Extracellular peroxidase reaction at hyphal tips of white-rot fungus <Emphasis Type="Italic">Phanerochaete crassa</Emphasis> WD1694 and in fungal slime

The distribution of an extracellular peroxidase reaction by white-rot fungus Phanerochaete crassa WD1694 was visualized by peroxidase activity staining. The extracellular peroxidase reaction occurred at the hyphal tips and in the fungal slime filling the gaps between the hyphae. We investigated whether the peroxidase reaction occurred from the hyphal tips or in the slime. The hyphal tips were observed by phase-contrast microscopy, which showed that slime did not exist around the hyphal tips. Time-course observation of hyphal tips showed that peroxidase staining became thick and intense at the tips that did not have fungal slime. Daily observation of the peroxidase staining revealed that the staining was first observed at the hyphal tips. Furthermore, strongly stained hyphae were observed in the stained slime. These results suggested that an active species that oxidizes a peroxidase substrate is first produced at the tips of the hyphae, and then occurs in the slime via diffusion when slime exists around the hyphae. Our results show that the extracellular peroxidase reaction that is important to lignin biodegradation by white-rot fungi occurs directly at the tips of the hyphae and in the slime. Part of this report was presented at the 50th Lignin Symposium, October 19–20, 2005, Nagoya, Japan     

Extracellular peroxidase activity at the hyphal tips of the white-rot fungus Phanerochaete crassa WD1694

The white-rot fungus Phanerochaete crassa WD1694 was cultivated and peroxidase activity staining was performed to determine the sites at which the extracellular peroxidase reaction actually occurs in vivo. Although the ligninolytic peroxidases were found in the culture filtrates, the culture medium did not show a color reaction. However, a particularly strong color reaction was observed on the hyphal tips. Visible spectra and absorbance of the staining were analyzed by microspectrophotometry, and the catalytic rates of the peroxidase reaction at the hyphal tips were calculated. The estimated catalytic rate of the peroxidase reaction at the hyphal tips peaked at 794 μM/min, expressed as the consumption rate of H₂O₂, on day 3 of the cultivation. Analysis of the extracellular enzyme eluted with 0.1% Tween 80 from the mycelium revealed that manganese peroxidase accounted for 89% of all the peroxidase activity measured. The results clearly showed the existence of the concentrated manganese peroxidase reaction around the hyphal tips of the organism.     

Glyoxal oxidase supplies hydrogen peroxide at hyphal tips and on hyphal wall to manganese peroxidase of white-rot fungus Phanerochaete crassa WD1694

Peroxidase activity staining localized at hyphal tips of white-rot fungus Phanerochaete crassa WD1694 that was cultivated in a shaken liquid culture containing unbleached kraft pulp was investigated. Manganese peroxidase was detected in culture solution, washing solution of mycelium, and mycelial extract. Glyoxal oxidase was detected only in mycelial extract and was not detected in culture solution. Addition of hydrogen peroxide generated peroxidase activity staining in the culture solution. Addition of catalase resulted in no staining in the culture of P. crassa WD1694, and the addition of methylglyoxal resulted in marked peroxidase activity staining at hyphal tips and on hyphal wall. In an optimized culture, glyoxal oxidase was produced in culture solution. Although the production of glyoxal oxidase and manganese peroxidase had a positive correlation, the secretion and the peak of glyoxal oxidase was observed 3 and 2 days later than those of manganese peroxidase. The N-terminal sequence of purified glyoxal oxidase had very high homology with that of P. chrysosporium. These results elucidated the hydrogen peroxide supply system in lignin biodegradation by white-rot fungi, i.e., while remaining on the hyphal cell wall, glyoxal oxidase provides hydrogen peroxide to manganese peroxidase that had diffused into the culture solution beforehand.     

Screening of wood-rotting fungi for kraft pulp bleaching by the Poly R decolorization test and biobleaching of hardwood kraft pulp byPhanerochaete crassa WD1694

From among 419 wood-rotting fungi 10 were selected by the Poly R decolorization test, and their ability to bleach hardwood kraft pulp was assayed. Of the 10 selected, 6 fungi (i.e.,Phanerochaete crassa WD1694 and F150;Pleurotus pulmonarius PSC-H, PSC-M, and PSC-T;and Pleurotus species A119) showed much higher bleaching ability thanPhanerochaete chrysosponum BKMF1767 orTrametes versicolor WD1670, both of which are well-known high ligninolytic fungi.P. crassa WD1694 had the highest bleaching ability among the selected strains, and it increased the pulp brightness from 28 to 54, with a corresponding decrease in kappa number from 16 to 6 after 10 days of cultivation and alkali extraction. MnP was a predominant ligninolytic enzyme ofP. crassa WD1694 during the biobleaching.This research was presented in part at the 46th Annual Meeting of the Japan Wood Research Society, Kumamoto, April 1996     

Genomic gene encoding manganese peroxidase from a white-rot fungus Phanerochaete crassa WD1694

The gene encoding manganese peroxidase of a white-rot fungus Phanerochaete crassa WD1694 was cloned and sequenced. Four genomic clones were sequenced in which 3 clones were existed as alleles. The analysis of intron–exon structures divided the 4 clones into three subfamilies that corresponded to mnp2 and mnp3 of Phanerochaete chrysosporium, and a new subfamily possessing only five introns. The purified P. crassa WD1694 MnP consisted of 4 isozymes with same molecular weight, same N-terminal sequence, and different pI. N-terminal sequence of deduced protein of P. crassa mnpB3 gene was identical to those of 4 MnP isozymes; however, the other 3 mnp genes had different N-terminal sequence. The molecular weight of encoded mature protein of mnpB3 gene and purified MnP had a gap that could be difference between MnP proteins encoded by single gene. The results suggested that 4 MnP isozymes of P. crassa WD1694 arose from single gene.     

Electron-dense particles in wood decayed by Ganoderma applanatum

Summary Hemlock sawdust samples degraded by Ganoderma applanatum showed no electron-dense particles either in hyphae or in wood cell walls after aldehyde/OsO₄ fixation. After KMnO₄ fixation at early stage of attack, particles were in hyphae, hyphal sheath and wood cell walls. In samples prepared by a cytochemical technique which localizes cellulase activity at the ultrastructural level, particles were in hyphae, hyphal sheath and wood cell walls. The smallest diameter range of the particles lay between 3 and 7 nm which corresponds to the size of cellulases. Larger diameter particles were peresent which are probably aggregates of the smaller units. We believe that particles present in hyphal cytoplasm and hyphal sheath are cellulolytic enzymes. Whether particles present in attacked wood cell walls are enzymes or degradation products cannot be determined by this study. Nevertheles, the particles reveal the decay pattern in wood by the white-rot fungus G. applanatum.     

Electron microscopic observations of sooty moulds on crape myrtle leaves

Sooty moulds on crape myrtle leaves were investigated using light and electron microscopy. The adaxial leaf surface was distinctly covered with soot‐like masses of dark brown hyphae and conidia. The main characteristics of the sooty moulds included stauroconidia, conidial clusters, hyphal degeneration and extracellular melanin depositions. Some conidia were round, measured ~10 μm in diameter, and mostly one‐septate; others were branched and multiseptate (stauroconidia). Based on their morphology, the sooty moulds were determined to consist of several component fungal species belonging to genera such as Antennaria, Metacapnodium and Tripospermum. Enclosed in electron‐dense melanin layers, hyphae and conidial clusters had concentric bodies exhibiting electron‐transparent cores and electron‐dense shells with fibrillar sheaths. Concentric bodies are hypothesized to function as multilayer lipid‐encapsulated nanobubbles or eukaryotic gas vesicles for cytoplasmic volume control. Intrahyphal hyphae possessed electron‐dense cytoplasm and lipid globules. These results suggest that sooty moulds are equipped with melanized cell walls, multicelled resting structures, hyphal regeneration, intrahyphal growth and gas vesicles to adapt to their xeric phylloplane environment. Condensed and elongated starch granules in the chloroplasts of mesophyll tissues may indicate the acclimation of the sooty leaf regions to light reduction and temperature increase.     

绿僵菌侵染光肩星天牛幼虫过程的透射电镜观察

应用透射电镜观察并研究光肩星天牛幼虫感染绿僵菌后体壁和中肠发生的病理变化。结果表明:接菌后16h,寄主表皮层中观察到少量菌丝段,至24h菌丝已普遍侵入,芽管在穿透过程中表现了机械压力和酶活性。36～48h菌丝即可穿透体壁进入血腔。菌丝和菌丝段经血淋巴循环侵入各器官组织。48～72h,随着菌丝和菌丝段在肠壁细胞中迅速大量增殖,肠壁组织逐渐被破坏,中肠微绒毛脱落,肠壁细胞形成空泡,围食膜被分解消失,最终中肠组织崩溃、解体。     

Penetration and infection processes in japanese cedar twig blight caused byStromatinia cryptomeriae

The diversity of host invasion mechanisms of the causal pathogen of Japanese cedar twig blight (Stromatinia cryptomeriae) was elucidated through detailed investigations of the disease cycle. Scanning electron microscopy (SEM) showed that ascospores began to germinate, and invade male strobilus tissue, within 24 h after arriving on the strobili. This involved direct penetration of the cuticle, and examination by SEM showed that cuticle degeneration had occurred around the point of penetration. A mucilage-like-substance was also observed around the tips of the germ tubes. Meanwhile field studies showed that incipient mycelial mat emerged from the bases of male strobilus in early June, and ceased growing onto the surface of the twig at the beginning of July. Macroscopically, the mycelial mat began to shed in mid-July, and disappeared completely during the summer season by the end of August. Immediately after the mat disappeared the first necrotic symptoms became evident on the twigs. This suggested strongly that these mats were involved in lesion formation. Observations of the mat’s behavior on the surface of twigs with light and differential interference microscopy, and SEM, showed thatS. cryptomeriae had two modes of invasion,i.e. stomatal and cuticular invasion. Hyphae from the mat were able to enter twig tissues through the stomata. Additionally, mycelial mat infected host tissue directly by hyphal penetration of the cuticle at the axes of cedar twigs. The mat was able to grow along the twig surface, then hyphae forming mat entered the host tissue and caused necrotic lesions. This paper describes the mechanisms of infection on both a strobilus and twig utilized by the pathogen of Japanese cedar twig blight.     

Anatomische Untersuchungen zur Infektionsbiologie von Sderoderris lagerbergii Gr. [Brunchorstia pinea (Karst.) von Höhn]1

Anatomical studies on the infection biology of Scleroderris lagerberii (Brunchorstia pinea) . Infection of Pinus nigra shoots by B. pinea was studied by means of both ordinary light and fluorescent microscopy. Following spore germination, the mycelium lives under and between the leaf scales, subsequently invading the leaf scales directly. The hyphae can grow through periderms, later invading the xylem via the rays.     

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

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 I_δ 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.     

Histology of Pinus maximinoi cones infected by Cronartium conigenum1

Apparently healthy and C. conigenum infected female strobili of Pinus maximinoi, collected in Guatemala, were histopathologically studied by light microscopy. Scales from infected cones were fused and fertile scales had aborted seeds. Infected cone scales lacked fibre cells in the cortex, which are abundant in healthy cone scales. Intercellular spaces of cortex contained abundant hyphae compared to phloem and xylem. The cell walls of infected tissues in the ovules were darkly stained, while those of comparable healthy tissue were colourless under Pianeze's IIIB stain. Infected ovules of different developmental stages with nucellus, or nucellus plus spongy tissue, were observed. The cells of each tissue type contained many haustoria and did not develop into female gametophyte and embryo. Ovule colonization by hyphae appeared after pollination and subsequently caused their early abortion.     

Rasterelektronenmikroskopische Abbildungen von Hyphen des Birkenporlings (Piptoporus betulinus) im Holz von Betula pendula

Scanning electron micrographs of the hyphae of Piptoporus betulinus in timber of Betula pendula. Hyphae of Piptoporus betulinus (Bull, ex Fr.) Karst. were photographed in naturally infected timber of Betula pendula Roth by means of scanning electron microscopy. The hyphae had an average diameter of 3μm and a cell wall thickness between 0.4 and 0.7 μm. The dissolution of the cell wall by specific fungal enzymes can be extremely well studied using scanning electron microscopy.     

Infection of red palm weevil,<Emphasis Type="Italic"> Rhynchophorus ferrugineus</Emphasis>, by spore-forming bacilli indigenous to its natural habitat in Egypt

A search for means of biologically controlling the red palm weevil (RPW) Rhynchophorus ferrugineus resulted in the isolation of several microbial cultures belonging to bacteria, actinomycetes and fungi. Among the obtained cultures, three potent spore-forming bacilli were further isolated from natural habitats associated with insect-damaged date palms in Egypt. The isolated bacterial cultures, (strains 73, 15 and 27) were identified as variants of Bacillus sphaericus, B. megaterium and B. laterosporus, respectively. Under standard bioassay conditions, the mortalities of the second-instar larvae of the target insect induced by the three isolated bacilli variants ranged between 40 and 60%. The most active culture (strain no. 73) was identified as B. sphaericus. This isolate formed spherical endospores and crystalline endotoxin comparable to those of B. sphaericus standard strain 2362, which is pathogenic to mosquito larvae, as shown by electron microscopy. However, the amino acid composition of the spore-endotoxin complexes was markedly different.     

In vitro interactions in artificial and wood‐based media between fungi colonizing stumps of Sitka spruce

Interspecific interactions between fungi that colonize stumps of Picea sitchensis in Scotland were tested in dual cultures on Norkrans agar, spruce sawdust and in autoclaved blocks cut from roots. Isolates were ranked according to competitive ability on the different media, based on their ability to overgrow competitors or to form deadlock interactions. On the defined medium, Phaeolus schweinitzii was the species most able to overgrow competitors, followed by Stereum sanguinolentum and Heterobasidion annosum; Resinicium bicolor was the least able to overgrow competitors on this medium. By contrast, R. bicolor was the most competitive on spruce sawdust medium. Deadlocking interactions were formed most often in dual cultures on Norkrans agar. Observation of hyphal interactions on Norkrans agar under the microscope identified several different response types including growth of thin hyphae compared to control cultures, hyphal coiling, vacuolation of hyphae, hyphal lysis of one competitor and deposition of crystals in the agar. Hymenomycetes caused varying amounts of decay in autoclaved root blocks. Resinicium bicolor was able to replace other species in most co‐inoculations. Stereum sanguinolentum appeared to be the least competitive species in root block inoculations, being replaced by Melanotus proteus and R. bicolor, although interactions with H. annosum varied widely. These results indicate that substrate has a marked effect on interspecific fungal interactions, with wood‐based, particularly intact woody tissues closely matching competitive behaviour displayed in the field.     

Morphological evidences for onion-induced growth inhibition of Trichophyton rubrum and Trichophyton mentagrophytes

The antifungal activity of onion (Allium cepa L.) on two important dermatophytes, Trichophyton rubrum and Trichophyton mentagrophytes, with special reference to morphological aspects was studied. Growth of both fungi was found to be strongly inhibited by aqueous onion extract (AOE) as a dose-dependent manner. The extract showed fungicidal effect for both fungi at concentrations >3.12% (v/v). The fungus T. mentagrophytes was more affected by the onion as compared to T. rubrum at all concentrations used. Morphological effects of onion exposure were examined in correlation with fungal growth. Corresponding to the growth inhibition, light and electron microscopy observations revealed morphological anomalies in hyphal compartments. The results demonstrated that AOE targets the cell membrane of the fungi as breaking down of both inner and outer membranes with consequent extrution of materials into the surrounding medium. Cytoplasmic membranes and other membranous structures of organelles, such as nuclei and mitochondria, were also disrupted. In correlation to the fungal growth, morphological alterations occurred to a less content for T. rubrum compared with T. mentagrophytes. The hyphae of T. rubrum were found to be mainly affected by converting to resistant forms, i.e., chlamidospores as a consequence of phenotype switching response to AOE. Plasmolysis accompanied by an almost complete depletion and disorganization of cytoplasmic structures were found to be the final event which led to cell death. Ultrastructural evidences obtained from this study strongly support that morphological changes of T. rubrum and T. mentagrophytes caused by AOE are associated with its fungistatic and fungicidal activities. With respect to the morphological results and the preliminary data on fungal biochemistry, a mechanism of action by interacting of AOE with thiol (-SH) groups present in essential compartments of the fungal cells was postulated.     

Carbon dioxide concentration and nitrogen input affect the C and N storage pools in Amanita muscaria-Picea abies mycorrhizae

We studied the influence of elevated atmospheric CO2 concentration ([CO2]) on the vacuolar storage pool of nitrogen-containing compounds and on the glycogen pool in the hyphal sheath of Amanita muscaria (L. ex Fr.) Hooker-Picea abies L. Karst. mycorrhizae grown with two concentrations of ammonium in the substrate. Mycorrhizal seedlings were grown in petri dishes on agar containing 5.3 or 53 mg N l(-1) and exposed to 350 or 700 microl CO2 l(-1) for 5 or 7 weeks, respectively. Numbers and area of nitrogen-containing bodies in the vacuoles of the mycorrhizal fungus were determined by light microscopy linked to an image analysis system. The relative concentration of nitrogen in the vacuolar bodies was measured by electron energy loss spectroscopy (EELS). Glycogen stored in the cytosol was determined at the ultrastructural level by image analysis after staining the sections (PATAg test). Shoot dry weight, net photosynthesis and relative amounts of N in vacuolar bodies were greater at the higher N and CO2 concentrations. The numbers and areas of vacuolar N-containing bodies were significantly greater at the higher N concentration only at ambient [CO2]. In the same treatment the percentage of hyphae containing glycogen declined to nearly zero. We conclude that, in the high N/low [CO2] treatment, the mycorrhizal fungus had an insufficient carbohydrate supply, partly because of increased amino acid synthesis by the non-mycorrhizal rootlets. When [CO2] was increased, the equilibrium between storage of glycogen and N-containing compounds was reestablished.     

A histological investigation of Oriental beech wood decayed by Pleurotus ostreatus and Trametes versicolor

Chemical, light and electron microscopic studies were carried out on wood of Oriental beech (Fagus orientalis Lipsky) decayed by the white‐rot fungi Pleurotus ostreatus and Trametes versicolor for 30, 60 and 120 days according to the modified European standard EN 113. Mass loss as well as lignin, cellulose and carbohydrate content were determined before and after fungal attack. There were no significant differences of wood mass loss and chemical composition between both fungi at the end of incubation. After each incubation period, small specimens were stained for microscopic studies. The micromorphology of fungal cell wall degradation was rather similar for both fungi. Both decreased the cell wall thickness to the same extent. The accumulation of hyphae as well as the rupture of cell walls was also similar. The occurrence of hyphae, cavities in the pits and vessel walls followed nearly the same patterns. The parenchyma cells were completely destroyed. Altogether, both fungi produced a simultaneous white rot in Oriental beech wood.     

Visualizing the early infection of Agathis australis by Phytophthora agathidicida,using microscopy and fluorescent in situ hybridization

Phytophthora agathidicida (PTA) causes a root rot and collar rot of New Zealand kauri (Agathis australis). This study developed techniques to visualize early infection of kauri by PTA in deliberately inoculated seedlings. Conventional light microscopy was carried out on cleared and stained roots using trypan blue to observe PTA structures. Additionally, scanning electron microscopy (SEM) was used to study the PTA root structures at a higher resolution. A fluorescent in situ hybridization assay (FISH) was developed using a PTA‐specific probe to label PTA structures in planta. Infection progression in roots of 2‐year‐old kauri inoculated with PTA at 5, 10, 16 and 20 days post‐inoculation (d.p.i.) was compared using these three approaches. Light microscopy identified no Phytophthora‐like structures in the control treatments. In PTA‐inoculated plants, lignitubers were produced 5 d.p.i. in cortical cells. Infection was localized after 5 days, but as the infection progressed (up to 20 d.p.i.), the ‘degree’ of root infection increased, as did the number of replicates in which structures were observed. SEM provided higher resolution images; again, no PTA structures were observed in the negative control material examined. The slide‐based FISH‐specificity assay successfully hybridized with PTA hyphae. Fluorescence was observed using 330–380 nm excitation and an emission filter at 420 nm (DAPI), with PTA nuclei fluorescing a bright greenish‐yellow. Cross‐reactivity was not observed when the assay was applied to six other non‐target Phytophthora species. Successful hybridization reactions occurred between the primer and PTA structures in planta. Applying this FISH assay has allowed clear differentiation of the intracellular and intercellular structures of PTA. The technique can be applied to longer term studies or analysis of ex situ inoculation studies aiming to elucidate differential host‐responses to the pathogen. Additionally, the technique could be applied to study the interactions with other fungal endophytes (e.g. mycorrhizal fungi), which could be assessed for biocontrol potential as part of the integrated management of the disease.     

Micromorphological characteristics of bamboo (<Emphasis Type="Italic">Phyllostachys pubescens</Emphasis>) fibers degraded by a brown rot fungus (<Emphasis Type="Italic">Gloeophyllum trabeum</Emphasis>)

The decay pattern in bamboo fibers caused by a brown rot fungus, Gloeophyllum trabeum, was examined by microscopy. The inner part of the polylaminate secondary wall was degraded, while the outer part of the secondary wall remained essentially intact. Degradation in bamboo fiber walls without direct contact with the fungal hyphae was similar to wood decay caused by brown rot fungi. Degradation in polylaminate walls was almost confined to the broad layers whereas the narrow layers appeared resistant. The p-hydroxylphenyl unit lignin in middle lamella, particularly in the cell corner regions, was also degraded. The degradation of lignin in bamboo fibers was evidenced by Fourier transform infrared spectra. The present work suggests that the decay of bamboo fiber walls by G. trabeum was influenced by lignin distribution in the fiber walls as well as the polylaminate structures.