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     

Phylogenetic analysis of oyster mushrooms (<Emphasis Type="Italic">Pleurotus</Emphasis> spp.) based on restriction fragment length polymorphisms of the 5′ portion of 26S rDNA

Polymorphism analysis of the 5 portion of 26S rDNA from 34 Pleurotus strains (12 intersterility groups) collected mainly from Asia was performed. By combining the restriction fragment length polymorphism (RFLP) patterns obtained from digestions with seven restriction enzymes, the 34 Pleurotus strains were assigned to 11 RFLP types. Ten RFLP patterns corresponded with biological species, but one pattern was found in intersterility groups I (P. ostreatus complex), II (P. pulmonarius complex), and VIII (P. eryngii). The phylogenetic tree suggests that Pleurotus species have evolved in two patterns based on 26S rDNA RFLP data. One major cluster comprising the P. ostreatus clade is separated by relatively short branches, suggesting that the P. ostreatus complex, the P. pulmonarius complex, P. eryngii, and P. nebrodensis (intersterility groups I, II, VIII, and IX, respectively), share a recent common ancestor. RFLP data did not distinguish the species in the intersterility groups I, II, and VIII. The other major cluster apparently divided into five sublevel clusters in early stages of evolution and these clades split into terminal nodes in late stages of evolution: the P. calyptratus-salmoneostramineus clade, the P. cornucopiae-ulmarius clade, the P. dryinus clade, the P. corticatus clade, and the P. cystidiosus-smithii clade.     

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.     

The biological species of oyster mushrooms (<Emphasis Type="Italic">Pleurotus</Emphasis> spp.) from Asia based on mating compatibility tests

Mating compatibility of 25 Pleurotus species collected mainly from Asia was tested by either the Mon–Mon mating or the Di–Mon mating tests. The results showed 5 intersterility groups (P. ostreatus, P. pulmonarius, P. cornucopiae, P. cystidiosus, and P. salmoneostramineus complexes) and 7 independent interincompatible species (P. calyptratus, P. corticatus, P. dryinus, P. eryngii, P. nebrodensis, P. smithii, and P. ulmarius). The P. ostreatus complex includes P. ostreatus, P. ostreatus var. columbinus, P. djamor, and P. flabellatus. The P. pulmonarius complex has 7 taxa: P. pulmonarius, P. eugrammus, P. eugrammus var. brevisporus, P. sajor-caju, P. sapidus, P. sp. florida, and P. opuntiae. The P. cornucopiae complex includes its variant P. cornucopiae var. citrinopileatus. The P. cystidiosus complex includes P. abalonus. The P. salmoneostramineus complex includes 3 pink-colored mushrooms: P. salmoneostramineus, P. ostreatoroseus, and P. rhodophyllus. According to mating compatibility tests, 12 biological species were identified from among 25 Pleurotus species.     

Isolation and sequence analysis of the promoter and an allelic sequence of the iron-sulfur protein subunit gene from the white-rot fungusPleurotus ostreatus

We have isolated a structural gene ofsdil, which encodes the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (EC 1.3.99.1), from a white-rot basidiomycete,Pleurotus ostreatus. Here we report isolation of the promoter region ofsdil and an allelic sequence encoding the second-type cDNA fragment isolated in the former experiments. The nucleotide sequence analysis of the promoter region revealed the existence of putative CAAT and TATA boxes, which permits us to develop an expression system in this species. The Southern blot analysis and the restriction fragment length polymorphism assay using monokaryotic strains demonstrated that no family genes tosdil exist in the haploid genome ofP. ostreatus. Moreover, a genetic analysis to detect a linkage between thesdil genotypes and flutolanil resistance in the mutantP. ostreatus strains was also developed.     

A comparison between decay patterns of the white‐rot fungus Pleurotus ostreatus in chestnut–leaved oak (Quercus castaneifolia) shows predominantly simultaneous attack both in vivo and in vitro

In this research, we examined decay patterns occurring in Quercus castaneifolia wood under natural conditions compared with controlled decay in vivo. Pleurotus ostreatus‐infected oak wood was obtained from the Sari forests in the north of Iran. The species causing decay was verified as P. ostreatus using rDNA‐ITS sequencing of pure cultures from infected sapwood. In addition to P. ostreatus, two wood‐inhabiting Ascomycota, Trichoderma harzianum and T. lixii, were present. Mass loss in oak sapwood samples exposed to P. ostreatus for 60 days was around 10 per cent. Samples were prepared from both naturally decayed wood and wood decayed under controlled conditions and examined using microscopy. P. ostreatus was found to produce a simultaneous white‐rot decay pattern in both conditions.     

Influence of wood species on the sawdust-based cultivation ofPleurotus abalonus andPleurotus eryngii

Mycelial growth and fruit body formation ofPleurotus abalonus andP. eryngii cultured on various sawdust-based substrates from different wood species were investigated. Growth onCryptomeria japonica substrate resulted in good mycelial growth and a high yield of fruit bodies.Larix kaempferi substrate was unsuitable for the cultivation of these mushrooms. The fruit body formation rate correlated with mycelial growth from all the wood species tested. Although differences were found for mycelial growth and fruit body formation on various wood species, there were no wood species that were completely unsuitable exceptL. kaempferi. These results show that a wide range of wood species can be used for the cultivation ofP. abalonus andP. eryngii.     

Reutilization of culture wastes ofPleurotus ostreatus andPhollota nameko for cultivation ofLyophyllum decastes

Possible reutilization of fresh and aged culture wastes of mushrooms for cultivatingLyophyllum decastes was investigated, although bark compost has commonly been used as a substrate for cultivating this fungus. The culture wastes studied were obtained after harvestingPleurotus ostreatus andPholiota nameko mushrooms. Mycelia ofL. decastes grew in the media containing both the fresh culture waste ofP. nameko and bark compost. However, it did not grow in the medium containing only the fresh culture waste ofP. nameko or in any media containing the fresh culture waste ofP. ostreatus. The mycelial growth inhibition in the fresh culture wastes ofP. ostreatus might be caused by the water-soluble inhibitors present. Mycelia ofL. decastes grew in all the media with aged culture wastes of bothP. ostreatus andP. nameko, which had been left outdoors for 6 months, regardless of whether bark compost was mixed. Fruit bodies were produced on all the tested media with aged culture wastes of both mushrooms, which had been left outdoors for a year. The aged culture waste ofP. nameko gave greater yields than the bark compost. This investigation shows that the aged culture wastes ofP. ostreatus andP. nameko could be reutilized for producingL. decastes mushrooms.This study was presented in part at the 47th annual meeting of Japan Wood Research Society, Kochi, April 1997     

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     

Isolation of cDNA and genomic fragments encoding the major manganese peroxidase isozyme from the white rot basidiomycetePleurotus ostreatus

We have isolated the cDNA and genomic sequences encoding the major isozyme of manganese peroxidase, MnP3, from the white rot basidiomycetePleurotus ostreatus strain IS1. The genemnp3 is interrupted by 10 introns and encodes a mature protein of 357 amino acid residues with a 26-amino-acid signal peptide. The amino acid residues known to be involved in peroxidase function and those that form the Mn-binding site in thePanerochaete chrysosporium MnP isozyme are conserved in MnP3. Comparison of the deduced primary structure of MnP3 with those of other peroxidases from various white rot fungi suggested that MnPs fromP. ostreatus andTrametes versicolor belong to a subgroup that is more similar to the lignin peroxidases than MnPs fromP. chrysosporium orCeriporiopsis subvermispora.     

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.     

Individual variation in low boiling point monoterpenes emitted from hinoki (Chamaecyparis obtusa) needles

Variations in the compositions of low-boilingpoint (LBP) monoterpenes in needle samples of 50 hinoki (Chamaecyparis obtusa) trees were investigated using the headspace technique. Considerable compositional variations were revealed, especially in sabinene composition. The sabinene composition varied from 4.9% to 78.0% of the total LBP monoterpenes.-Pinene, myrcene, and limonene also showed considerable variations (9.0%–32.7%, 5.5%–22.6%, 3.6%–29.0% respectively). Analysis of the monoterpene composition allowed definition of four chemotypes based on the contingency table test. No correlation was observed between tree size and LBP monoterpene composition, indicating that the compositional variation in LBP monoterpene exists genetically in this population of hinoki.Part of this paper was presented at the 11th annual meeting of the Chugoku Shikoku branch of the Japan Wood Research Society, Matsue, September 1999     

Promotive effect of the hot water-soluble fraction from corn fiber on vegetative mycelial growth in edible mushrooms

Corn fiber (CNF) is an abundant by-product of the wet corn milling process used to produce corn starch. In light of the need to recycle organic wastes, the effects of adding a hot water-soluble fraction (HWSF) from CNF to a medium on the vegetative mycelial growth of nine edible mushrooms such as Lentinula edodes and Pholiota nameko were investigated. The results showed that the mycelial growth of these fungi was markedly increased (1.4–9.5 times that of the control) by adding 5%–20% CNF-HWSF to the medium. These promotive effects were also apparent on mycorrhizal mushrooms, such as Tricholoma matsutake (3.3-fold) and Lyophyllium shimeji (3.7-fold). The promotive effects on mycelial growth were shown in the low-molecular-weight fractions (molecular weight <500 daltons) prepared from CNF-HWSF. The promotive actions were more effective on slow-growing mushrooms (L. edodes and P. nameko) than on rapidly growing mushrooms (Pleurotus ostreatus and Flammulina velutipes). The results obtained in this experiment suggest that CNF-HWSF can be used as a promotive substance for cultivating edible mushrooms.Part of this work was presented at the 4th meeting of the Japanese Society of Mushroom Science and Biotechnology, Hiroshima, 2000; and at the 2nd meeting of the Far East Asia for Collaboration on Edible Fungi Research, Tottori, 2002     

Provenance variation in growth characters of four subtropical pine species planted in Mexico

In 1995 a species/provenance test was established in the state of Puebla, Mexico to assess and compare the growth of Pinus greggii var. australis, P. maximinoi, P. patula and P. tecunumanii, including 10, 5, 11 and 4 provenances of each species, respectively. Each provenance test lot consisted of seed from 7 to 10 individual trees collected in natural stands. Range-wide samples were included in the test for P. greggii var. australis and P. patula. Material of P. maximinoi and P. tecunumanii from Guatemala, Honduras and Nicaragua, as well as Mexico was established in the test. The test site is in a mountainous area at 1440m elevation, near 20°N latitude. At 5 years of age significant differences were seen at both the species and provenance levels in survival, total height, diameter and volume. The overall test survival was 93%. Variance among species was three time greater than among provenances, comprising 25–33% of the total variation. P. maximinoi had the greatest height, diameter and volume (62dm³) followed by P. tecunumanii (53dm³). P. greggii var. australis had an average volume of 43dm³ and P. patula produced 30dm³. Although P. maximinoi had the lowest survival rate, the San Jeronimo provenance was the most productive of all 30 provenances tested; 98% survival and 73dm³ volume. This was equivalent to 2.8cm annual diameter increment and 2m in height growth per year. Significant growth differences among provenances within species indicate that potential gain exists using provenance selection.     

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.     

Study on biodegradated ability of thirteen edible fungi to straw

The biodegradated abilities of 13 edible fungi to straw were studied. The results showed that all the experimental fungi except Tricholama mongolicum had definite biodegradated abilities to the lignin and cellulose of straw. The Ideal fungus for straw degradation was screened out as Pleurotus ostreatus, which showed a higher degradation ability for lignin (17.86%) and lower degradation rate for cellulose (2.24%), with a Selection Factor (SF) of 7.97. The degradation rates of lignin and cellulose for other fungi ranged from 2.30% to 16.54% and 5.60% to 17.32%, respectively, and the SF was very low in range of 0.14 to 2.24.The ratio of colony‘s diameters to the color-zone (d1/d2) and SF are negative correlation, with a correlation coefficient of -0.1476.     

Chemical constituents from the rice fermented with the edible mushroom Pleurotus eryngii and their quinone oxidoreductase 1 inducing effect

The fruiting bodies or mycelia of mushrooms have been used as food and food-flavoring material for centuries due to their nutritional and medicinal values and the diversity of their bioactive components. The present research was the first to study the chemical components in rice fermented with the edible mushroom Pleurotus eryngii and the quinone oxidoreductase 1 inducing effect of these compounds. Through chemical investigation, one new compound, ((6S,7S)-6,7-dihydroxy-6-methyl-2-(3-methylbutanoyl)-4,5,6,7-tetrahydrobenzofuran-3-yl)methyl acetate (1) and eight known compounds (2–9) were isolated from the P. eryngii-fermented rice. All of these compounds were isolated from rice fermented with the edible mushroom P. eryngii for the first time. Their structures were elucidated by MS and NMR data analyses. Alternariol-5-O-methyl ether (2) showed strong quinone oxidoreductase 1 inducing effect with an IR value of 2.58 at the concentration of 20 μg/ml. The content of adenosine (8) in the fermented rice (175.64 μg/g) is much higher than that of non-fermented rice (14.38 μg/g).     

Prevalence and incidence of the root-inhabiting fungi, Fusarium, Cylindrocarpon and Pythium, on container-grown Douglas-fir and spruce seedlings in British Columbia

Surveys were made at the end of the 1990 and 1991 growing seasons for root-inhabiting fungi in the genera Fusarium, Cylindrocarpon and Pythium from the roots of one year-old container-grown Douglas-fir and spruce seedlings grown under greenhouse conditions. In the 1990 survey of four nurseries, it was found that 61–97% of both Douglas-fir and spruce roots were colonized with Fusarium, Cylindrocarpon or Pythium. There were significantly (p0.05) more Douglas-fir roots than spruce roots colonized by Fusarium at all nurseries, however, there were significantly (p0.05) more spruce roots than Douglas-fir roots colonized by Cylindrocarpon and Pythium. Root colonization of Douglas-fir and spruce by the three fungal genera during 1991 varied from 0–82% at three nurseries, however, only at a south coastal nursery was there significantly (p0.05) more spruce than Douglas-fir roots colonized by Cylindrocarpon. Significantly more seedlings were infected in 1990 than in 1991. In 1991, there were few significant differences between Douglas-fir and spruce, in the percentage of seedlings with colonized roots and in the percentage of growth medium colonized by the fungi. However, there were significant differences between nurseries.     

Leaf quality of selected hedgerow species at two canopy ages in the derived savanna zone of West Africa

Prunings in hedgerow intercropping systems are a potential source of organic matter and their quality is an important characteristic driving decomposition and nutrient release. To determine the potential range of residue characteristics and the impact of canopy age on those, selected characteristics of Leucaena leucocephala, Gliricidia sepium, and Senna siamea hedgerow leaf residues were determined for young (5–10 weeks) and old (39–47 weeks) hedgerow canopies in six sites, representative in terms of soil for the derived savanna zone of West-Africa. The N content of the Leucaena and Senna residues decreased with age (from 4.8 to 3.9% and from 4.1 to 3.0%, respectively, while the N content of the Gliricidia residues remained constant (3.9%). The P content of all species decreased with age (from 0.30 to 0.20%, on average). The lignin content of the Leucaena residues increased, and their polyphenol content decreased with age, while for the Senna residues only an increase in polyphenol content with canopy age was evident. Neither the lignin nor the polyphenol content of the Gliricidia leaf residues was markedly affected by canopy age. For all species, the P and ADF content were positively correlated with the ash and lignin content, respectively. The N content of the Leucaena and Senna residues was positively correlated with their P content while the lignin content of the same species was negatively correlated with the polyphenol content. Because of the low variation in certain residue characteristics between sites, the presented data and equations could potentially be used to reasonably estimate those characteristics if no other information is available. For other characteristics, such as polyphenol contents, measurements are preferable in view of the relatively high variation encountered.This revised version was published online in November 2005 with corrections to the Cover Date.     

Litterfall,litter decomposition and the use of mulch of four indigenous tree species in the Atlantic lowlands of Costa Rica

Litterfall, forest-floor litter biomass and nutrients, short-term litter decomposition and the effects of leaf mulches on initial growth of maize were studied for four indigenous tree species with agroforestry potential:Stryphnodendron microstachyum Poepp. et Endl.(S. excelsum), Vochysia ferruginea Mart,Vochysia guatemalensis Donn. Sm. (V. hondurensis) andHyeronima alchorneoides (O), growing in a young experimental plantation in the Atlantic humid lowlands of Costa Rica. Total annual leaf litterfall was higher inV. ferruginea plots, followed byS. microstachyum, V. guatemalensis andH. alchorneoides; all with values comparable to those reported for other tree species grown in agroforestry combinations in humid tropical regions. Forest-floor litter accumulation was highest underV. ferruginea andV. guatemalensis. Both litterfall and forest-floor litter material had similar patterns in nutrient concentrations: N was higher inS. microstachyum, Ca was higher inV. guatemalensis, K was higher inH. alchorneoides; Mg was higher inV. guatemalensis andH. alchorneoides; H. alchorneoides andV. guatemalensis had the highest P.V. ferruginea litter decomposed more slowly, whileS. microstachyum apparently decomposed faster than the other species. The twoVochysia species showed increases in N and P concentration in decomposing litter after seven weeks in the field,H. alchorneoides showed an increase in litter N and a decrease in litter P, andS. microstachyum showed a net decrease in both N and P over the same time period. The patterns found in the litter bag study were confirmed by results obtained in a tethered-leaves experiment.S. microstachyum andV. ferruginea litters lost more weight when mixed in a 11 proportion than either of them alone. Maize seedlings growing in plots mulched withS. microstachyum andH. alchorneoides leaves showed greatest initial growth, confirming patterns found in decomposition and nutrient release studies. The results show that these species could be used in agroforestry combinations with different advantages according to the specific objectives desired, whether these are soil protection, nutrient recycling, or enhancement of the growth of associated crops.