Cloning and identification of partial DNA fragment for the <Emphasis Type="Italic">B</Emphasis> mating-type factor in <Emphasis Type="Italic">Lentinula edodes</Emphasis> using degenerate PCR

The edible fungus Lentinula edodes is a heterothallic homobasidiomycete whose mating is controlled by a bifactorial incompatibility mating system determined by two unlinked factors (the A and B mating-type factors). Although this mechanism is well accepted, there is a lack of understanding about its molecular basis, as the incompatibility factors have not been cloned and sequenced. In this study, by means of degenerate PCR we obtained one 773 bp DNA fragment cosegregating with B ₂ mating-type factor in L. edodes stock HL01. Sequencing analysis revealed that it belonged to a pheromone receptor, suggesting that the genetic basis for B factor in L. edodes is the same as in the two model mushroom species, Schizophyllum commune and Coprinus cinereus, the structure and function of whose B incompatibility factors have been studied in detail. So far as we know, this is the first report about the cloning of B mating factor in L. edodes.     

Occurrence ofPseudomonas tolaasii on fruiting bodies ofLentinula edodes formed onQuercus logs

A bacterial disease occurred on fruiting bodies ofLentinula edodes that formed outdoors onQuercus bedlogs during winter. The pathogen was identified asPseudomonas tolaasii based on morphological and bacteriological characteristics. Symptoms exhibited by infected fruiting bodies ranged from mild browning to severe necrotic cavities that characteristically developed in the cap tissue along the periphery of the attachment area to the stalk. The mode of symptom development was greatly influenced by the internal tissue structure of fruiting bodies. Multiplication of bacterial cells within the fruiting bodies was strictly intercellular and thus differed from previously reported bacterial disease ofL. edodes incited by an unidentified rod-shaped bacterium. The present strain ofP. tolaasii was capable of attacking theL. edodes mycelium in the inner bark and outer sapwood regions and caused lysis of heavily infected hyphae.Paper No. 301 of the Tottori Mycological Institute.     

A new biological function of Shiitake mushroom,Lentinula edodes, in a myco-heterotrophic orchid,Erythrorchis ochobiensis

The biological function ofLentinula edodes in a myco-heterotrophic orchid,Erythrorchis ochobiensis was examined, using one local variant each from Japan (JPN), Papua New Guinea (PNG) and New Zealand (NZ). All variants induced seed germination: PNG and NZ isolates were effective at 25°C and JPN isolate showed the highest germination rate at 30°C. Germinated seeds developed into plants and formed normal endomycorrhizas. Hence, it is concluded thatL. edodes has a perfect symbiotic potential withE. ochobiensis, though it has not been observed in the root of the orchid in the field.     

香菇复配氨基酸培养基优化及其对香菇菌丝生长生理的影响

[背景]L-氨基酸能够提供香菇生长发育的部分养分需求,利用外源添加氨基酸的方式对香菇生长基质进行优化,对于香菇产业的增产提质具有积极的发展意义.[目的]利用外源添加丙氨酸(Ala)、丝氨酸(Ser)和天冬酰胺(Asn)的方式改善培养基成分,并探究复配氨基酸促进香菇生长的可能代谢途径.[方法]在三因素三水平上,通过响应面...     

Estimation of viability of inner bark tissue of <Emphasis Type="Italic">Quercus serrata</Emphasis>, a substrate for log cultivation of <Emphasis Type="Italic">Lentinula edodes</Emphasis>, using the TTC assay method

In the log cultivation of Shiitake (Lentinula edodes), early colonization of this fungus is extremely retarded in living wood tissues, in particular in inner bark tissues. To estimate the viability of inner bark tissues of Quercus serrata, a substrate for log cultivation of Shiitake, we employed a colorimetric assay utilizing a tetrazolium salt (2,3,5-triphenyltetrazolium chloride, TTC) and investigated the relationships between degree of decrease in viability and increase in growth of L. edodes in the tissues. When the mixtures of different proportions of living and dead tissues were assayed, formazan production was proportional to the percentage of living tissues. When logs dried for various time periods were inoculated with L. edodes, the fungus grew more extensively in tissues with reduced formazan production. These results indicate that the TTC assay is a useful method for estimation of viability and thus can be used to decide the proper timing for inoculation of L. edodes.Contribution no. 372 from the Tottori Mycological Institute     

Effect of lignin-derived phenolic monomers on the growth of the edible mushrooms Lentinus edodes,Pleurotus sajor-caju and Volvariella volvacea

Pleurotus sajor-caju was generally more tolerant to lignin-related phenolic monomers and tannin derivatives than Lentinus edodes and the straw mushroom, Volvariella volvacea. Several phenols, at up to 5 mM, enhanced mycelial growth of P. sajor-caju. No clear pattern was evident when the effects of phenols and tannins on the growth of V. volvacea and L. edodes were compared, but the lower concentrations of 4-hydroxybenzaldehyde and vanillin which were tested were markedly more toxic to the straw mushroom. The distribution of phenolic monomers and tannin derivatives in the agricultural wastes used for mushroom cultivation may be an important growth determinant. However, the differences in the growth inhibition profiles of L. edodes, P. sajor-caju and V. volvacea suggest that, alone, the effect of these compounds on fungal growth is unlikely to account for the varying abilities of the three mushroom species to grow and fruit on a particular lignocellulosic substrate.     

Induction of apoptosis in cultured cells by extracts from shiitake (Lentinula edodes) mycelial culture broth

Extracts fromshiitake (Lentinula edodes) mycelial culture broth, by an organic solvent ethyl acetate, inhibited the proliferation of cultured cells. At lower concentrations (1.25–15 μg/ml), this inhibition, measured by the MTT assay, was dose- and cell line-dependent. Inhibition of tumor cells, such as Caski, SiHa, HeLa, HP-1 and A375, byL. edodes-436 extracts was stronger than inhibition of normal cells (3T3). At 20 μg/ml, the extracts induced changes in cell shape, DNA-fragmentation and the activation of caspase-3. The extracts also inhibited the binding of E2F protein to its promoter. The results suggest that extracts ofL. edodes culture broth contain substances that have the ability to induce apoptosis in the cultured cells.     

Parasitism ofLygus lineolaris eggs onCoronilla varia,solanum tuberosum,and three host weeds in southeastern Québec

Parasitism ofLygus lineolaris (Palisot de Beauvois) eggs by three species ofMymaridae, Anaphes iole Girault,Erythmelus miridiphagus Dozier andPolynema pratensiphagum Walley and one species ofScelionidae, Telenomus sp., was investigated at Ste-Anne-de-Bellevue, Québec. The maximum level of field parasitism ofL. lineolaris eggs by individual species was 15.4, 53.8, 70.0 and 16,7%, respectively. Parasitoids were retrieved from eggs ofL. lineolaris inserted in stems and branches ofAmaranthus retroflexus L.,Chenopodium album L.,Coronilla varia L.,Rumex obtusifolius L. andSolanum tuberosum L. Amaranthus retroflexus andS. tuberosum sustained a large population ofL. lineolaris and egg parasitism was high enough to consider it as a potential control factor.      

Hemagglutinating activity of the fungusLentinus edodes (Berk.) sing [Lentinus edodes (Berk.) Pegler]

The hemagglutinating (HA) activity of the submerged mycelium and the culture liquid (CL) of four strains ofLentinus edodes was studied. The HA activity of the CLs proved to be much higher than that of mycelia. The carbohydrate specificity of fungal agglutinating factors was determined. HA activity was investigated as a function of the inoculum size, cultivation temperature, and culture age. The agglutinating activity of different morphogenetic structures ofL. edodes F-249, including mycelium, brown mycelial mat (MM), primordia, and fruiting bodies, was studied. MM was found to possess the maximum HA activity, which can be explained by the possible involvement of agglutinins in the formation of MM, which is composed of glued hyphae.     

Efficient transformation of the medicinal mushroomGanoderma lucidum

Ganoderma lucidum is a well-known and important medicinal mushroom, but its genetic modification has not been reported. We developed an efficient procedure for isolation and regeneration of protoplasts fromG. lucidum. To construct a vector for high-level expression of heterologous genes inG. lucidum, the 1.4-kb regulatory region of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD) was isolated from the genomic DNA ofLentinus edodes, and theGPD promoter was fused to the β-glucuronidase (GUS) and bialaphos resistance (bar) genes. Using the resulting construct, p301-bG1, an efficient transformation system based on electroporation was established forG. lucidum. GUS expression was observed among transformants conferring bialaphos resistance, indicating that theL. edodes GPD promoter can be used for expression of exogenous genes inG. lucidum. We also studied green fluorescent protein (GFP) as another reporter for transformation ofG. lucidum. TheL. edodes GPD promoter was fused respectively to theGFP andbar genes, and the resulting construct, p301-bg, was introduced intoG. lucidum. StableGFP expression in transformants was detectable by fluorescence microscopy, suggesting the suitability ofGFP as a reporter system in transformation of this mushroom. This is the first report of an efficient transformation system forG. lucidum using different reporters, paving the way for genetic modification of this famous medicinal mushroom.     

Host habitat finding and host selection of theDrosophila parasitoidLeptopilina australis (Hymenoptera,Eucoilidae), with a comparison of the niches of EuropeanLeptopilina species

Summary Decaying petioles of giant hogweed,Heracleum mantegazzianum Sommier & Levier, are used as a breeding site by six species ofDrosophila and the drosophilidScaptomyza pallida. The most numerous parasitoid species associated with this community isLeptopilina australis. BecauseL. australis was previously unknown in western Europe, we present the characters to distinguish it form its close relativeL. clavipes. Experiments on host species selection and survival ofL. australis showed that this parasitoid mainly usesD. limbata as host. Olfactometer experiments showed thatL. australis is attracted by the odour of decaying hogweed stalks, especially when these contain larvae ofD. limbata. L. australis is also strongly attracted by the odour of stinkhorns, a habitat in which it has never been found in nature.D. phalerata is the dominant fly species in stinkhorns, and is not a host ofL. australis. We offer a possible functional explanation for this unexpected habitat choice, by showing thatD. transversa andD. kuntzei, both species found to breed in fungi, are also suitable hosts forL. australis. We also discuss habitat choice with regard to a proposed phylogeny of theLeptopilina species in temperate Europe. Finally, we discuss niche overlap ofL. australis with the otherLeptopilina species.     

A new alternative process for Kraft E1 effluent treatment

Lentinus edodes (UEC-2019 strain) was selected after screening 51 ligninolytic strains of fungi for their ability to decolorize phenolic industrial effluent with high content of lignin peroxidases, Mn-peroxidases and beta-glucosidases. This strain removed 73 % of color in theEucalyptus Kraft E1 effluent in 5 days without any additional carbon sources. A 13% mycelial adsorption was found. Correlation between mass loss, COD, TOC and decolorization was observed. When an effluent pre-irradiated (10 min) in the presence of ZnO was treated withL. edodes, a marked enhancement of the decolorization at 48 h was obtained.L. edodes is an active fungus in this pre-treatment and biobleaching process. The combined photo-biological decolorization procedure appears to be an efficient decontamination method with great potential in industrial effluent treatment.Abbreviation COD Chemical oxygen demand - TOC Total organic carbon     

Chromosome banding patterns in Lettuce species (Lactuca sect.Lactuca,Compositae)

Chromosome banding patterns obtained with C- and N- banding, and AgNO₃ staining were studied in somatic metaphase complements of fourLactuca species.L. sativa andL. serriola have almost identical chromosome morphology, andL. saligna differs only slightly from them, butL. virosa is quite distinct from the other species. A gross comparison of the banded karyotypes suggests a closer relationship ofL. saligna toL. sativa/serriola than toL. virosa. Our data agree with the results of previous crossing experiments in these species but conflict partly with recent RFLP data which indicate a closer phenetic relationship ofL. saligna toL. virosa than toL. sativa/serriola. Such a discrepancy may be explained assuming that domestication ofL. sativa/serriola resulted in an increased selection pressure on unique DNA sequences as demonstrated by the RFLP data. Differential evolution of specific heterochromatin classes (and presumably of highly repetitive DNA classes), as revealed by chromosome banding techniques was not linked to domestication. Thus the disparity in conclusions about relationship (in terms of genetic similarity) as based on the different experimental approaches reflects a non-parallel evolution of highly repetitive vs. unique DNA classes.     

Pollination biology of annual and perennialLeonotis species (Lamiaceae)

The pollination syndromes ofLeonotis (Pers.)R. Br. are discussed. Ornithophily accounts for most of the nutlets set in perennialLeonotis species but in the annual species [L. nepetifolia (L.)R. Br.] autogamy prevails.Leonotis species are predominantly pollinated by a variety of sunbirds, although bees are also involved. Nectar is sucrose-dominant in perennial species but hexose-dominant in the annual.     

Feeding behavior ofLemur catta andLemur fulvus

The feeding behavior of two sympatric species of lemurs, Lemur cattaand Lemur fulvus,was studied in an enclosure simulating a natural habitat at the Duke University Primate Center. L. fulvusspent less time feeding during the day than L. catta.But the former species ate more fruit and had longer feeding bouts on preferred food items than L. catta.They also had a shorter food passage time than L. cattaand their choice of resting places was more influenced by food distribution. Furthermore, the two lemur species ate parts of different plant species and showed different reactions to chemical plant components. According to these results, L. fulvusis a more conservative feeder than L. catta.These interspecific differences in feeding behavior may be one of a number of differences that allow the two species to coexist. In allopatry, however, L. fulvusmay also adopt feeding patterns similar to those of L. catta.But L. cattawas never found to change its feeding strategies in different areas. It may be this option of L. fulvusto adopt different feeding strategies in different situations that allows this species to have the widest range of all Malagasy lemurs. Duke University Primate Center Publication No. 259.     

New available SCAR markers: potentially useful in distinguishing a commercial strain of the superior type from other strains of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in China

At present, more than 100 strains of Lentinula edodes are cultivated on a commercial scale in China. A simple, reliable, and effective method to distinguish some commercial strains of the superior type from other commercial strains is very important for the Lentinula industry. In this study, 23 commercial strains of L. edodes cultivated widely in China at present were collected and analyzed with randomly amplified polymorphic DNA (RAPD) technique. Three informative dominant sequence characterized amplified region (SCAR) markers were developed by designing three pairs of specific SCAR primers from three sequenced differential RAPD bands, respectively. Based on the three SCAR markers, three different multiplex polymerase chain reaction (PCR) phenotypes were detected among the 23 studied commercial strains and in which a multilocus phenotype characterizing a commercial strain Cr02 of the superior type could potentially be used to distinguish this strain from the other 22 studied commercial strains. To our knowledge, this study is the first to describe the development of a multiplex PCR technique based on SCAR markers for detecting the molecular phenotypes among commercial strains of L. edodes in China.     

Highly repeated DNA analysis and systematics of the Lemuridae,a family of Malagasy prosimians

Hybridization of highly repeated DNA sequences ofEulemur fulvus mayottensis, Lemur catta, andVarecia has been performed on blots of different species of Lemuridae (L. catta, Hapalemur griseus, Varecia variegata variegata, V. v. rubra, E. macaco macaco, E. coronatus, E. mongoz, andE. rubriventer). The probe ofE. fulvus only hybridized with the differentEulemur species, whereas that ofVarecia hybridized with the two subspecies ofVarecia and that ofL. catta with bothL. catta andHapalemur. These results were used to confirm the classification ofVarecia in a separate genus and to review the separation of theL. catta/Hapalemur group from the other species ofEulemur. Comparison of the migration patterns from DNA fragments of these different species has been used to propose a cladogram of the differentEulemur species.     

Isolation and characterization of the gene encoding a chitin synthase with a myosin motor-like domain from the edible basidiomycetous mushroom, <Emphasis Type="Italic">Lentinula edodes</Emphasis>, and its expression in the course of fruit-body formation

We isolated and characterized the genomic and complementary DNAs encoding a chitin synthase from an edible basidiomycetous mushroom, Lentinula edodes. The gene (which we designated Lechs1) contains a large open reading frame encoding a polypeptide of 1937 amino acid residues. The open reading frame is interrupted by 14 small introns (49–116 bp). The gene product (LeChs1) consists of a myosin motor-like domain in its N-terminal half and a chitin synthase domain in its C-terminal half, analogous to the class V and VI chitin synthases of other filamentous fungi. Phylogenetic analysis demonstrated that LeChs1 is classified into class VI chitin synthases. Southern blot analysis indicated that Lechs1 is a single-copy gene per haploid genome and that L. edodes has no other highly homologous chitin synthase genes. Northern blot analysis revealed that Lechs1 is expressed throughout the whole stages of fruit-body formation of L. edodes, but its expression level gradually declines in a fruit body-maturation-dependent manner with highest expression in vegetative mycelia and fruit body at the early stage of maturation (immature fruit body). This is the first report on the isolation and characterization of the gene encoding a chitin synthase with a myosin motor-like domain from basidiomycetes.     

Responses of four arid zone grass species from varying habitats to drought stress

The effects of 4 or 8 drought cycles on four grass species,Cenchrus pennisetiformis, Leptochloa fusca, Panicum turgidum, andPennisetum divisum were assessed in a pot experiment. There were significant differences between the species in biomass production under water stress.C. pennisetiformis andP. turgidum produced significantly greater fresh and dry matter thanP. divisum and especially thanL. fusca. L. fusca had the lowest andP. divisum highest osmotic potentials compared with the other species after the completion of 4 or 8 drought cycles. Osmotic adjustment (difference between osmotic potential of droughted/rehydrated plants and control plants) was highest inL. fusca. The stomatal conductance was significantly decreased with increased drought stress inC. pennisetiformis. The elasticity ofC. pennisetiformis, P. turgidum andP. divisum increased with increase in number of drought cycles, whereas that ofL. fusca remained unchanged.L. fusca andP. turgidum had the lowest leaf hydration of all species after 8 drought cycles. The chlorophyllsa andb in all species remained unaffected by drought treatments. The proline content ofC. pennisetiformis andL. fusca increased significantly with increased drought stress, whereas that ofP. turgidum remained unaffected after 4 or 8 drought cycles.L. fusca synthesized great amount of leaf soluble proteins during 8 drought cycles, whereasP. divisum had low protein content after 4 drought cycles. The protein contents ofC. pennisetiformis andP. turgidum remained unaffected after 8 drought cycles. The leaf epicuticular wax ofL. fusca increased consistently with increased drought stress, but leaf wax ofP. divisum increased only at the highest drought stress and that ofC. pennisetiformis andP. turgidum increased after 4 drought cycles. On the basis of these results it was established thatC. pennisetiformis andP. turgidum were the most tolerant,P. divisum intermediate, andL. fusca the most sensitive to drought stress. The osmotic adjustment did not positively correlate with the degree of drought resistance.     

Effects of different levels of wheat bran, rice bran and maize powder supplementation with saw dust on the production of shiitake mushroom (Lentinus edodes (Berk.) Singer)

The cultivation of shiitake mushroom (Lentinus edodes) is increasing rapidly in Bangladesh due to its nutritional and medicinal importance with excellent flavor and longer shelf life. With the aim of increased production, we have cultivated L. edodes on saw dust (SD) supplemented with different levels (10%, 15%, 20%, 25%, 30%, 35% and 40%) of wheat bran (WB), rice bran (RB), maize powder (MP) and their combination (WB+RB+MP = 1:1:1) to investigate the growth, yield and quality of this mushroom. Most of the growth, yield and quality parameters varied significantly when mushrooms were cultivated with different levels of supplementation. The yield of mushroom was increased with the level of each supplementation upto a certain level, and then decreased. SD supplemented with 25% WB produced the highest number of fruiting bodies (34.8/500 g packet), highest biological yield (153.3/500 g packet), and biological efficiency (76.6%) of L. edodes. But the yield of the best quality mushroom was observed on SD with 40% WB supplementation; however, the qualities were not always supplementation dose dependent. In this study, we report that 25% WB supplementation with SD may be very effective for higher yield and 40% WB supplementation for better quality of L. edodes.