Cryptocotyle lata (Trematoda: Heterophyidae) Adult from a Korean Raccoon Dog,Nyctereutes procyonoides koreensis

Total 513 heterophyid flukes were collected from a carcass of wild Korean raccoon dog, Nyctereutes procyonoides koreensis, in Korea. With morphological and molecular characteristics, the flukes were identified to Cryptocotyle lata. The adult C. lata were minute, transparent, pentagonal, 522 μm long by 425 μm wide. Ceca extended into post-testicular region. Ventrogenital sac elliptical, 79 μm by 87 μm with genital pore and ventral sucker. Two testes semielliptical and slightly lobed, located in the posterior region, right testis 173 μm by 155 μm, left testis 130 μm by 134 μm. In a phylogenetic tree, the fluke specimen of this study was grouped with C. lata divergent from Cryptocotyle lingua. We report here N. procyonoides koreensis first as a natural definitive host of C. lata.     

Membrane Filtration of the Reiter Treponeme

A membrane filtration technique with commercially available membrane filters (Millipore Corp.) was effective for the removal of Reiter treponemes from liquids such as fluorescent-antibody conjugates, to which the organisms are added for adsorption. Reiter treponemes from an 8-day culture were not microscopically detectable in filtrates through membranes with a pore diameter of 0.45 μm, but treponemes were demonstrated in the filtrate by cultural methods. No organisms of the 8-day culture passed through a membrane filter having a pore size of 0.22 μm, as determined by microscopy and culture. Culture data indicated that a filter with a pore size of 0.1 μm was necessary to prevent passage of treponemes from 4-day cultures. It is recommended that a membrane filter with a pore size of 0.22 μm or smaller be used for the removal of Reiter treponemes from suspensions and that the age of the culture be considered in choosing filter pore size.     

Deficient SUMO Attachment to Flp Recombinase Leads to Homologous Recombination-dependent Hyperamplification of the Yeast 2 μm Circle Plasmid

Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 μm circle plasmid (2 μm). Here we show that accumulation of 2 μm in the SUMO pathway mutants siz1Δ siz2Δ, slx5Δ, and slx8Δ is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 μm. Characterization of this species from siz1Δ siz2Δ showed that it contains tandem copies of the 2 μm sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 μm–encoded Flp recombinase and the cellular homologous recombination repair (HRR) pathway. Importantly, reduced SUMO attachment to Flp is sufficient to induce formation of this species. Our data suggest a model in which Flp that cannot be sumoylated causes DNA damage, whose repair via HRR produces an intermediate that generates tandem copies of the 2 μm sequence. This intermediate may be a rolling circle formed via break-induced replication (BIR), because mutants defective in BIR contain reduced levels of the HMW form. This work also illustrates the importance of using cir° strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 μm amplification.     

Size of Suspended Bacterial Cells and Association of Heterotrophic Activity with Size Fractions of Particles in Estuarine and Coastal Waters

The size of bacteria and the size distribution of heterotrophic activity were examined in estuarine, neritic, and coastal waters. The data indicated the small size of suspended marine bacteria and the predominance of free-living cells in numerical abundance and in the incorporation of dissolved amino acids. The average per-cell volume of suspended marine bacteria in all environments was less than 0.1 μm³. Cell volume ranged from 0.072 to 0.096 μm³ at salinities of 0 to 34.3‰ in the Newport River estuary, N.C., and from 0.078 to 0.096 μm³ in diverse areas of the Gulf of Mexico. Thus, the free-living bacteria were too small to be susceptible to predation by copepods. In the Newport River estuary, ca. 93 to 99% of the total number of cells and 75 to 97% of incorporated tritium (from ³H-labeled mixed amino acids) retained by a 0.2-μm-pore-size filter passed through a 3.0-μm-pore-size filter. Although the amino acid turnover rate per cell was higher for the bacteria in the >3.0-μm size fraction than in the <3.0-μm size fraction, the small number of bacteria associated with the >3.0-μm size particles resulted in the low relative contribution of attached bacteria to total heterotrophic activity in the estuary. For coastal and neritic samples, collected off the coast of Georgia and northeast Florida and in the plume of the Mississippi River, 56 to 98% of incorporated label passed through a 3.0-μm-pore-size filter. The greatest activity in the >3.0-μm fraction in the Georgia Bight was at nearshore stations and in the bottom samples. Our data were consistent with the hypothesis that resuspension of bottom material is an important factor in influencing the proportion of heterotrophic activity attributable to particle-associated bacteria.     

Margollus bokanicus n. sp. from Iran,the Fourth Species of a Rare Nematode Genus (Dorylaimida,Tylencholaimellidae)

Margollus bokanicus n. sp., collected from natural habitats in Khorasaneh district, Bokan, West Azarbaijan province, Iran, is described. Morphological and morphometric data are provided as well as drawings and light microscopy illustrations. The new species is characterized by a medium size body length (0.60 to 0.73 mm), labial and postlabial sclerotizations, lip region 7-μm wide, offset by constriction and long neck (167 to 207 μm), long pharyngeal basal bulb (27 to 36 μm) or 16% to 17% of total neck length, female genital system monodelphic–opisthodelphic, anterior branch reduced to a uterine sac (26–29 μm) or 1.1 to 1.3 times the body diameter, long posterior uterus (25–28 μm) or 1.1 to 1.3 times the body diameter, V = 40 to 47, cylindroid female tail (17 to 24 μm, c = 31 to 38, c’ = 1.1 to 1.4), and males unknown. This taxon is easily distinguishable from other Margollus species by its smaller general size and more posterior vulva. A compendium of Margollus species is also presented.     

Physicochemical characterization of mitochondrial DNA from soybean

Mitochondrial DNA (mtDNA) of soybean (Glycine max L.) was isolated and its buoyant density was contrasted with that of nuclear (nDNA) and chloroplast (ctDNA) DNA. Each of the three DNAs banded at a single, characteristic buoyant density when centrifuged to equilibrium in a CsCl gradient. Buoyant densities were 1.694 g/cm³ for nDNA and 1.706 g/cm³ for mtDNA. These values correspond to G-C contents of 34.7 and 46.9%, respectively. Covalently closed, circular mtDNA molecules were isolated from soybean hypocotyls by ethidium bromide-cesium chloride density gradient centrifugation. Considerable variation in mtDNA circle size was observed by electron microscopy. There were seven apparent size classes with mean lengths of 5.9 μm (class 1), 10 μm (class 2), 12.9 μm (class 3), 16.6 μm (class 4), 20.4 μm (class 5), 24.5 μm (class 6), and 29.9 μm (class 7). In addition, minicircles were observed in all preparations. Partially denatured, circular mtDNA molecules with at least one representative from six of the seven observed size classes were mapped. In class 4, there appear to be at least three distinct denaturation patterns, indicating heterogeneity within this class. It is proposed that the mitochondrial genome of soybean is distributed among the different size circular molecules, several copies of the genome are contained within these classes and that the majority of the various size molecules may be a result of recombination events between circular molecules.     

Brine Assemblages of Ultrasmall Microbial Cells within the Ice Cover of Lake Vida,Antarctica

The anoxic and freezing brine that permeates Lake Vida''s perennial ice below 16 m contains an abundance of very small (≤0.2-μm) particles mixed with a less abundant population of microbial cells ranging from >0.2 to 1.5 μm in length. Fluorescent DNA staining, electron microscopy (EM) observations, elemental analysis, and extraction of high-molecular-weight genomic DNA indicated that a significant portion of these ultrasmall particles are cells. A continuous electron-dense layer surrounding a less electron-dense region was observed by EM, indicating the presence of a biological membrane surrounding a cytoplasm. The ultrasmall cells are 0.192 ± 0.065 μm, with morphology characteristic of coccoid and diplococcic bacterial cells, often surrounded by iron-rich capsular structures. EM observations also detected the presence of smaller unidentified nanoparticles of 0.020 to 0.140 μm among the brine cells. A 16S rRNA gene clone library from the brine 0.1- to 0.2-μm-size fraction revealed a relatively low-diversity assemblage of Bacteria sequences distinct from the previously reported >0.2-μm-cell-size Lake Vida brine assemblage. The brine 0.1- to 0.2-μm-size fraction was dominated by the Proteobacteria-affiliated genera Herbaspirillum, Pseudoalteromonas, and Marinobacter. Cultivation efforts of the 0.1- to 0.2-μm-size fraction led to the isolation of Actinobacteria-affiliated genera Microbacterium and Kocuria. Based on phylogenetic relatedness and microscopic observations, we hypothesize that the ultrasmall cells in Lake Vida brine are ultramicrocells that are likely in a reduced size state as a result of environmental stress or life cycle-related conditions.     

Description and SEM Observations of Meloidogyne sasseri n. sp. (Nematoda: Meloidogynidae), Parasitizing Beachgrasses

Meloidogyne sasseri n. sp. is described and illustrated from American beachgrass (Ammophila breviliffulata) originally collected from Henlopen State Park and Fenwick Island near the Maryland state line in Delaware, United States (6). Its relationship to M. graminis, M. spartinae, and M. californiensis is discussed. Primary distinctive characters of the female perineal pattern were a high to rounded arch with shoulders, widely spaced lateral lines interrupting transverse striations, a sunken vulva and anus, and coarse broken striae around the anal area. Second-stage juvenile body length was 554 μm (470-550), stylet length 14 μm (13-14.5), tail length 93 μm (83-115), tapering to a finely rounded terminus. Male stylet length 20 μm (19-21.5), spicule length 33 μm (30-36). Scanning electron microscope observations provided additional details of perineal patterns and face views of the female, male, and J2 head. Wheat, rice, oat, Ammophila sp., Panicum sp., bermudagrass, zoysiagrass and St. Augustinegrass were tested as hosts. Distribution of the species was the coasts of Delaware and Maryland. The common name "beachgrass root-knot" is proposed for M. sasseri n. sp.     

Potential Importance of Fish Predation and Zooplankton Grazing on Natural Populations of Freshwater Bacteria

The rates of ingestion of natural bacterial assemblages by natural populations of zooplankton (>50 μm in size) were measured during a 19-day period in eutrophic Frederiksborg Slotssø, Denmark, as well as in experimental enclosures (containing 5.3 m³ of lake water). The fish and nutrients of the enclosures were manipulated. In enclosures without fish, large increases in ingestion by zooplankton >140 μm in size were found (up to 3 μg of C liter⁻¹ h⁻¹), compared with values less than 0.3 μg of C liter⁻¹ h⁻¹ in the enclosures with fish and in the open lake. Daphnia cucullata and D. galeata dominated the community of zooplankton of >140 μm. Ingestion rates for zooplankton between 50 and 140 μm decreased after a period of about 8 days, in all enclosures and in the lake, to values below 0.1 μg of C liter⁻¹ h⁻¹. On the last 2 sampling days, somewhat higher values were observed in the enclosures with fish present. The >50-μm zooplankton ingested 48 to 51% of the bacterial net secondary production in enclosures without fish, compared to 4% in the enclosures with added fish. Considering the sum of bacterial secondary production plus biomass change, 35 to 41% of the available bacteria were ingested by zooplankton of >50 μm in the enclosures without fish, compared with 4 to 6% in the enclosures with added fish and 21% in the open lake. Fish predation reduced the occurrence of zookplankton sized >50 μm and thus left a large proportion of the available bacteria to zooplankton sized <50 μm. In fact, there were 4.6 × 10³ to 5.0 × 10³ flagellates (4 to 8 μm in size) ml⁻¹ in the enclosures with fish added as well as in the lake, compared with 0.5 × 10² to 2.3 × 10² ml⁻¹ in the enclosures without fish. This link in the food chain was reduced when fish predation on zooplankton was eliminated and a direct route of dissolved organic matter, via the bacteria to the zooplankton, was established.     

Parvatrema duboisi (Digenea: Gymnophallidae) Life Cycle Stages in Manila Clams,Ruditapes philippinarum,from Aphae-do (Island), Shinan-gun,Korea

Life cycle stages, including daughter sporocysts, cercariae, and metacercariae, of Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974 (Digenea: Gymnophallidae) have been found in the Manila clam Ruditapes philippinarum from Aphae-do (Island), Shinan-gun, Jeollanam-do, Korea. The daughter sporocysts were elongated sac-like and 307–570 (av. 395) μm long and 101–213 (av. 157) μm wide. Most of the daughter sporocysts contained 15–20 furcocercous cercariae each. The cercariae measured 112–146 (av. 134) μm in total length and 35–46 (av. 40) μm in width, with 69–92 (av. 85) μm long body and 39–54 (av. 49) μm long tail. The metacercariae were 210–250 (av. 231) μm in length and 170–195 (av. 185) μm in width, and characterized by having a large oral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, strongly suggesting P. duboisi. The metacercariae were experimentally infected to ICR mice, and adults were recovered at day 7 post-infection. The adult flukes were morphologically similar to the metacercariae except in the presence of up to 20 eggs in the uterus. The daughter sporocysts and metacercariae were molecularly (ITS1–5.8S rDNA-ITS2) analyzed to confirm the species, and the results showed 99.8–99.9% identity with P. duboisi reported from Kyushu, Japan and Gochang, Korea. These results confirmed the presence of various life cycle stages of P. duboisi in the Manila clam, R. philippinarum, playing the role of the first as well as the second intermediate host, on Aphae-do (Island), Shinan-gun, Korea.     

Effects of Condensed Tannins on Endoglucanase Activity and Filter Paper Digestion by Fibrobacter succinogenes S85

The effect of condensed tannins from birdsfoot trefoil (Lotus corniculatus L.) on the cellulolytic rumen bacterium Fibrobacter succinogenes S85 was examined. Condensed tannins inhibited endoglucanase activity in the extracellular culture fluid, at concentrations as low as 25 μg ml^-1. In contrast, cell-associated endoglucanase activity increased in concentrations of condensed tannins between 100 and 300 μg ml^-1. Inhibition of endoglucanase activity in both the extracellular and the cell-associated fractions was virtually complete at 400 μg of condensed tannins ml^-1. Despite the sharp decline in extracellular endoglucanase activity with increasing concentrations of condensed tannins, filter paper digestion declined only moderately between 0 and 200 μg of condensed tannins ml^-1. However, at 300 μg ml^-1, filter paper digestion was dramatically reduced and at 400 μg ml^-1, almost no filter paper was digested. F. succinogenes S85 was seen to form digestive grooves on the surface of cellulose, and at 200 μg ml^-1, digestive pits were formed which penetrated into the interior of cellulose fibers. Cells grown with condensed tannins (100 to 300 μg ml^-1) possessed large amounts of surface material, and although this material may have been capsular carbohydrate, its osmiophilic nature suggested that it had arisen from the formation of tannin-protein complexes on the cell surface. The presence of electron-dense extracellular material suggested that similar complexes were formed with extracellular protein.     

Reproductive Development of Verutus volvingentis (Tylenchida: Heteroderidae)

Verutus volvingentis Esser, 1981 deposits eggs in the rhizosphere without a gelatinous matrix. Ecdysis was not observed to occur in the egg. Spicular primordia in the rectal area of a second-stage larva were well defined. One larva increased in width from 28.2 μm to a maximum of 51.7 μm after 176.5 hours of feeding, prior to the second ecdysis. It then decreased steadily in width to 33.3 μm, at which time it had molted to a fully developed male. Males leave the third-stage larval integument embedded in the root following final ecdysis. The unique feature of female development was the occurrence of large vaginal primordial cells. Male and female development took from 6 to 15 days and 17 days, respectively.     

Description of the Kona Coffee Root-knot Nematode,Meloidogyne konaensis n. sp.

Meloidogyne konaensis n. sp. is described from coffee from Kona on the island of Hawaii. The perineal pattern of the female is variable in morphology, the medial lips of the female are divided into distinct lip pairs, and the excretory pore is 2-3 stylet lengths from the base of the stylet. Mean stylet length is 16.0 μm, and the knobs gradually merge with the shaft. The knobs are indented anteriorly and rounded posteriorly and the dorsal esophageal gland orifice (DEGO) is long, 3.5-7 μm. The morphology of the stylet of the male is the most useful diagnostic character, with 6-12 large projections protruding from the shaft. One medial lip may be divided into distinct lip pairs. A large intestinal caecum often extends nearly to the level of the DEGO. Mean juvenile length is 502 μm, mean stylet length is 13.4 μm, and mean tail length is 58 μm. The tail may be distinctly curved ventrally and the phasmids are located in the ventral incisure about one anal body width posterior to the anus.     

Paurodontella parapitica n. sp. (Nematoda: Hexatylina,Sphaerularioidea) from Kermanshah Province,Western Iran

Paurodontella parapitica n. sp., collected from the rhizosphere of an apple tree in Kermanshah province, western Iran, is described. The new species is characterized by a body length of 505 to 723 µm (females) and 480 to 600 µm (males), lip region continuous by depression; 7 to 8 μm broad, 3 to 4 µm high, stylet length 7 to 9 µm or 1 to 1.3 times the lip region diameter, short postuterine sac of 4 to 6 μm long, lateral fields with five to six incisures; outer incisures crenated and inner incisures weakly crenated, excretory pore situated 90 to 100 µm from anterior end; functional males common in the population, with spicules 24 to 26 μm long. Tail of both sexes similar, almost straight and elongate-conoid. The new species resembles in morphology and morphometrics to four known species of the genus, namely P. apitica, P. minuta, P. myceliophaga, and P. sohailai. The results of phylogenetic analyses based on sequences of D2/D3 expansion region of 28S rRNA gene revealed this genus is polyphyletic in four different clades in Tylenchid.     

Population Size and Distribution of Rhizobium leguminosarum bv. trifolii in Relation to Total Soil Bacteria and Soil Depth

Bacterial cells small enough to pass through 0.4-μm-pore-size filters made up 5 to 9% of the indigenous bacterial population in 0- to 20-cm-depth samples of Abiqua silty clay loam. Within the same soil samples, cells of a similar dimension were stained with fluorescent antibodies specific to each of four antigenically distinct indigenous serogroups of Rhizobium leguminosarum bv. trifolii and made up 22 to 34% of the soil population of the four serogroups. Despite the extensive contribution of small cells to these soil populations, no evidence of their being capable of either growth or nodulation was obtained. The density of soil bacteria which could be cultured ranged between 0.5 and 8.5% of the >0.4-μm direct count regardless of media, season of sampling, or soil depth. In the same soil samples, the viable nodulating populations of biovar trifolii determined by the plant infection soil dilution technique ranged between 1 and 10% of the >0.4-μm direct-immunofluorescence count of biovar trifolii. The <0.4-μm cell populations of both total soil bacteria and biovar trifolii changed abruptly between the 10- to 15-cm and 15- to 20-cm soil depth increments, increasing from 5 to 20% and from 20 to 50%, respectively, of their direct-count totals. The increase in density of the small-cell population corresponded to a significant increase in soil bulk density (1.07 to 1.21 g cm⁻³). The percent contribution of the <0.4-μm direct count to individual serogroup totals increased with soil depth by approximately 2-fold (39 to 87%) for serogroups 17 and 21 and by 12-fold (6 to 75%) for serogroups 6 and 36.     

Revision of the Genus Paratylenchus Micoletzky, 1922, and Descriptions of New Species. Part I of 3 Parts

A revision of part of the genus Paratylenchus is reported covering those species with stylets averaging < 22 μm. Thirteen new species are described and further observations are given on the morphology and relationships of nine other species. Paratylenchus gahriciis transferred to the genus Hemicriconemoides. This article is the first of three parts: the second will treat Paratylenchus spp. with stylets averaging 24-40 μm (and include a key to the species described in parts 1 and2), the third will include species with stylets >40μm, respectively.     

Meloidogyne californiensis n. sp. (Nemata: Meloidogyninae), Parasitic On Bulrush,Scirpus robustus Pursh

Meloidogyne californiensis n. sp. is described and illustrated from bulrush Scirpus robustus in California. LM and SEM studies revealed that this species differs from other known species in the genus Meloidogyne especially by the prominent posterior cuticular protuberances in the female, the distinct shape of the perineal pattern which is marked by one prominent stria in the perineum, indistinct lateral lines, many broken discontinuous striae on both sides of the arch, and the excretory pore being located posterior to stylet base. Second-stage juveniles 448-628 μm long, stylet length 11-13 μm, styler delicate, with small knobs sloping posteriorly, cephalic region with 2 or 3 annuli, and inflated rectum. Males vary greatly in size (712-1,952 μm), stylet length 18-28 μm (mean 22 μm), cephalic region slightly set off the body with two or three annuli, spear heavy with massive rounded knobs, lateral field marked by four areolated incisures as seen by SEM.     

Fulvifomes nonggangensis and F. tubogeneratus (Hymenochaetales,Basidiomycota): Two New Species from Southern China Based on Morphological and Molecular Evidences

Two new species of Fulvifomes are described from specimens collected in rainforests of Nonggang Nature Reserve of southern China, based on morphological characteristics and molecular phylogenetic analysis of the internal transcribed spacer (ITS) and nuclear large subunit ribosomal DNA (nLSU) sequences. Fulvifomes nonggangensis sp. nov. is characterized by perennial, sessile and solitary basidiocarps, applanate pileus, small cystidioles of 9.9–15.4 × 2.9–3.5 μm, large pores of 5–6 per mm, a dimitic hyphal system, and broadly ellipsoid basidiospores of 4.3–5.3 × 3.3–4.2 μm. F. tubogeneratus sp. nov. is characterized by perennial, sessile, and imbricate basidiocarps, a duplex context, small pores of 7–8 per mm, a dimitic hyphal system, and ovoid to subglobose basidiospores of 5.72 × 5.00 μm.     

Mycobacterium gilvum Illustrates Size-Correlated Relationships between Mycobacteria and Acanthamoeba polyphaga

Mycobacteria are isolated from soil and water environments, where free-living amoebae live. Free-living amoebae are bactericidal, yet some rapidly growing mycobacteria are amoeba-resistant organisms that survive in the amoebal trophozoites and cysts. Such a capacity has not been studied for the environmental rapidly growing organism Mycobacterium gilvum. We investigated the ability of M. gilvum to survive in the trophozoites of Acanthamoeba polyphaga strain Linc-AP1 by using optical and electron microscopy and culture-based microbial enumerations in the presence of negative controls. We observed that 29% of A. polyphaga cells were infected by M. gilvum mycobacteria by 6 h postinfection. Surviving M. gilvum mycobacteria did not multiply and did not kill the amoebal trophozoites during a 5-day coculture. Extensive electron microscopy observations indicated that M. gilvum measured 1.4 ± 0.5 μm and failed to find M. gilvum organisms in the amoebal cysts. Further experimental study of two other rapidly growing mycobacteria, Mycobacterium rhodesiae and Mycobacterium thermoresistibile, indicated that both measured <2 μm and exhibited the same amoeba-mycobacterium relationships as M. gilvum. In general, we observed that mycobacteria measuring <2 μm do not significantly grow within and do not kill amoebal trophozoites, in contrast to mycobacteria measuring >2 μm (P < 0.05). The mechanisms underlying such an observation remain to be determined.     

Production and Distribution of Endoglucanase, Cellobiohydrolase, and β-Glucosidase Components of the Cellulolytic System of Volvariella volvacea, the Edible Straw Mushroom

The edible straw mushroom, Volvariella volvacea, produces a multicomponent enzyme system consisting of endo-1,4-β-glucanase, cellobiohydrolase, and β-glucosidase for the conversion of cellulose to glucose. The highest levels of endoglucanase and cellobiohydrolase were recorded in cultures containing microcrystalline cellulose (Avicel) or filter paper, while lower but detectable levels of activity were also produced on carboxymethyl cellulose, cotton wool, xylitol, or salicin. Biochemical analyses of different culture fractions in cultures exhibiting peak enzyme production revealed that most of the endoglucase was present either in the culture filtrate (45.8% of the total) or associated with the insoluble pellet fraction remaining after centrifugation of homogenized mycelia (32.6%). Cellobiohydrolase exhibited a similar distribution pattern, with 58.9% of the total enzyme present in culture filtrates and 31.0% associated with the pellet fraction. Conversely, most β-glucosidase activity (63.9% of the total) was present in extracts of fungal mycelia whereas only 9.4% was detected in culture filtrates. The endoglucanase and β-glucosidase distribution patterns were confirmed by confocal laser scanning microscopy combined with immunolabelling. Endoglucanase was shown to be largely cell wall associated or located extracellularly, with the highest concentrations being present in a region 1 to 2 μm wide immediately adjacent to the outer surface of (and possibly including) the hyphal wall and extending 60 to 70 μm from the hyphal tip. Immunofluorescence patterns indicated little if any intracellular endoglucanase. Most β-glucosidase was located intracellularly in the apical area extending 60 to 70 μm below the hyphal tip, although enzyme was also evident in the extracellular region extending approximately 15 μm all around the hyphal tip and trailing back along the length of the hypha. The regions of the hypha located some distance from the apical region appeared to be devoid of intracellular β-glucosidase, and the enzyme appears to be associated almost exclusively with, or located on the outside surface of, the hyphal wall.