Autophagy During Conidiation and Conidial Germination in Filamentous Fungi

Filamentous fungi form aerial hyphae on solid medium, and some of these differentiate into conidiophores for asexual sporulation (conidiation). In the filamentous deuteromycete, Aspergillus oryzae, aerial hyphae are formed from the foot cells and some differentiate into conidiophores, which are composed of vesicles, phialides and conidia. Recently, we isolated the yeast ATG8 gene homologue Aoatg8 from A. oryzae, and visualized autophagy by the expression of an EGFP (enhanced green fluorescent protein)–AoAtg8 fusion protein and DsRed2 protein in this fungus. Furthermore, by constructing the Aoatg8 deletion and conditional mutants, we demonstrated that autophagy functions during the process of differentiation of aerial hyphae, conidiation and conidial germination in A. oryzae. Here, we discuss the contribution of autophagy towards the differentiation and germination processes in filamentous fungi.

Addendum to:

Functional Analysis of the ATG8 Homologue Aoatg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

T. Kikuma, M. Ohneda, M. Arioka and K. Kitamoto

Eukaryot Cell 2006; 5:1328-36     

Use of intercellular washing fluid to investigate the secreted proteome of the rice–<Emphasis Type="Italic">Magnaporthe</Emphasis> interaction

Early interactions between invading penetration hyphae of the pathogenic fungus Magnaporthe oryzae and rice cells occur at the apoplast, the free diffusional space outside the plasma membrane of leaves. After initial colonization, intercellular hyphae are again in intimate contact with the rice apoplast. While several studies have looked at proteomics in rice–Magnaporthe interactions, none have focused on apoplast localized proteins. We adjusted a protocol for intercellular washing fluids (IWF) to rice leaves infected with Magnaporthe oryzae for proteomic analysis. In our IWF extract, we identified several proteins associated with compatible or incompatible pathogen interactions. Three DUF26 domain proteins were identified as changing in abundance 12 h after inoculation, confirming DUF26 domain-containing proteins are among early, pathogen stress-responsive proteins induced by infection with Magnaporthe oryzae. A Magnaporthe cyclophilin, previously identified as a virulence factor was also identified in the intercellular washing fluid.     

Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase

Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions.     

Actinomycetales bacteria from a spruce stand: Characterization and effects on growth of root symbiotic and plant parasitic soil fungi in dual culture

The rhizosphere, the narrow zone of soil around living roots, is characterized by root exudates which attract soil microorganisms. Most importantly, certain soil fungi establish symbiotic interactions with fine roots which enhance nutrient availability for the plant partner (mycorrhiza). The establishment of such a symbiosis can be affected by soil bacteria. In this study we isolated Gram-positive soil bacteria from the rhizosphere of a spruce stand rich with fly agaric (Amanita muscaria) fruiting bodies. Using a coculture technique in Petri dishes, bacterial isolates were characterized by their effect on the growth of fungal hyphae. A group of bacterial strains were identified which significantly promoted growth of fly agaric hyphae. One of these strains was shown to additionally inhibit growth of pathogenic fungi such as Armillaria obscura (wide host range) and Heterobasidion annosum (causes wood decay in conifers). Taxonomic characterization of the effective bacterial isolates by their morphological appearance, by the analysis of diaminopimelic acid, cell wall sugars, and DNA sequencing (16S rDNA) identified them as actinomycetes, some of which are not yet contained in data banks.     

Microscopic observations on the interaction of the mycoparasite Verticillium biguttatum with Rhizoctonia solani and other soil-borne fungi

The mycoparasitic interactions of Verticillium biguttatum with Rhizoctonia solani and with a variety of other soil-borne fungi were investigated in dual cultures. V. biguttatum interacted with various soil fungi by appressed growth along the host hyphae and infrequent penetrations. Intracellular growth and subsequent sporulation, however, only occurred with R. solani, a few binucleate Rhizoctonia and Ceratobasidium spp., and Sclerotinia sclerotiorum. Effective mycoparasitism on sclerotia was restricted to those belonging to R. solani.Electron-microscopic observations revealed that V. biguttatum can penetrate the host cell with infection tubes. This process is probably mediated by enzymatic hydrolysis of the cell wall. Subsequently, trophic hyphae develop within the host cytoplasm, ultimately resulting in death of the host cell.     

AoAtg26, a putative sterol glucosyltransferase,is required for autophagic degradation of peroxisomes,mitochondria, and nuclei in the filamentous fungus Aspergillus oryzae

Autophagy is a conserved process in eukaryotic cells for degradation of cellular proteins and organelles. In filamentous fungi, autophagic degradation of organelles such as peroxisomes, mitochondria, and nuclei occurs in basal cells after the prolonged culture, but its mechanism is not well understood. Here, we functionally analyzed the filamentous fungus Aspergillus oryzae AoAtg26, an ortholog of the sterol glucosyltransferase PpAtg26 involved in pexophagy in the yeast Pichia pastoris. Deletion of Aoatg26 caused a severe decrease in conidiation and aerial hyphae formation, which is typically observed in the autophagy-deficient A. oryzae strains. In addition, cup-shaped AoAtg8-positive membrane structures were accumulated in the Aoatg26 deletion strain, indicating that autophagic process is impaired. Indeed, the Aoatg26 deletion strain was defective in the degradation of peroxisomes, mitochondria, and nuclei. Taken together, AoAtg26 plays an important role for autophagic degradation of organelles in A. oryzae, which may physiologically contribute to the differentiation in filamentous fungi.     

Fungal lysis by a soil bacterium fermenting cellulose

Recycling of plant biomass by a community of bacteria and fungi is fundamental to carbon flow in terrestrial ecosystems. Here we report how the plant fermenting, soil bacterium Clostridium phytofermentans enhances growth on cellulose by simultaneously lysing and consuming model fungi from soil. We investigate the mechanism of fungal lysis to show that among the dozens of different glycoside hydrolases C. phytofermentans secretes on cellulose, the most highly expressed enzymes degrade fungi rather than plant substrates. These enzymes, the GH18 Cphy1799 and Cphy1800, synergize to hydrolyse chitin, a main component of the fungal cell wall. Purified enzymes inhibit fungal growth and mutants lacking either GH18 grow normally on cellulose and other plant substrates, but have a reduced ability to hydrolyse chitinous substrates and fungal hyphae. Thus, C. phytofermentans boosts growth on cellulose by lysing fungi with its most highly expressed hydrolases, highlighting the importance of fungal interactions to the ecology of cellulolytic bacteria.     

Beyond the rhizosphere: growth and function of arbuscular mycorrhizal external hyphae in sands of varying pore sizes

Research on nutrient acquisition by symbiotic arbuscular mycorrhizal (AM) fungi has mainly focused on the root–fungus interface and less attention has been given to the growth and functioning of external hyphae in the bulk soil. The growth and function of external hyphae may be affected by unfavourable soil environments, such as compacted soils in which pores may be narrow. The effects of pore size on the growth of two AM fungi (Glomus intraradices and G. mosseae) and their ability to transport ³³P from the bulk soil to the host were investigated. Trifolium subterraneum L. plants were grown individually in `single arm cross-pots' with and without AM fungi. The side arm was separated from the main compartment by nylon mesh to prevent root penetration. It contained three zones: 5 mm of soil:sand mix (HC1); 25 mm of media treatment (HC2); and 20 mm of ³³P-labelled soil (HC3). There were four media treatments; soil and three types of quartz sand with most common continuous pore diameters of 100, 38 and 26 m. AM plants had similar growth and total P uptake in all treatments. However, plants grown with G. intraradices contained almost three times more ³³P than those grown with G. mosseae, indicating G. intraradices obtained a greater proportion of P at a distance from the host roots. Differences in P acquisition were not correlated with production of external hyphae in the four media zones and changes in sand pore size did not affect the ability of the fungi studied to acquire P at a distance from the host roots. Production of external hyphae in HC2 was influenced by fungal species and media treatment. Both fungi produced maximum amounts of external hyphae in the soil medium. Sand pore size affected growth of G. intraradices (but not G. mosseae) and hyphal diameter distributions of both fungi. The results suggest that not only are G. mosseae and G. intraradices functionally complementary in terms of spatial phosphorus acquisition, they are also capable of altering their morphology in response to the soil environment.     

Hyphal heterogeneity in Aspergillus oryzae is the result of dynamic closure of septa by Woronin bodies

Hyphae of higher fungi are compartmentalized by septa. These septa contain a central pore that allows for inter‐compartmental and inter‐hyphal cytoplasmic streaming. The cytoplasm within the mycelium is therefore considered to be a continuous system. In this study, however, we demonstrate by laser dissection that 40% of the apical septa of exploring hyphae of Aspergillus oryzae are closed. Closure of septa correlated with the presence of a peroxisome‐derived organelle, known as Woronin body, near the septal pore. The location of Woronin bodies in the hyphae was dynamic and, as a result, plugging of the septal pore was reversible. Septal plugging was abolished in a ΔAohex1 strain that cannot form Woronin bodies. Notably, hyphal heterogeneity was also affected in the ΔAohex1 strain. Wild‐type strains of A. oryzae showed heterogeneous distribution of GFP between neighbouring hyphae at the outer part of the colony when the reporter was expressed from the promoter of the glucoamylase gene glaA or the α‐glucuronidase gene aguA. In contrast, GFP fluorescence showed a normal distribution in the case of the ΔAohex1 strain. Taken together, it is concluded that Woronin bodies maintain hyphal heterogeneity in a fungal mycelium by impeding cytoplasmic continuity.     

Influence of arbuscular mycorrhizal fungi on soil structure and aggregate stability of a vertisol

The influence of arbuscular mycorrhizal (AM) fungi on aggregate stability of a semi-arid Indian vertisol was studied in a pot experiment in which Sorghum bicolor (L.) was grown as test plant for 10 weeks. Pasteurized soil inoculated with AM fungi was studied with pasteurized and unpasteurized soils as references. A part of the soil in each pot was placed in nylon mesh bags to separate effects of roots and hyphae. The sorghum plants were planted outside the mesh bags which permitted AM hyphae to enter while excluding roots. Aggregate stability of the soil was determined by wet-sieving and turbidimetric measurements. Development of the AM fungi was quantified as colonized root length and external hyphal length. Soil exposed to growth of roots and hyphae (outside mesh bags) showed aggregates with larger geometric mean diameter (GMD) in pasteurized soil inoculated with AM fungi than in pasteurized uninoculated soil. There was no significant difference in GMD of the inoculated, pasteurized soil and the unpasteurized soil. No significant effects of inoculation or plant growth were found in pasteurized soil exposed to hyphal growth only (inside the mesh bags). However, the unpasteurized soil had significantly higher GMD than the pasteurized soil, irrespective of plants and inoculum. Turbidimetric measurements of soil exposed to roots and hyphae (outside mesh bags) showed the highest aggregate stability for the inoculated pasteurized soil. These results demonstrate that AM fungi contribute to the stabilization of soil aggregates in a vertisol, and that the effect is significant after only one growing season. The effect was associated with both AM hyphae and the stimulation of root growth by AM fungi. The contribution from plant roots and AM hyphae to aggregate stability of different size fractions is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Arbuscular mycorrhizal fungi stimulate organic phosphate mobilization associated with changing bacterial community structure under field conditions

The extraradical hyphae of arbuscular mycorrhizal fungi (AMF) harbour and interact with a microbial community performing multiple functions. However, how the AMF‐microbiome interaction influences the phosphorus (P) acquisition efficiency of the mycorrhizal pathway is unclear. Here we investigated whether AMF and their hyphal microbiome play a role in promoting organic phosphorus (P) mineralizing under field conditions. We developed an AMF hyphae in‐growth core system for the field using PVC tubes sealed with membrane with different size of pores (30 or 0.45 μm) to allow or deny AMF hyphae access to a patch of organic P in root‐free soil. AMF and their hyphae associated microbiome played a role in enhancing soil organic P mineralization in situ in the field, which was shown to be a function of the change in bacteria community on the hyphae surface. The bacterial communities attached to the AMF hyphae surface were significantly different from those in the bulk soil. Importantly, AMF hyphae recruited bacteria that produced alkaline phosphatase and provided a function that was absent from the hyphae. These results demonstrate the importance of understanding trophic interactions to be able to gain insight into the functional controls of nutrient cycles in the rhizosphere.     

Interactions Between Hyphosphere-Associated Bacteria and the Fungus <Emphasis Type="Italic">Cladosporium herbarum</Emphasis> on Aquatic Leaf Litter

We investigated microbial interactions of aquatic bacteria associated with hyphae (the hyphosphere) of freshwater fungi on leaf litter. Bacteria were isolated directly from the hyphae of fungi from sedimented leaves of a small stream in the National Park “Lower Oder,” Germany. To investigate interactions, bacteria and fungi were pairwise co-cultivated on leaf-extract medium and in microcosms loaded with leaves. The performance of fungi and bacteria was monitored by measuring growth, enzyme production, and respiration of mono- and co-cultures. Growth inhibition of the fungus Cladosporium herbarum by Ralstonia pickettii was detected on leaf extract agar plates. In microcosms, the presence of Chryseobacterium sp. lowered the exocellulase, endocellulase, and cellobiase activity of the fungus. Additionally, the conversion of leaf material into microbial biomass was retarded in co-cultures. The respiration of the fungus was uninfluenced by the presence of the bacterium.     

Suppression of nematophagous fungi by enchytraeid worms: a field exclosure experiment

The feeding biology of Enchytraeus crypticus and other enchytraeids is poorly understood as is their effect on nematophagous fungi. Because enchytraeids had been associated with nematophagous fungi in the field and had suppressed these fungi in soil microcosms, we tested the hypothesis that exclusion of enchytraeids, largely E. crypticus, would improve establishment of certain nematophagous fungi in field plots. The fungi, Hirsutella rhossiliensis and Monacrosporium gephyropagum, are being studied as potential control agents of plant-parasitic nematodes and were formulated as hyphae in alginate pellets. The pellets were mixed into soil without enchytraeids and placed in cages (PVC pipe, 80 cm³ volume) with fine (20 μm) or coarse (480 μm) mesh; cages were buried 15 cm deep in field plots and then recovered after 6–52 days. When fine mesh was used, enchytraeids were excluded and the fungi increased to large numbers. When coarse mesh was used, enchytraeid numbers in cages increased rapidly and the fungi did poorly. Although mesh also affected other potential fungivores, including collembolans and large dorylaimid nematodes, we suspect that enchytraeids were more important because large numbers were consistently found in cages with coarse mesh soon after the cages were placed in soil. Organisms smaller than enchytraeids (bacteria, fungi, and protozoa) also appeared to be important because the fungi did better in heat-treated soil than in non-heat-treated soil, regardless of mesh size. The rapid increase in enchytraeid numbers in cages with hyphal pellets and coarse mesh was probably caused by movement of enchytraeids toward the pellets with hyphae: increase in enchytraeid numbers was minimal when movement into cages was blocked (or when cages contained pellets without hyphae). Overall, the data were consistent with the hypothesis that enchytraeids, or other meso- or macrofauna, contributed to suppression of nematophagous fungi in our field plots. Received: 22 April 1997 / Accepted: 16 June 1997     

Influence of two legume species on hyphal production and activity of two arbuscular mycorrhizal fungi

 Two arbuscular mycorrhizal (AM) fungi (Glomus mosseae and G. intraradices) were compared for abundance of intraradical and soil-borne hyphae in association with Astragalus sinicum, a small-seeded, and Glycine max, a large-seeded legume. A. sinicum was more responsive than G. max to mycorrhizal formation, especially at early growth stages. Biomass allocation was greater in roots than shoots for mycorrhizal A. sinicum, while the opposite was true for G. max. Hyphal development in root and soil compartments was estimated by trypan blue staining and after staining for succinate dehydrogenase (SDH) or alkaline phosphatase (ALP) activity. Total fungal abundance increased steadily in roots and soil with time to a maximum 8 weeks after planting. SDH- and ALP-active AM hyphae increased in roots during plant growth but decreased in soil at later harvests. Mycorrhizal root mass in A. sinicum and G. max increased about 14-fold and 2.5-fold, respectively, but total length of soil hyphae produced per plant differed little, so that the pattern of AM soil to root abundance of the two fungi varied considerably with the host plant. Accepted: 23 July 1997     

Autophagy vitalizes the pathogenicity of pathogenic fungi

Plant pathogenic fungi utilize a series of complex infection structures, in particular the appressorium, to gain entry to and colonize plant tissue. As a consequence of the accumulation of huge quantities of glycerol in the cell the appressorium generates immense intracellular turgor pressure allowing the penetration peg of the appressorium to penetrate the leaf cuticle. Autophagic processes are ubiquitous in eukaryotic cells and facilitate the bulk degradation of macromolecules and organelles. The study of autophagic processes has been extended from the model yeast Saccharomyces cerevisiae to pathogenic fungi such as the rice blast fungus Magnaporthe oryzae. Significantly, null mutants for the expression of M. oryzae autophagy gene homologs lose their pathogenicity for infection of host plants. Clarification of the functions and network of interactions between the proteins expressed by M. oryzae autophagy genes will lead to a better understanding of the role of autophagy in fungal pathogenesis and help in the development of new strategies for disease control.     

Extramatrical structures of hydrophobic and hydrophilic ectomycorrhizal fungi

The extramatrical mycelia of Suillus bovinus, Rhizopogon luteolus and R. vinicolor, all examples of hydrophobic (ho), mat-forming mycorrhizal fungi, were examined while associated with their hosts in the unsterilized rhizoscope, and efforts were made to produce and examine similar structures in vitro. Comparisons were made with four hydrophilic (hi) mycorrhizal fungi, Thelephora terrestris, Cenococcum geophilum, Laccaria laccata and Hebeloma crustuliniforme. The ho fungi formed linear structures (coarse, rhizomorph-like cords, with vessels in the center) and fans, both in the rhizoscope and in vitro. The same was seen in mycorrhizal mycelia in forest soils. These cords did not themselves give rise to the fans peripherally, and were not proper rhizomorphs, but were created continuously from single exploring air hyphae in the preexisting fan. Thus the ho exploring hyphae aggregated into strands, which grew in thickness only when no suitable, exploitable substrate was found. The assembly of hyphae creating ho cords was seen in the air as well as on inert hydrophilic (glass) or hydrophobic (plastic) surfaces, but never in water. It is hypothesized that the ho cell wall surface glues hyphae together while cords are formed. Water disturbed strands and mantles already formed. The ho exploring hyphae could also create ho mycelial patches (as in a mat) at the water-air interface of a number of substrates. The periphery of these patches seemed to be composed of shorter exploiting hyphae penetrating different water-soaked substrates. Exploring, aerial hyphal tips of the ho fungi were shown to excrete water droplets from openings in the ho cell wall surface, both in vitro and in the rhizoscope. In the rhizoscope, droplet excretion was apparently directly governed by photosynthesis in the shoot of the seedling. It is proposed that the drop exudation represents a kidney-like function of the extramatrical hyphae and a bridge to drier soil particles to initiate nutrient uptake by the hyphae. The ecological function of the different extramatrical structures of ho fungi are discussed. The ho cords or hyphae may translocate water only in the vessels or symplastically and not in the cell walls. The ho property may be essential among the S-selected (stress-tolerant) factors in these forest fungi. The transfer from water-repelling exploring structures into more hi exploiting structures in water contact with surrounding soil debris is, therefore, of great importance. The hi fungi did not form rhizomorph-like strands, in most cases, but an extending hyphal mycelium, representing foraging, exploring and exploiting structures at the same time. In the field, short strands may be found. On the hi fungi droplets were also produced but readily fused into a water sheath around the hypha. The hyphae thus tended to wick water via the cell wall.     

Contribution by two arbuscular mycorrhizal fungi to P uptake by cucumber (Cucumis sativus L.) from32P-labelled organic matter during mineralization in soil

An experiment was set up to investigate the role of arbuscular mycorrhiza (AM) in utilization of P from organic matter during mineralization in soil. Cucumber (Cucumis sativus L.) inoculated with one of two AM fungi or left uninoculated were grown for 30 days in cross-shaped PVC pots. One of two horizontal compartments contained 100 g soil (quartz sand: clay loam, 1:1) with 0.5 g ground clover leaves labelled with³²P. The labelled soil received microbial inoculum without AM fungi to ensure mineralization of the added organic matter. The labelling compartment was separated from a central root compartment by either 37 m or 700 m nylon mesh giving only hyphae or both roots and hyphae, respectively, access to the labelled soil. The recovery of³²P from the hyphal compartment was 5.5 and 8.6% for plants colonized withGlomus sp. andG. caledonium, respectively, but only 0.6 % for the non-mycorrhizal controls. Interfungal differences were not related to root colonization or hyphal length densities, which were lowest forG. caledonium. Both fungi depleted the labelled soil of NaHCO₃-extractable P and³²P compared to controls. A 15–25% recovery of³²P by roots was not enhanced in the presence of mycorrhizas, probably due to high root densities in the labelled soil. The experiment confirms that AM fungi differ in P uptake characteristics, and that mycorrhizal hyphae can intercept some P immobilization by other microorganisms and P-sorbing clay minerals.     

Cellular interactions between arbuscular mycorrhizal fungi and rhizosphere bacteria

Summary We have investigated whether direct physical interactions occur between arbuscular mycorrhizal (AM) fungi and plant growth promoting rhizobacteria (PGPRs), some of which are used as biocontrol agents. Attachment of rhizobia and pseudomonads to the spores and fungal mycelium ofGigaspora margarita has been assessed in vitro and visualized by a combination of electron and confocal microscopy. The results showed that both rhizobia and pseudomonads adhere to spores and hyphae of AM fungi germinated under sterile conditions, although the degree of attachment depended upon the strain.Pseudomonas fluorescens strain WCS 365 andRhizobium leguminosarum strains B556 and 3841 were the most effective colonizers. Extracellular material of bacterial origin containing cellulose produced around the attached bacteria may mediate fungal/bacterial interactions. These results suggest that antagonistic and synergistic interactions between AM fungi and rhizosphere bacteria may be mediated by soluble factors or physical contact. They also support the view that AM fungi are a vehicle for the colonization of plant roots by soil rhizobacteria.Abbreviations AM arbuscular mycorrhiza - PGPR plant growth promoting rhizobacteria - CBH cellobiohydrolase - DAPG 2,4-(diacetyl-phloroglucinol - TY triptone-yeast - LB Lauria-Bertani Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Soil salinity delays germination and limits growth of hyphae from propagules of arbuscular mycorrhizal fungi

Colonisation of plant roots by some arbuscular mycorrhizal (AM) fungi is reduced in the presence of sodium chloride (NaCl), probably due to a direct effect of NaCl on the fungi. However, there appear to be differences between the fungi in their ability to colonise plants in the presence of NaCl. This experiment tested the hypothesis that propagules of different isolates and species of AM fungi from saline and nonsaline soils would differ in their ability to germinate and grow in the presence of NaCl in the soil solution. Spores or pieces of root colonised by a range of AM fungi were incubated between filters buried in soil to which NaCl had been added at concentrations of 0, 150 or 300 mM in the soil solution. At regular intervals, filters were removed from the soil and both the percentage of propagules which had germinated and the length of proliferating hyphae were determined. Germination of spores of AM fungi studied was delayed in the presence of NaCl, but the fungi differed in the extent to which germination was inhibited. Two isolates of Scutellospora calospora reached maximum germination in 300 mM NaCl, but neither of two isolates of Acaulospora laevis germinated in the presence of NaCl. Germination of spores of the other fungi, including some isolated from saline soil, fell between these extremes. For some fungi, the specific rate of hyphal extension was reduced by NaCl. For others, the specific rate of growth was similar in the presence of NaCl to that in the control treatment, but overall production of hyphae was reduced in the NaCl treatments because germination was reduced.