Hyphal Growth Rate Research Articles

Cultural characteristics and extraction of the fungal pigment phleichrome from the phytopathogenic fungusCladosporium phlei

The cultural characteristics of the fungusCladosporium phlei were assessed in order to develop an improved method for the production of the fungal pigment, phleichrome, which is an intermediate in the production of a photodynamic therapeutic agent. The growth ofC. phlei, as measured by the hyphal growth rate and increase in biomass, varies significantly depending on the culture media utilized (V8 juice-based medium proved optimal for both growth rate and biomass increase). How-ever, even on a V8 juice plate, the growth ofC. phlei occurred slowly and in a limited fashion, in that the colony covered only 75% of the agar surface after more than 4 weeks of cultivation at 20°C. Supplementations of glucose, fructose, galactose, and sucrose increased both hyphal expansion and mass production, whereas supplementations of other carbon sources, including glycerol and sorbitol, exerted no detectable effects. The effect of inorganic nitrogen supplementation was negligible, whereas organic nitrogen evidenced significant effects, with enhanced growth with malt extract and growth inhibition with yeast extract and tryptone. Sporulation was enhanced under conditions of continuous light, and a minimum of 103 spores per mL of liquid media was found to be necessary for the optimal mass increase. A simple extraction procedure was established in order to isolate the deep red pigment which was subsequently identified as phleichrome via NMR analysis. WhenC. phlei was cultured on V8 medium containing 5% glucose and 2% malt extract, the quantity of mycelial mass was estimated as 20.6 g (dry weight) per liter of culture. The expected phleichrome yields from the mycelia and culture filtrates were estimated to be 43 and 2 mg/L, repectively.

Growth rate of Aspergillus nidulans hyphae is independent of a prominent array of microtubules

Roles for the microtubule (MT) cytoskeleton in fungal growth include mitosis and nuclear migration but otherwise are less clearly understood. Confocal microscopy was used to quantify MT abundance and growth rate in hyphae of a haploid Aspergillus nidulans strain containing green fluorescent protein (GFP)-α-tubulin. There was no correlation between growth rate and MT abundance for 112 growing hyphae in an untreated population. However, 109 nongrowing hyphae from the same group had lower average MT abundance. Results for untreated cells were compared with cells treated for 30–120 min with the MT drugs benomyl and taxol, the actin drug latrunculin B, and with solvents used for the drug treatments. Compared with their respective controls, MT abundance was significantly increased by dimethyl sulfoxide (DMSO), significantly reduced by benomyl, and moderately increased by latrunculin, but was unaffected by ethanol. In the same cells, growth rates were significantly increased by ethanol and taxol, significantly reduced by latrunculin, and unaffected by DMSO. Average hyphal growth rate in the first 60 min following 1 μg/ml benomyl treatment was statistically similar to untreated cells, despite the absence of visible MTs after 2 min of treatment. However, growth rate was significantly reduced by 2.5 μg/ml benomyl over the same time period, implying additional effects at the higher concentration. For individual hyphae in each treatment, growth rates varied over short time periods; treatment with 0.1% ethanol substantially increased this variability. Growth rates of taxol-treated hyphae decreased following fluorescence observation, suggesting a possible application to cancer chemotherapy. Overall, there was no correlation between cytoplasmic MT abundance and A. nidulans growth rate within 2 h of cytoskeletal drug or solvent treatment.

Population balance modeling of the conidial aggregation of Aspergillus niger

Numerous biotechnological production processes are based on the submerse cultivation of filamentous fungi. Process design, however, is often hampered by the complex growth pattern of these organisms. In the morphologic development of coagulating filamentous fungi, like Aspergillus niger, conidial aggregation is the first step of filamentous morphogenesis. For a proper description of this phenomenon it is necessary to characterize conidial populations. Kinetic studies performed with an in-line particle size analyzer suggested that two distinct aggregation steps have to be considered. The first step of conidial aggregation starts immediately after inoculation. Both the rate constants of formation and disintegration of aggregates have been determined by measuring the concentration of conidia at the beginning of the cultivation and the concentration of particles at steady state during the first hours of cultivation. In contrast to the first aggregation step, where the collision of conidia is presumed to be responsible for the process, the second aggregation step is thought to be initiated by germination of conidia. Growing hyphae provide additional surface for the attachment of non- germinated conidia, which leads to a strong decrease in particle concentration. The specific hyphal length growth rate and the ratio of particle concentration to the growing adhesion hyphal surface are decisive matters of the second aggregation step. Both aggregation steps can be described by population dynamics and simulated using the program package PARSIVAL (PARticle SIze eVALution) for the treatment of general particle population balances.

The Aspergillus nidulans pkcA gene is involved in polarized growth, morphogenesis and maintenance of cell wall integrity

The protein kinase C (PKC) family participates in maintaining integrity and growth of fungal cell walls. However, the precise molecular role of these proteins in the filamentous fungi remains unknown. In this work, pkcA, the gene encoding the PKC homolog in the filamentous fungus Aspergillus nidulans, was cloned and its function analyzed using a conditional alcA-PKC mutant strain. Repression of pkcA expression resulted in increased conidial swelling, decreased rates of hyphal growth, changes in the ultrastructure of the cell wall and increased sensitivity to antifungal agents. These results suggest that the protein encoded by pkcA is involved in key aspects of cell morphogenesis and cell wall integrity.

A chitinase gene, chiB, involved in the autolytic process of Aspergillus nidulans

Chitinases are thought to be involved in the morphogenesis and autolysis of filamentous fungi. We cloned a gene (chiB) encoding a class V chitinase from Aspergillus nidulans. ChiB expressed in Escherichia coli had chitin-hydrolyzing activity, indicating that chiB encoded a chitinase. Deletion of chiB affected neither germination efficiency nor hyphal growth rate, but considerably reduced the intracellular and extracellular chitinase activities. The decrease in hyphal dry weight during autolytic phase was slower in the mutant than in the wild-type strain. Western blot analysis indicated that the quantity of ChiB significantly increased when the wild-type mycelia were starved for carbon sources, a condition that induced hyphal autolysis. These results suggest that chiB plays an important role in the autolytic process in A. nidulans.

A New Subgroup of Rhizoctonia AG-D, AG-D III, Obtained from Japanese Zoysia Grass Exhibiting Symptoms of a New Disease.

Isolates of an unidentified Rhizoctonia sp. (UN isolates) were obtained from Japanese zoysia grass (Zoysia japonica Steud) that exhibited symptoms of a new sheath rot disease. UN isolates were binucleate and showed hyphal fusion with tester isolates of Rhizoctonia anastomosis group (AG)-D. Those isolates were compared with isolates of subgroups I and II of Rhizoctonia AG-D based on cultural morphology, hyphal growth rate at different temperatures, anastomosis frequency, pathogenicity, and sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA genes (rDNA-ITS region). The mycelial color of UN isolates was light yellow which differs from AG-D I but is similar to AG-D II. Sclerotia of UN isolates were dark brown in color and larger in size (1 to 3 mm in diameter) than those of AG-D subgroup I (1 mm in diameter), whereas isolates of AG-D II produced white mycelial clamps 4 to 5 mm in size. Hyphal growth rate of UN isolates was slower than that of two AG-D subgroups at several temperatures, especially 25°C. In pathogenicity tests on Japanese zoysia grass, UN isolates showed moderate disease severity and lower pathogenicity than isolates of AG-D subgroups I and II. Sequences of the rDNA-ITS region within UN isolates were almost homologous, but had lower homology with subgroups AG-D I or II. Phylogenetic trees constructed using ITS sequences showed that UN isolates formed an individual cluster that differed from the clusters of the two subgroups. We propose that UN isolates are a new subgroup of Rhizoctonia AG-D, subgroup III, and the name of the disease is "spring-rot" on Japanese zoysia grass.

HIV-1 anti-retroviral drug effect on the C. albicans hyphal growth rate by a Bio-Cell Tracer system

Declining incidence of oropharyngeal candidosis and opportunistic infections over recent years can be attributed to the use of highly active anti-retroviral therapy (HAART). Infection with C. albicans generally involves adherence and colonization of superficial tissues. During this process, budding yeasts are able to transform to hyphae and penetrate into the deep tissue. Using the biocell tracer system, C. albicans hyphal growth was dynamically observed at the cellular level. Ritonavir was effective in the inhibition of hyphal growth with growth rate of 0.8 mm/min. This study showed the in vitro effect of HIV anti-retroviral drug on the growth rate of the C. albicans hyphae.

Analysis of Morphological Relationship Between Micro‐ and Macromorphology of Mortierella Species Using a Flow‐Through Chamber Coupled with Image Analysis

Using a flow-through chamber coupled with image analysis, the morphological parameters of 11 Mortierella species were quantified, and the relationship between micro- and macromorphology was investigated. On potato-dextrose-agar plates, 5 species formed rose petal-like colonies, 3 formed large round colonies, and 3 formed donut-like colonies. By observing micromorphology in a flow-through chamber, fungi were divided into 3 groups, classified according to morphological parameters: (i) a group with a high branch formation rate (q(b): tip/microm/h) and a low tip extension rate (q(tip): microm/tip/h); (ii) a group with a low branch formation rate and a high tip extension rate; and (iii) a group intermediate between the former and the latter groups. In suspension culture, group (i) fungi formed a hyphal bundle with a pulpy pellet-like morphology and a pellet core. In contrast, group (ii) fungi showed an aggregation of hyphae without the pellet core. In a narrow-specific hyphal growth rate (mu(l)) range (0.35-0.45 h(-1)), a higher branch formation rate led to increased hyphal branching, resulting in the formation of a hyphal bundle with a pulpy pellet-like morphology and a pellet core. When the branch formation rate was lower than 2 x 10(-3) tips/microm/h, the mycelia formed less branched but longer hypha. Our study surmises that a micromorphology consisting of a high hyphal growth rate (0.4 h(-1)), low tip extension rate (20 tips/microm/h), and high branch formation rate (8 x 10(-3) tips/microm/h) forms the suitable macromorphology for arachidonic acid production.

Recovery and characterization of asexual recombinants ofMagnaporthe grisea

Eleven nitrate non-utilizing (nit) mutants were recovered from six field isolates ofMagnaporthe grisea that had different degrees of sensitivity to the blasticide kitazin P (iprobenfos, IBP). Allnit mutants were resistant to chlorate and there were no significant differences in hyphal growth rate, conidial production, sensitivity to IBP, and pathogenicity betweennit mutants and their parent isolates.nit phenotypes and IBP-resistance were used as two independent genetic markers to study asexual recombination inM. grisea. Asexual recombinants were recovered from the heterokaryotic mycelium of three inter-strain pairings, namely, DY2-3(nitl-LR) + A7-3 (nitM-S); A7-3 (nitM-S) + F4-2 (nitl-MR); and F4-2 (nitl-MR) + DY2-4 (nitA-LR), at a frequency of 7.31%, 14.00% and 8.63%, respectively. The growth rate, conidial production and pathogenicity of asexual recombinants were similar to those of parental strains. We concluded that asexual recombination resulting from hyphal fusion might contribute to variability inM. grisea.

Influence of mechanical stress and surface interaction on the aggregation of Aspergillus niger conidia

Productivity of fungal cultures is closely linked with their morphologic development. Morphogenesis of coagulating filamentous fungi, like Aspergillus niger, starts with aggregation of conidia, also denominated as spores. Several parameters are presumed to control this event, but little is known about their mode of action. Rational process optimization requires models that mirror the underlying reaction mechanisms. An approach in this regard is suggested and supported by experimental data. Aggregation kinetics was examined for the first 15 h of cultivation under different cultivation conditions. Mechanical stress was considered as well as pH-dependent surface interaction. Deliberations were based on a two-step aggregation mechanism. The first aggregation step is only affected by the pH-value, not by the fluid dynamic conditions in the bioreactor. The second aggregation step, in contrast, depends on the pH-value as well as on agitation and aeration induced power input. For the given experimental set-up, agitation had much more influence than aeration. In addition, hyphal growth rate was determined to be the driving force for the second aggregation step.

A Pattern Recognition Tool for Quantitative Analysis of In Planta Hyphal Growth of Powdery Mildew Fungi

The development of fungal pathogens can be quantified easily at the level of spore germination or penetration. However, the exact quantification of hyphal growth rates after initial, successful host invasion is much more difficult. Here, we report on the development of a new pattern recognition software (HyphArea) for automated quantitative analysis of hyphal growth rates of powdery mildew fungi on plant surfaces that usually represent highly irregular and noisy image backgrounds. By using HyphArea, we measured growth rates of colonies of the barley powdery mildew, Blumeria graminis f. sp. hordei, on susceptible and induced-resistant host plants. Hyphal growth was not influenced by the resistance state of the plants up to 48 h postinoculation. At later time points, growth rate increased on susceptible plants, whereas it remained restricted on induced-resistant plants. This difference in hyphal growth rate was accompanied by lack of secondary haustoria formation on induced-resistant plants, suggesting that induced resistance in barley against Blumeria graminis is caused mainly by reduced penetration rates of primary as well as secondary appressoria leading, finally, to fewer and less-developed fungal colonies. No evidence was found for reduced nutrient-uptake efficiency of the primary haustoria in induced-resistant leaves, which would be expected to have resulted in reduced hyphal growth rates during the first 48 h of the interaction.

A fungal metallothionein is required for pathogenicity of Magnaporthe grisea.

The causal agent of rice blast disease, the ascomycete fungus Magnaporthe grisea, infects rice (Oryza sativa) plants by means of specialized infection structures called appressoria, which are formed on the leaf surface and mechanically rupture the cuticle. We have identified a gene, Magnaporthe metallothionein 1 (MMT1), which is highly expressed throughout growth and development by M. grisea and encodes an unusual 22-amino acid metallothionein-like protein containing only six Cys residues. The MMT1-encoded protein shows a very high affinity for zinc and can act as a powerful antioxidant. Targeted gene disruption of MMT1 produced mutants that show accelerated hyphal growth rates and poor sporulation but had no effect on metal tolerance. Mmt1 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. Mmt1 appears to be distributed in the inner side of the cell wall of the fungus. These findings indicate that Mmt1-like metallothioneins may play a novel role in fungal cell wall biochemistry that is required for fungal virulence.

Oxygen flux magnitude and location along growing hyphae of Neurospora crassa

Oxygen fluxes were mapped at the growing apices and along mycelial hyphal segments of the ascomycete Neurospora crassa. High spatial resolution was obtained using micro-oxygen probes (2–3 μm tip diameters) and the self-referencing technique to maximize the sensitivity of oxygen flux measurements. As expected, oxygen influx was inhibited by cyanide, although oxygen influx (and hyphal growth) resumed with the induction of an alternate oxidase activity. Along hyphal segments, variations in oxygen influx were not correlated with location, near or far from septa, and varied over time along the same hyphal segment. Growing hyphae had a region of maximal oxygen influx greater than 10 μm behind the hyphal tip, the oxygen influx was correlated with hyphal growth rate. The region of maximal oxygen influx did not correspond with mitochondrial density, which is maximal (about 30% of hyphal volume) 5–10 μm behind the tip. Therefore, tip-localized mitochondria do not contribute to the respiratory requirements of the growing hypha. The tip-localized mitochondria may function in clearing calcium from the cytoplasm, although a decline in chlortetracycline flourescence after cyanide inhibition could also be due to ATP-depletion due to inhibition of actively respiring mitochondria. Alternatively, the growing tip may be the site of mitochondrial biogenesis.

Time series analysis demonstrates the absence of pulsatile hyphal growth.

Hyphal tip growth has been previously reported as pulsatile, defined as regularly alternating fast and slow rates of extension. The growth of pollen tubes, and hyphae of Neurospora crassa and Saprolegnia ferax were analysed using high spatial and temporal resolution. By using long (100-500 s) records of growth rate, sampled every second, it was possible to apply rigorous statistical analysis of the time series. As previously demonstrated, pollen tubes can show pulsatile growth, detectable with this system. In contrast, hyphal growth rates do not show any evidence of pulsatile growth; instead, growth rates appear to fluctuate randomly. It is concluded that pulsatile growth is not a common feature of hyphal tip growth.

Calcium gradient dependence of Neurospora crassa hyphal growth.

A tip-high cytoplasmic calcium gradient has been identified as a requirement for hyphal growth in the fungus Neurospora crassa. The Ca2+ gradient is less steep compared to wall vesicle, wall incorporation and vesicular Ca2+ gradients, but this can be explained by Ca2+ diffusion. Analysis of the relation between the rate of hyphal growth and the spatial distribution of tip-localized calcium indicates that hyphal growth rates depend upon the tip-localized calcium concentration. It is not the steepness of the calcium gradient, but tip-localized calcium and the difference in tip-localized calcium versus subapical calcium concentration which correlate closely with hyphal growth rate. A minimal concentration difference between the apex and subapical region of 30 nM is required for growth to occur. The calcium concentration dependence of growth may relate directly to biochemical functions of calcium in hyphal extension, such as vesicle fusion and enzyme activation during cellular expansion. Initiation of tip growth may rely upon random Ca2+ motions causing localized regions of elevated calcium. Continued hyphal expansion may activate a stretch-activated phospholipase C which would increase tip-localized inositol 1,4,5-trisphosphate (IP3). Hyphal expansion, induced by mild hypoosmotic treatment, does increase diacylglycerol, the other product of phospholipase C activity. This is consistent with evidence that IP3-activated Ca2+ channels generate and maintain the tip-high calcium gradient.

Functional analysis of Ras in Colletotrichum trifolii

Ras is a small monomeric GTP binding protein that transduces signals for growth and differentiation of eukaryotic organisms. Previously, a unique ras gene, designated Ct-ras, was cloned from the alfalfa fungal phytopathogen, Colletotrichum trifolii. Expression of Ct-Ras in mouse fibroblast cells (NIH3T3) demonstrated that Ct-ras is functionally similar to the mammalian ras genes since activating mutations of Ct-ras caused oncogenic phenotypes in nu/nu mice, including tumors. In C. trifolii, activated ‘oncogenic’ Ras (Val2) induced abnormal hyphal proliferation, defects in polarized growth and significantly reduced differentiation such as conidiation and appressorium formation in a nutrient dependent manner. Gene disruption of ct-ras was lethal. To further evaluate the function of Ct-Ras in C. trifolii, three different approaches were used: overexpression of cytosolic Ras by CAAX box deletion; expression of dominant negative Ct-Ras T22N; and antisense ct-ras expression. Results showed that suppression of Ct-Ras activity significantly decreases fungal germination frequencies and hyphal growth rates. Taken together, these data suggest involvement of Ct-Ras in regulation of fungal cell growth and differentiation.

Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae).

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.

Relationships Between Blueberry Flower Age, Pollination, and Conidial Infection byMonilinia vaccinii-corymbosi

ABSTRACT Monilinia vaccinii-corymbosi infects open blueberry flowers via the gynoecial pathway, leading to mummification of the developing fruit. To determine the effect of flower age on infection, stigmata were inoculated with conidia of M. vaccinii-corymbosi between 0 and 5 days after anthesis, fungal growth rates through the stylar canal were measured in detached flowers in the laboratory, and fruit disease incidence was determined in plants grown in the greenhouse. Hyphal growth rates were greatest in flowers inoculated on the day of anthesis, declined linearly with increasing flower age at inoculation (r = 0.921; P < 0.0001; n = 12), and were unaffected by the presence or absence of pollen applied at the time of inoculation. In greenhouse-grown plants, the percentage of infected fruit decreased exponentially with increasing flower age at inoculation (R = 0.878; P = 0.0057; n = 10), with disease incidence ranging from 76.4% for flowers inoculated on the day of anthesis to 15.5% for those inoculated 4 days later. Fruit disease incidence in the greenhouse was linearly correlated with hyphal growth rates in detached flowers (r = 0.985; P < 0.0001; n = 9), justifying the use of detached flowers when investigating gynoecial infection by M. vaccinii-corymbosi. In separate experiments, the effects of timing and sequence of pollination and inoculation on hyphal growth rates through the stylar canal and on disease incidence were investigated. Application of pollen to detached flowers 1 or 2 days before inoculation reduced hyphal growth rates by between 14.0 and 42.9% compared with flowers that received pollen and conidia simultaneously. Similarly, reductions in fruit disease incidence by between 9.5 and 18.3% were observed on greenhouse-grown plants for pollination-to-inoculation intervals ranging from 1 to 4 days. These results document that newly opened flowers are most susceptible to infection by M. vaccinii-corymbosi and that fruit disease incidence is reduced if pollination occurs at least 1 day before inoculation. Strategies that lead to early pollination of newly opened flowers may be useful for managing mummy berry disease in the field.

The role of water in solid-state fermentation

Solute diffusion as well as cell absorption occurs in aqueous medium. Thermodynamics and kinetic parameters relative to water motion in solid media are successively examined with regard to their consequence on the solid-state fermentation (SSF). Specific attention is focused on fungal physiology: • at microscopic level through the influence of cell water flux on the maintenance of a constant cell turgor pressure, through the constant branching orientation and hyphal growth rate; • at macroscopic level through the influence of the hydration on the maintenance of a radial growth rate, on the evolution of germination, sporulation and metabolic activity of a fungal colony. Interactions between water status, oxygen supply and heat removing from SSF are finally pointed out.

An IQGAP-related protein, encoded by AgCYK1, is required for septation in the filamentous fungus Ashbya gossypii

In filamentous ascomycetes hyphae are compartmentalized by septation in which the cytoplasm of the compartments are interconnected via septal pores. Thus, septation in filamentous fungi is different from cytokinesis in yeast like fungi. We have identified an Ashbya gossypii orthologue of the Saccharomyces cerevisiae CYK1 gene which belongs to the IQGAP-protein family. In contrast to S. cerevisiae disruption of AgCYK1 yields viable mutant strains that exhibit wildtype-like polarized hyphal growth rates. In the Agcyk1 mutant cortical actin patches localize to growing hyphal tips like wildtype, however, mutant hyphae are totally devoid of actin rings at presumptive septal sites. Septation in wildtype results in the formation of chitin rings. Agcyk1 mutant hyphae are aseptate and do not accumulate chitin in their cell walls. Agcyk1 mutant strains are completely asporogenous indicating that septation is essential for the formation of sporangia in A. gossypii. AgCyk1p-GFP localizes to sites of future septation as a ring prior to chitin depositioning. Furthermore, decrease in Cyk1p-ring diameter was found to be a prerequisite for the accumulation of chitin and septum formation.