synchronised plant and endophyte growth. This characteristic pattern of growth was maintained when both Epichloe and Neotyphodium spp. were introduced by seedling inoculation into new host species. Hyphae, with few exceptions, grew rapidly as leaves grew and ceased when leaf growth ceased. This pattern of growth offers an explanation for the characteristic appearance of hyphae in leaf sheaths of host grasses; seldom branched and for all species other than N. occultans, aligned parallel to the leaf axis. Hyphal growth of a second group of endophytic fungi, referred to as p-endophytes, was not regulated in the same way, with growth continuing as leaves aged. This pattern of growth gives rise to high concentrations of branched ramifying hyphae in old leaf sheaths. Although host genotype did not effect the basic pattern of hyphal growth of Epichloe/Neotyphodium endophytes, it strongly influenced the concentration and distribution of hyphae throughout leaves. Examination by TEM revealed no evidence that penetration of hyphae into dense tissue was aided by the secretion of pectic enzymes that loosen the middle lamella connecting host cells. Instead it appeared that penetration occurred by hyphae physically pushing between cells. The absence of enzymatic loosening of the middle lamella during penetration could explain why hyphae of these endophytes apparently do not elicit host defence reactions in natural associations. However, subtle interactions between the hyphae of Epichloe/Neotyphodium and host cells in natural associations were observed.

Rapid and sensitive bioassay to study signals between root exudates and arbuscular mycorrhizal fungi

A sensitive bioassay was developed to provide a way to detect chemical signals from host plants which induce changes in hyphal growth patterns of germinated spores of arbuscular mycorrhizal (AM) fungi. The assay can be used to test host root exudates, as well as particulate fractions (root cap border cells and root mucilage), for their ability to affect AM fungal growth. Hyphal branching, induced by various host root components, can be detected as early as 4 h although results of the bioassay were usually determined after 16 to 24 h. The type of branching pattern observed was dose-dependent.

A ras homologue of Neurospora crassa regulates morphology.

In order to study the role of signal transduction pathways in the regulation of morphology in Neurospora crassa, we cloned and characterized a ras homologue, termed NC-ras2. The predicted protein product of this gene is composed of 229 amino acid residues and contains all the consensus sequences shared by the ras protein family. The gene is located in linkage group V. An NC-ras2 disruptant showed morphological characteristics very similar to those of the smco7 mutant, which also maps to linkage group V. Nucleotide sequence analysis revealed that the smco7 mutant harbored a single base deletion in the NC-ras2 gene, which is predicted to result in the truncation of the protein product. Introduction into the smco7 mutant of an NC-ras2 clone yielded stable transformants with a wild-type phenotype. The smco7 mutant exhibited very slow hyphal growth and the rate of conidial formation was approximately one two-hundredth of wild type. The smco7 mutation causes both the changes in the pattern of hyphal growth and the defects in cell wall synthesis. Both the diameter and the length of the apical compartment were shorter in the hyphae of the smco7 mutant. These results suggest that NC-ras2 is identical to smco7, and that the signal transduction pathway mediated by the NC-ras2 protein regulates the apical growth of hyphae, cell wall synthesis, and conidial formation in N. crassa.

Morphology of intercellular hypha of downy mildew fungus in Japanese radish.

Morphological studies on Peronospora parasitica Pers. ex Fr. was carried out in order to understand its behavior in tissues of Japanese radish. Fungal growth in leaf, petiole, hypocotyl and root of a susceptible cultivar, Shirokubi-miyashige and a resistant cultivar, Heian-tokinashi was investigated. In leaf, the hypha turned and twisted irregularly in the intercellular spaces of spongy parenchyma, and usually formed one haustorium in one host cell. However in root tissue where the parenchyma cells were large and much closer together, the hypha was smooth and formed one to several haustoria in one host cell. Hyphal growth pattern in petiole and hypocotyl was similar to that in root tissue. Fungal growth rate in root slices of resistant and susceptible cultivar was already significantly different on the 3rd day after inoculation, while the shape of its intercellular hypha and haustorium was almost the same. Prior to haustorium formation, a hitherto undescribed structure, leaf-like structure (LL structure), was formed from the intercellular hypha in narrow space between root parenchyma cells. It is flat in shape of ca. 6μm in thickness, covering the surface of host cell. Size of the LL structure was proportional to that of host cells. The LL structure formed various type of haustoria, ranging 0 to 25 numbers in one cell. The number of the LL structures depended on the hyphal density in the tissue.

Cyclic AMP control of hierarchical growth pattern of hyphae in Neurospora crassa

Wild-type Neurospora crassa shows a clear hyphal dominance growth pattern with a hierarchy of hyphal diameters containing wide main hyphae, narrow primary branch hyphae, and still narrower secondary branches. Mutants ( cr-1), lacking cyclic AMP, lack this size hierarchy when grown in medium without cyclic AMP. When grown in media containing sufficient cyclic AMP or 8-bromocyclic AMP to correct other morphological aberrations, these mutants still lack hierarchical growth. When grown in the presence of very high levels (3 m M) of 8-bromocyclic AMP, there is a long delayed formation of hyphae of different diameters. It is suggested that differences in cyclic AMP concentrations among the hyphae of wild type may be central to the production of the hierarchical growth pattern.

A model for the germ tube formation and mycelial growth form ofCandida albicans

A model based on morphological and ultrastructural evidence is presented which illustrates a novel and hitherto undescribed pattern of germ tube formation and hyphal growth in early and mature colonies of Candida albicans. Accordingly, most of the cytoplasm within the parent yeast cell migrates into and forward with the extending germ tubes and leaves behind an extensively vacuolated yeast cell. Growing hyphae similarly are subtended by migrating "slugs' of protoplasm and leave behind vacuolated intercalary compartments. The vacuolated cell compartments apparently must first regenerate their protoplasmic contents before producing branches or secondary germ tubes. This model is used to explain certain unusual features of the growth kinetics of the filamentous form of this organism.

Single-spore isolate variation: the effect on varietal designation in Phytophthora megasperma

Eight single zoospore isolates (SSI) were collected from each of 35 parent isolates of Phytophthora megasperma from 14 hosts. Morphological characters, hyphal growth patterns, and growth rate at several temperatures were measured and compared to assess variation of characters among the SSI of each parent. Oogonia, usually with paragynous antheridia, were formed by 94% of the SSI. The number of oogonia formed and the proportion of paragynous to amphigynous antheridia varied among SSI. Some SSI differed significantly from their parent in all characters measured. Oogonium (and oospore) diameter and sporangium shape were relatively consistent, but sporangium size varied widely. Parent isolates could not easily be classified as P. megasperma var. megasperma or P. megasperma var. sojae by oogonium diameter. Of the 35 parent isolates, 43% produced some SSI with oogonia averaging &lt; 45 μm and some with oogonia averaging &gt; 45 μm, the spore diameter used for varietal separation. Basing varieties on oogonium diameter is not justified because of such differences among SSI of a single parent and between wild-type isolates. The results supported a revised description of P. megasperma.

Chemotropism by isolated ring traps ofDactylella doedycoides

Uninflated ring traps ofD. doedycoides were mechanically detached from the mycelium and placed on agar near dead or moribund nematodes. Some rings were then induced to inflate by exposure to chlorobutanol. All produced hyphae which grew towards the prey, eventually differentiating into feeding hyphae which digested the carcass if the worm had been killed by dessication, or enveloping it, if it had been killed by heat. The pattern and dynamics of hyphal growth differed in inflated and uninflated rings.

Utilization of keratinophilic material by selected Trichophyton terrestre spaceflight phenotypes

Phenotype strains of Trichophyton terrestre Durie & Frey produced variations in hyphal growth patterns and conidial production when subjected to human hair collected from a single source. The strains were grown from wild type conidia irradiated in space with selected wavelengths of ultraviolet light.