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.     

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     

Selective staining and visualization of hyphal sheath of a white-rot fungus <Emphasis Type="Italic">Phanerochaete crassa</Emphasis> WD1694 with phloxine B

The hyphal sheath is a morphological feature of many kinds of fungi. Although the fine structures of the sheath have been studied in detail by a number of electron microscopy techniques, the function and physiology of the hyphal sheath are not yet clarified. One reason for this is that the hyphal sheath is a colorless, mucilaginous, and delicate material so that it is not easily identified. We developed a simple method to visualize and identify the hyphal sheath of the white-rot fungus Phanerochaete crassa WD1694. The small mycelial pellets in shaken liquid cultures of P. crassa WD1694 were stained directly with phloxine B. Both the hyphae and the hyphal sheath that filled the gaps between each of the hyphae were visualized and observed by light microscopy. The stained hyphae were further studied by transmission electron microscopy, atomic force microscopy, and fl uorescence microscopy. Based on these observations, we confirmed that the staining of the hyphae was also due to the presence of the hyphal sheath that closely covered the fungal cell wall. These results clearly showed that the hyphal sheath was selectively stained with phloxine B and could be observed and identified by conventional light microscopy. Part of this report was presented at the 50th Lignin Symposium, Nagoya, October 2005     

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.     

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     

Reaction of manganese peroxidase ofBjerkandera adusta with synthetic lignin in acetone solution

The reaction of manganese peroxidase (MnP) of the white-rot fungusBjerkandera adusta with synthetic lignin dehydrogenation polymer, DHP) in acetone medium was investigated. Gel-permeation chromatography of the DHP treated by MnP demonstrated depolymerization of syringyl DHP in the reaction mixture containing 70% acetone; moreover, concomitant repolymerization occurred to give highly polymerized products. Guaiacyl DHP was only repolymerized by MnP in the same acetone solution without giving degradation products. Addition of ascorbic acid to reaction mixtures containing acetone resulted in preferential depolymerization of syringyl DHP.Part of this report was presented at the meeting of Kansai Branch, Japan Society for Bioscience, Biotechnology, and Agrochemistry in Kagawa, October 1996     

Polyethylene degradation by manganese peroxidase in the absence of hydrogen peroxide

A possible role of Tween 80 in the polyethylene degradation by manganese peroxidase (MnP) and the basis of the MnP action in the absence of hydrogen peroxide were investigated. The MnP activity in the system was retained its maximum level for 6 days in the presence of Tween 80. Tween 20 and CHAPSO stabilized MnP in the system similarly to Tween 80, and these surfactants also promote the polyethylene degradation. The system containing malonate buffer, Mn(II), and MnP produced Mn(III) in the absence of hydrogen peroxide, but the effect of Tween 80 addition on Mn(III) production in the absence of hydrogen peroxide was small. The results show that Mn(III) is generated by the MnP action initiated and amplified by the decomposition of malonate by Mn(III) and that a surfactant such as Tween 80 is required to stabilize MnP in the system.This study was presented in part at the 42nd lignin symposium, Sapporo, October 1997 and the 43rd lignin symposium, Fuchu, Tokyo, October 1998     

In vitro reduction of manganese dioxide by a ferrireductase system from the white-rot fungus <Emphasis Type="Italic">Phanerochaete sordida</Emphasis> YK-624

Reduction of manganese dioxide is demonstrated for an in vitro ferrireductase system that includes NADPH-dependent ferrireductase and the iron-binding compound (IBC) isolated from the white-rot fungus Phanerochaete sordida YK-624. The Fe(II)–IBC complex was more effective in reducing manganese dioxide to Mn(II) than were complexes of Fe(II) and organic acids of low molecular weight such as nitrilotriacetate, although IBC also reduced manganese dioxide to Mn(II) in the absence of Fe(II). The generated Fe(III)–IBC complex was a better substrate for NADPH-dependent ferrireductase than were other ferric chelates, suggesting that the Fe(III)–IBC complex is reduced to an Fe(II) complex by NADPH-dependent ferrireductase. Moreover, production of the Fe(III)–IBC complex by the reduction of manganese dioxide in a reaction system containing Fe(II) and IBC was observed to be coupled to reduction of the Fe(III)–IBC complex by NADPH-dependent ferrireductase. These results indicate that the ferrireductase system of P. sordida YK-624 plays an important role in the reduction of manganese dioxide, which is necessary for the production and function of manganese peroxidase.     

Comparisons of hyphal growth of Raffaelea quercivora among four Japanese Fagaceae species

Japanese oak wilt causes widespread oak mortality in Japan. Possible differences in susceptibility to the causal fungus, Raffaelea quercivora, may be due to vessel arrangements in host trees. To clarify whether constitutive defence mechanisms including vessel arrangements or induced defence mechanisms are the main determinants of host susceptibility, we inoculated the fungus into living seedlings or sterilized stem segments of four Japanese fagaceous species. In seedlings, water conductance was assessed with dye. In both seedlings and stem segments, hyphal growth was examined by fluorescence microscopy. In seedlings, the area of non‐conductive sapwood in stem cross sections and hyphal growth differed significantly among species. In the susceptible species Quercus crispula and Quercus serrata, hyphal growth was significantly and positively correlated with the proportion of non‐conductive sapwood. In stem segments, hyphal growth was not significantly different among species or between vessel arrangements and was similar to or greater than that in seedlings. These results suggest that the extent of sapwood colonization by R. quercivora could be used as a marker for susceptibility and that susceptibility is determined mainly by induced defence responses.     

杏鲍菇和乳白耙齿菌锰过氧化物酶活性规律的研究

采用LNAS培养基在4种不同培养液中,对白腐菌杏鲍菇和乳白耙齿菌在28℃下进行静止培养,得到了不同时间内的培养液,用紫外-可见分光光度计检测了在470 nm处对于2,6-DMP的氧化作用后光密度值的变化情况,作为锰过氧化物酶（MnP）产生和活性大小的依据,从而获得了杏鲍菇和乳白耙齿菌锰过氧化物酶与培养基的成分和酶作用底物的关系。结果表明,杏鲍菇和乳白耙齿菌均可产生MnP,对比4种成分不同的培养基酶活力测定结果,在培养液内添加酶的底物木屑和2,6-DMP后可提高MnP的分泌量。以前的研究认为：Mn2＋是MnP产生的必要因子。而本试验研究结果表明,Mn2＋并不是杏鲍菇和乳白耙齿菌产生MnP所必需的。论文为进一步利用这两种真菌产木质素降解酶,以及为进一步深入研究这两种真菌MnP对木质素及其它异生物质降解的作用机理提供基础的酶学研究。     

不同植物材料浸出液对双孢蘑菇生长的影响

采用平板培养和摇瓶发酵培养的方法,通过在加入不同浓度的银杏叶、竹叶、板栗壳、蒜汁、生姜汁和橘子皮等6种植物材料的浸出液培养观察双孢蘑菇生长试验,初步研究和探讨了含这6种浸出液的培养基对双孢蘑菇菌丝生长势、胞外蛋白含量、胞内多糖及过氧化物酶活性等生长参数的影响。结果表明：与对照相比,双孢蘑菇在添加上述6种浓度为1-3g/L浸出液中生长时,6种水浸出液对菌丝生长势、菌落直径和过氧化物酶活性都有促进作用,且加入的浸出液浓度不同促进程度也不同。加入银杏叶浸出液促进菌丝生长效果最明显,使菌丝干重高达432mg/100mL;加入1g/L生姜浸出液使胞内多糖和过氧化物酶活性都达到最高值,分别为12.38mg/mL和40.9U,是对照中胞内多糖含量和过氧化物酶活性的3倍和40倍。从生产成本和经济效益方面考虑,水浸提物优于乙醇浸提物,可采用在常规培养基中添加2g/L银杏叶水浸出液将有利于双孢蘑菇的菌丝生长。     

Variations in virulence and hyphal growth of four Raffaelea quercivora isolates within Quercus crispula

Mass mortality of fagaceous trees caused by Japanese oak wilt has occurred widely in Japan. Although virulence of the causal fungus, Raffaelea quercivora, appeared to differ among isolates, its relation to the fungal growth within trees was unknown. To clarify the differences in fungal virulence against susceptible Quercus crispula, we examined fungal growth of four R. quercivora isolates within trees and the resulting virulence. In our study, the isolates were multiple‐inoculated in seedlings and single‐inoculated in twigs of mature trees. In the multiple‐inoculation test, mortality rates were examined by the observation of external symptoms. In the single‐inoculation test, water conductance and hyphal growth within the trees were examined by applying aqueous dyes and fluorescence microscopy, respectively. Mortality rates, the proportion of the cross‐sectional area comprising non‐conductive sapwood and horizontal hyphal growth differed significantly among the isolates. Univariate logistic regression analyses showed that both the proportion of non‐conductive sapwood and hyphal growth were significantly positively related to mortality rates. For three isolates, hyphal growth was significantly positively correlated with the proportion of non‐conductive sapwood. These results suggested that the virulence against Q. crispula varies among R. quercivora isolates and that the extent of fungal colonization of the tree determines fungal virulence.     

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.     

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.     

RETRACTED ARTICLE: Abundance of Collembola collected from ectomycorrhizal hyphal mats and fruiting bodies of Tricholoma matsutake

Collembolans collected from hyphal mat soil and fruit bodies of Tricholoma matsutake were examined to investigate whether mycophagous microarthropods are a potential insect pest of this fungus in forest soils. The number of collembolans collected in hyphal mat soil did not differ significantly from that in adjacent nonmat soil. Fungal materials contained in the gut of collembolans consisted mostly of hyphal fragments of dematiaceous fungi and unknown basidiomycetes. There were few collembolans on the fruit bodies of T. matsutake, which has the largest fruit body of the fungi at the study site. Our findings suggested that collembolans are not significant feeders on the hyphal mat and fruit body of T. matsutake. An erratum to this article is available at .     

Evaluation of Pinus radiata seed treatments to control Fusarium circinatum: effects on seed emergence and disease incidence

In this study, the effects of hot water (HWT), hydrogen peroxide and fungicides on the incidence of Fusarium circinatum on artificially inoculated Pinus radiata seeds were evaluated. Fifteen commercial fungicide formulations were screened in vitro for inhibitory activity on mycelial growth and conidial germination of F. circinatum. With half‐maximal effective concentration (EC₅₀) lower than 0.5 ppm, fluazinam, imazalil and tebuconazole were the most effective fungicides on mycelial growth, while captan, mancozeb or pyraclostrobin were the most effective (EC₅₀ < 0.3 ppm) on conidial germination. Based on the results obtained, imazalil, fluazinam, mancozeb and pyraclostrobin were selected for further testing. The effects of HWT, hydrogen peroxide and fungicide treatments on seed emergence and the incidence of F. circinatum were assessed. Seed treatments with fungicides prior to sowing were less effective and inconsistent in reducing the incidence of F. circinatum on seedlings. In contrast, hot water and hydrogen peroxide treatments significantly reduced F. circinatum contamination on P. radiata seeds with an overall disease incidence lower than 0.8% on seedlings. Furthermore, subsequent application of fungicides on seedlings did not improve the effectiveness of HWT. These results, therefore, suggest that hot water is a better alternative to hydrogen peroxide and fungicides as Pinus seed treatment against F. circinatum and could easily be implemented as standard in commercial nurseries to control the spread of the pitch canker disease.     

Stomatal conductance, growth and root signaling in young oak seedlings subjected to partial soil drying

Leaf conductance, water relations, growth, and abscisic acid (ABA) concentrations in xylem sap, root apices and leaves were assessed in oak seedlings (Quercus robur L.) grown with a root system divided between two compartments and subjected to one of four treatments: (a) well watered, WW; (b) half of root system exposed to soil drying and half kept well watered, WD; (c) whole root system exposed to drought, DD; and (d) half of root system severed, RE. Sharp decreases in plant stomatal conductance, leaf water potential, hydraulic conductance and leaf growth were observed during DD treatment. No significant differences in plant leaf water potential and stomatal conductance were detected between the WW and WD treatments. Nevertheless, the WD treatment resulted in inhibition of leaf expansion and stimulation of root elongation only in the well-watered compartment. Abscisic acid concentrations did not change in leaves, root tips, or xylem sap of WD- compared to WW-treated plants. Increased concentrations of ABA were observed in xylem sap from DD-treated plant roots, but the total flux of ABA to shoots was reduced compared to that in WW-treated plants, because of decreases in transpiration flux. Similar plant responses to the WD and RE treatments indicate that the responses observed in the WD-treated plants were probably not triggered by a positive signal originating from drying roots.     

Regeneration of protoplasts from hyphal strands ofVolvariella volvacea

A series of experiments on the preparation and regeneration of protoplasts from hyphal strands ofVolvariella volvacea (Bull. ex. Fr.) Singer were conducted with the aim of optimizing the conditions for its efficient regeneration. One commercial (Vvcl) and two wild (EAAC-0001 and EAAC-0002) strains ofV. volvacea from the Philippines were used and subjected to varying conditions to determine the most efficient means for regeneration of their protoplasts. The effects of age and type of strain, pH, type and concentration of osmotic stabilizer, enzymatic composition, treatment time, temperature, reciprocal frequency during enzymatic lysis of the cell wall, and centrifugation conditions were investigated. Results showed that the three strains ofV. volvacea had varying responses in terms of yield, size, and ability of their protoplasts to regenerate into the protoplast regeneration medium. Among the three strains, EAAC-0002 had the highest rate of regeneration. The 5-day-old culture ofV. volvacea, when subjected to a combination of 2% Novozyme 234 and 0.2% chitinase in 0.6M mannitol (pH 6.0) for 3 h at 30°C, 90 strokes/min and centrifuged at 1100 g for 10 min; produced an efficient yield of protoplasts with a relatively high regeneration rate.Part of this paper was presented at the 47th annual meeting of the Japan Wood Research Society, Kochi, Japan, April 2–5, 1997     

Change in oxidation state of manganese atoms in kraft pulp during biological bleaching with white-rot fungusPhanerochaete sordida YK-624

Change in the oxidation state of manganese atoms in unbleached hardwood kraft pulp (UKP) during the biological bleaching of UKP withPhanerochaete sordida YK-624 was determined by the electron spin resonance (ESR) method. The spectrum of Mn(II), which reveals hyperfine splitting, was not observed in the ESR analysis of UKP, but the spectrum for manganese dioxide was observed. After fungal treatment of UKP withP. sordida YK-624, the spectrum of Mn(II) was detected. The reduction of manganese dioxide was triggered by the increase in NADPH-dependent ferrireductase activity. It is concluded that the manganese dioxide dominant in UKP was reduced byP. sordida YK-624 to Mn(II), which stimulates the production and function of manganese peroxidase.     

Iron-binding compounds produced by white-rot fungusPhanerochaete sordida YK-624

Iron-binding compounds were isolated from a culture ofPhanerochaete sordida YK-624 and were found to bind to Fe(III) preferentially compared with Fe(II). Two iron-binding compounds were purified to near-homogeneity with gel permeation chromatography. Hydrolysis of the iron-binding compounds with 6N hydrochloric acid gave ninhydrin-negative products. The molecular weight of these compounds was 3–5 kDa. These compounds may play an important role in the reduction of extracellular manganese dioxide to Mn(II) by intracellular ferrireductases for lignin degradation by manganese peroxidase.