Physiological considerations for efficient mycelial colonization of Philippine strains ofVolvariella volvacea

The nutritional and physical requirements for the efficient mycelial colonization ofVolvariella volvacea (Bull. ex. Fr.). Singer were elucidated with the percentage mycelial colonization and density as references. This investigation was limited to the evaluation of two commercial strains (designated Vvc1 and Vvc2) and two wild strains (designated EAAC-0001 and EAAC-0002) ofV. volvacea from the Philippines with the aim of providing baseline data on their physiological requirements. The four strains ofV. volvacea had varying preferences for carbon. Vve1 preferred polysaccharides (starch and cellulose), whereas Vvc2 grew luxuriantly at a relatively rapid rate in sugar alcohol (sorbitol). The two wild strains preferred starch as a carbon source. In terms of nitrogen utilization, soytone, peptone, and glycine supported efficient mycelial colonization of the four strains. The vitamin utilization test revealed that ascorbic acid, calcium pantothenate, and biotin are good sources. The mycelial growth performances of the strains were also evaluated on six dehydrated mycological media. Efficient colonization of Vvc1, Vvc2, and EAAC-0002 with dense mycelial growth was noted in mycological agar. EAAC-0001, on the other hand, grew more efficiently in malt extract agar. The Philippine strains ofV. volvacea grew luxuriantly when incubated at 35°C and pH 8.0 under dark and sealed conditions. Moreover, the relatively higher moisture content (70%) of the oolong tea leaf formulation favorably stimulated efficient mycelial colonization. Under optimum physiological conditions, Vvc1, Vvc2, and EAAC-0002 were fast-growing strains, whereas EAAC-0001 was a moderately growing type.     

Plantlet regeneration from leaf protoplasts ofPopulus euphratica

Protoplasts were isolated from the leaves of sterile plants ofPopulus euphratica Oliv. by using 1% Cellulase “Onozuka” RS and 0.25% Pectolyase Y-23 in 0.6m of mannitol solution. Protoplasts were cultured in modified Murashige and Skoog's (MS) medium which contained no ammonium ions but was supplemented with BAP (6-benzylaminopurine), 2,4-D (2,4- dichlorophenoxy-acetic acid), and 1% sucrose at the cell density of 9×10⁴/ml. Cell divisions occurred in every culture medium, especially in the medium containing 0.5 mg/l of BAP and 0.1 mg/l of 2,4-D, in which callus was successfully induced by successive culture through cell cluster formation. Shoots were regenerated from the callus, and their growth was enhanced on 1/2 MS medium containing 0.8 mg/l of BAP. Finally, shoots were rooted and plantlets were regenerated on 1/2 MS medium without a hormone. A part of this paper was presented at the 106th Annual Meeting of the Jpn. For. Soc. (1995).     

Screening of heterozygous DNA markers in shiitake (Lentinula edodes) using de-dikaryotization via preparation of protoplasts and isolation of four meiotic monokaryons from one basidium

A suitable screening method for heterozygous DNA markers in shiitake,Lentinula edodes (Berk.) Pegler, is reported. Monokaryons were derived from a dikaryon by de-dikaryotization via protoplast formation. Compatibility of the monokaryons was determined by pairwise culture on agar plates. We selected the primers to amplify polymorphic fragments among the original strain (Hokken600H600) and two monokaryons (H600PP-39 and H600PP-67) showing compatibility. A total of 135 fragments were selected as specific random amplified polymorphic DNAs (RAPDs) resulting from 56 primers of the 147 primers tested. Furthermore, we tested whether the polymorphic fragments segregated into 22 among four strains isolated from a basidium. Most of the polymorphic fragments (about 97.8%) showed 22 segregation among the four strains. We concluded that the polymorphic fragments were heterozygous if they were detected in either of the monokaryons (H600PP-39 and H600PP-67) and segregated to 22 among four meiotic strains (H600B-1,-2, -3, and -4). A total of 132 heterozygous DNA markers were therefore selected from a dikaryon of shiitake (Hokken600H600).Part of this report was presented at the 8th Annual Meeting of Mushroom Science and Biotechnology and the 47th Annual Meeting of the Japan Wood Research Society, Kochi, 1997     

Karyotype analysis of interspecific fusants of basidiomycetes by pulsed-field gel electrophoresis

The purpose of this research was to analyze the karyotype of the interspecific fusants of twoPleurotus species. Auxotrophic mutants derived from the cultivated strain ofP. ostreatus andP. cornucopiae were used. Protoplasts were fused electrically, and the fusants were selected under auxotrophic complementation. Esterase isozyme analysis showed that several fusants had isozyme bands originating from both parental strains, and others had unilateral isozyme bands. The fusant that had expressed isozyme bands of both parental strains showed chromosomal DNA bands of both of the parental strains in pulsedfield gel electrophoresis analysis. Despite the above results, the chromosomal composition of the fusants obtained by the pulsed-field gel electrophoresis did not exhibit all of the bands of both fusion parents.     

Preparation and crossing of mating-capable monokaryons via protoplasting of the dikaryotic mycelia of a mycorrhizal mushroom, <Emphasis Type="Italic">Lyophyllum shimeji</Emphasis>

In order to develop a novel method of obtaining monokaryons for a mycorrhizal fungus, Lyophyllum shimeji, monokaryotization of dikaryotic stock culture via protoplast formation and regeneration was performed using 12 dikaryotic stocks. From 6 dikaryotic stocks, a total of 120 monokaryons were isolated, and their mating compatibility was tested. Mating-compatible monokaryons were successfully derived from a dikaryotic stock (NBRC 100325), and monokaryons of only 1 mating type relative to the parental dikaryons were isolated from another 3 strains (MH01710, OK2L-1, and HY7L-1). We successfully prepared monokaryotic stocks via protoplast monokaryotization, a technique that can be used to identify biological species of L. shimeji. This technique could be used for breeding various mycorrhizal mushrooms, including Tricholoma matsutake, for which the preparation of monospore cultures is extremely difficult. Part of this study was presented at the 11th Annual Meeting of the Japanese Society of Mushroom Science and Biotechnology (Asahikawa), September 2007     

Large electro-fused protoplasts ofPopulus alba selected by a micromanipulator: Techniques and some characteristics of cells and their regenerants

An improved method for selection of large electro-fused protoplasts ofPopulus alba by a micromanipulator was developed. The conditions for electric cell fusion treatment were optimized. For the best result, protoplasts with a cell density of 5 × 10⁵/mL were treated with an alternate current (1 MHz, 200 V/cm) and pulsed with a direct current (2 kV/cm) for 100μs in 2.5 mM CaCl₂ and 0.55 M mannitol. The electo-fused protoplasts were cultured in NH₄NO₃-free Murashige and Skoog’s medium containing 0.6 M of mannitol, 0.09 M sucrose, 1μM of 2,4-dichlorophenoxyacetic acid and 0.1μM of benzyladenine, the same medium used for protoplast culture, but at a very low cell density of 5–10 × 10²/mL in a well of a 96-well culture plate. When cell aggregates derived from individual fused protoplasts were transferred to fresh medium with 0, 0.3 or 0.6 M mannitol, large colonies developed. In the shoot differentiation medium, the reaction of the calluses derived from large fused protoplasts towards the growth regulators differed from the non-fused ones. In medium containing 1μM each of naphthalene acetic acid andN-(2-chloro-4-pyridyl)-N′-phenylurea, growth of callus from electro-fused ones was not reduced by much compared to the control, but shoot differentiation was inhibited. Gibberellic acid (0.1–10μM) was beneficial to shoot regeneration; however, irregularly shaped leaves appeared at high gibberellic acid concentrations. Shoots regenerated were rooted in Murashige and Skoog’s medium containing 4μM of indole-3-butyric acid. Some plantlets obtained had a varied morphology. Based on the characteristics of growth, some cells derived from electro-fused protoplasts appear to be physiologically different from the non-fused one.     

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 .     

Introduction of mitochondrial DNA from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> into <Emphasis Type="Italic">Pleurotus pulmonarius</Emphasis> by interspecific protoplast fusion

Interspecific protoplast fusion between two monokaryotic strains (a methionine auxotrophic and chloramphenicol-resistant Pleurotus ostreatus strain and a wild-type strain of Pleurotus pulmonarius) was carried out to introduce mitochondrial DNA (mtDNA) from P. ostreatus into P. pulmonarius. Because mycelial colonies regenerated on minimum medium containing chloramphenicol only after the treatment of protoplast electrofusion, the regenerants were regarded as protoplast fusants. The fusants isolated from regenerated colonies were uninucleate, and their resistance of chloramphenicol seemed to be a stable trait. The fusants mated with P. pulmonarius but not with P. ostreatus, and showed almost identical random amplified polymorphic DNA (RAPD) patterns to that of the parental P. pulmonarius monokaryon. The sizes of mitochondrial genomes were estimated to be 81.5 kb for P. ostreatus monokaryon, 54.9 kb for P. pulmonarius monokaryon, and 84.5 or 86.0 kb for the fusants. BamHI and EcoRI digest patterns of the fusant mtDNAs were almost the same as the parental P. ostreatus monokaryon. From the above results, the fusants seemed to carry nuclei derived from the monokaryon of P. pulmonarius and mtDNA including the chloramphenicol-resistance gene from the P. ostreatus monokaryon, suggesting that the P. ostreatus mtDNA had been introduced into P. pulmonarius cells by interspecific protoplast fusion. Contribution No. 383 of the Tottori Mycological Institute     

Production of protoplasts from cultures of <Emphasis Type="Italic">Ophiostoma picea</Emphasis>

A method is described for producing viable protoplasts from germinating conidia (germlings) of Ophiostoma picea. The use of MgSO₄-based osmotic stabilizers significantly improved protoplast release, as did the use of 20-h-old germlings. Protoplast release rates also increased with higher enzyme concentration and incubation times, but there were associated negative effects, including reduced regeneration. The results indicate that the use of young germlings, low lytic enzyme levels, and short exposure periods produced the most viable protoplasts.     

Regeneration growth in different light environments of mixed species, multiaged, mountainous forests of Romania

Growth of regenerating trees in different light environments was studied for the mountainous, mixed-species forests in the Carpathian Mountains of Romania. The primary species in these mixtures were silver fir (Abies alba Mill.), European beech (Fagus sylvatica L.) and Norway spruce (Picea abies (L.) Karst). Seedlings/saplings of these species were selected and measured in different stands from two different geographical locations. Regenerating trees were measured for height and diameter growth during the summer of 2002. For each seedling/sapling, percentage of above canopy light (PACL) and stand basal area (BA) were used to assess available and occupied growing space respectively. Regeneration growth was compared against these two variables and regression relationships were developed. Using these models, we predicted the dynamics of regeneration as both growth and species composition. Our results showed that in low-light environments (PACL<20–35%; BA>30 m²/ha), shade tolerant fir and beech clearly outcompeted the spruce. Therefore, in dense stands, spruce could be eliminated by the shade tolerant species. For intermediate levels of cover (PACL=35–70%; BA=15–35 m²/ha) the spruce grew at comparable rates as the beech and fir. All three species showed similar growth rates in open conditions (PACL>80–90%; BA<15–20 m²/ha) with the spruce having a tendency to outgrow the others. However, in terms of establishment, such conditions favor spruce and inhibit fir and beech.     

Preliminary studies on the early quality identification ofAuricularia auricular

In production industry ofAuricularia auricular, the varieties quality is most important impact factor on output. For evaluating the early quality of the edible fungus, 9 varieties ofAuricularia auricular (Au9, CF09, CF05, 29, 916, chang10, chang7, Au.Japanese and 8808) were cultured on the medium consists of potato dextrose agar (PDA) and sawdust to test their mycelium growth rate, endurance to high temperature, resistance to mildew, assimilation of nutriment, and resistance to drought. The result showed that the mycelium of Chang 10, CF09, 29 and Au.Japanese varieties had the eminent characteristics such as short lifespan, stronger assimilation of nutriment, and endurance to high temperature and steady growth. These four varieties are determined as superiority varieties ofAuricularia auricular in accordance with the research results. Foundation item: The research was supported by Science Fund of Northeast Forestry University (2004). Biography: Li Ling (1964-), female, Engineer in Northeast Forestry University, Harbin 150040, P. R. China. Responsible editor: Zhu Hong     

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.     

Transmission of mitochondrial plasmids in protoplast cell fusion between compatible monokaryons of <Emphasis Type="Italic">Lentinula edodes</Emphasis>

Electrophoretic analysis of the transmission pattern of mitochondrial plasmids in protoplast cell fusion between compatible monokaryons of Lentinula edodes indicates that three of the four plasmids carried in parental monokaryons are effectively transferred and replicated in the protoplast fusants. The two monokaryons, 1158a and 1569a, carried different plasmids that could be distinguished by a single restriction digest. Electrophoresis of intact plasmids and restriction analyses indicate that all but one of the fusants carry three of the four possible plasmids, indicating that transmission of plasmids in protoplast fusions is principally biparental in L. edodes. Thus, heterocytoplasmic cells of L. edodes can be effectively constructed by protoplast cell fusion. In addition, plasmids of the same homology group cannot coexist in the heteroplasmic cells after protoplast cell fusion. Contribution No. 382 of the Tottori Mycological Institute     

Antimicrobial activity of some medicinal plants from India

The results of a preliminary antimicrobial screening of the methanol extracts of Zingiber officinale, Asteracantha longifolia, Citrus acida, Salacia microsperma and Tinospora cordifolia are reported.     

我国樟子松人工林天然更新研究进展

樟子松人工林天然更新问题的研究一直在进行,但人工林内幼苗数量过少及难以发育为幼树的问题一直难以彻底解决。文中综述了影响我国沙地樟子松人工林天然更新能力的因素,如光照、温度、水分、土壤环境、生物因子及人工营林技术等的研究概况。在研究过程中发现,樟子松人工林的遗传多样性与土壤微生物对樟子松人工林天然更新影响方面的研究尚少有报道,或可以为解决樟子松人工林天然更新问题提供更深入的思路。     

蒙古沙冬青子叶节诱导培养再生植株的研究

[目的]建立蒙古沙冬青子叶节丛生芽再生体系。[方法]以蒙古沙冬青子叶节为外植体,研究种子萌发培养基中6-BA浓度、子叶节萌发天数、丛生芽诱导的基本培养基及6-BA浓度对丛生芽诱导的影响和外源激素对丛生芽伸长及生根的影响。[结果]表明:(1)添加6-BA的萌发培养基与不添加的相比能够显著促进子叶节的生长及子叶节丛生芽的诱导,且6-BA浓度在2.0 mg·L-1时,诱导率最高可达73.3%,平均芽数2.26个;(2)种子萌发天数对子叶节丛生芽的诱导有显著影响,萌发7 d的子叶节丛生芽诱导率最高,诱导率为74.7%,但与萌发9、11 d的子叶节丛生芽诱导率差异不显著;(3)MS和B5培养基对丛生芽诱导的影响差异不显著,但B5抑制褐化的效果显著好于MS;(4)采用1.0 mg·L-16-BA+0.3 mg·L-1IAA激素组合有利于丛生芽伸长,伸长率为60.5%;(5)不同浓度生长素组合均能促进丛生芽幼苗生根,1.0 mg·L-1IBA生根率最大,生根数最多。[结论]最佳蒙古沙冬青子叶节丛生芽再生体系为:以在MS+2.0 mg·L-16-BA培养基中黑暗培养7 11 d的幼苗子叶节为外植体,在B5+1.0mg·L-16-BA培养基中诱导丛生芽,待丛生芽长约0.3 0.5 cm后,转入B5+1.0 mg·L-16-BA+0.3 mg·L-1IAA培养基中进行伸长培养,当丛生芽伸长至2 3 cm时,单株切下转入1/2 B5+1.0 mg·L-1IBA培养基中生根,该体系与愈伤组织培养相比能缩短培养时间,改善褐化严重、玻璃化等问题,可为蒙古沙冬青的扩繁及进一步开展遗传转化奠定基础。     

A new norlignan from Taxodium ascendens

A new norlignan, (2R,3R,4S,5S)-2,4-bis(4-hydroxyphenyl)-3,5-dihydroxy-tetrahydropyran (1), together with 9 known compounds were isolated from the branches and leaves of Taxodium ascendens. Their structures were mainly determined on the basis of MS, IR, 1D and 2D NMR spectral evidences. Methanol extract showed inhibitory activity on carbonic anhydrase II with an IC₅₀ value of 4.27 µg/ml, acetone extract and methanol extract inhibited activity of cathepsin B with IC₅₀ values of 2.12 and 3.71 µg/ml, respectively.     

Effect of Regeneration Method on RAPD-Based Genetic Variation of Cyclobalanopsis glauca (Fagaceae)

Cyclobalanopsis glauca is a dominant species of evergreen broad-leaved forests in mainland China. This study compares the genetic variation of an artificially regenerated population with its donor population and two other wild populations, by using RAPD markers. A total of 74 clear, reproducible bands were scored for 12 RAPD primers; 72 were polymorphic (P = 97.3%). AMOVA revealed that most genetic variation was within populations and only 10.35% was among populations. Various measures indicated that there is no difference in genetic diversity between the planted and the original populations. Φ_ST between the planted offspring population and the donor population was larger than those between the planted and other two natural populations, indicating that artificial regeneration might lead to biased genetic composition, given that temporal differentiation is usually lower than spatial differentiation. This divergence may be due to unequal seed production among the maternal individuals and viability differences among seeds.     

Cultivation of <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> on umbrella plant (<Emphasis Type="Italic">Cyperus alternifolius</Emphasis>) substrate

Mycelial growth and mushroom yields of three strains of Pleurotus eryngii produced on wheat bran-supplemented umbrella plant (Cyperus alternifolius) substrate were assessed using surface brightness, bromophenol blue color reactions, ergosterol and glucosamine contents, and water potential as indicators of strain performance. Mycelial growth was 31%–46% greater, depending on strain, on the umbrella plant substrate compared with the mushroom industry standard sugi (Cryptomeria japonica) substrate. Mushroom yields on the first flush were 20%–23% higher, depending on strain, on the plastic bottle-contained umbrella plant substrate. However, yields on the second break were lower from the umbrella plant substrate. Because many growers in Japan only harvest one flush, production of P. eryngii on umbrella plant substrate may offer commercial producers an alternative basal ingredient to diminishing supplies of sugi sawdust.Part of this report was presented at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, April 2002.     

Changes in carbohydrase activities during vegetative growth and development of fruit-bodies of Hypsizygus marmoreus grown in sawdust-based culture

Carbohydraseproduction of Hypsizygus marmoreus cultured on sawdust rice bran medium by bottle cultivation was investigated to elucidate the carbohydrase utilized as the growth substrate for the fruit-body formation of this fungus. Among the extracellular enzymes assayed, xylanase showed the highest activity. This activity greatly increased during the end of vegetative mycelial growth and fruit-body formation. Among the cellulases. CM-cellulase showed higher activity than avicelase. The activities of -1,3-glucanase, amylase, and chitinase were low. Among the intracellular enzymes, both xylanase and amylase showed higher activity levels than the other carbohydrases. In contrast, -1,3-glucanase, avicelase, and chitinase activities in mycelia were considerably lower. These results suggest that xylanase, CM-cellulase, and amylase play an important role in mycelial maturation and fruit-body growth of H. marmoreus.