Improvement of yield of Pleurotus eryngii var. eryngii by substrate supplementation and use of a casing overlay

Improved yield and biological efficiency (BE) of Pleurotus eryngii var. eryngii were achieved by supplementation of substrate with a commercial delayed-release nutrient and use of a casing overlay. Yield increases of 14% were achieved from cased substrates that were supplemented at time of casing with delayed-release nutrient (Remo’s). Use of a casing layer enhanced yield by 141% over non-cased substrates. When casing and substrate supplementation were combined, yield increased 179% over non-cased/non-supplemented substrates. Mushrooms harvested from cased substrates were darker in color and solids contents were lower compared to non-cased substrates. An additional break of mushrooms was harvested from non-cased “spent” substrate by fragmenting and re-supplementing the substrate prior to the application of a casing overlay. Three production methods were compared for their effect on mushroom yield: “standard”, “casing” and “casing after first break”. Casing of the substrate before first break (“casing” production method) resulted in the highest yield and biological efficiency.     

Interaction between gfp-tagged Pseudomonas tolaasii P12 and Pleurotus eryngii

Pseudomonas sp., (formerly reported as strain P12) which produces brown blotch disease symptoms on Pleurotus eryngii, has been identified as P. tolaasii based on its biochemical, physiological properties and 16S rDNA sequence analysis. This pathogen is able to infect basidiocarps when surface-inoculated on mushroom casing soil. However, infected basidiocarps develop the brown blotch disease symptoms when the pathogen concentration in the fruiting body tissues is higher than 10(4) cfu/g d.w. Using gfp-tagged cells and confocal laser scanning microscopy, it was possible to show that the pathogen has the ability to tightly attach to the hyphae of Pleurotus eryngii.     

白灵侧耳漆酶分离纯化及其酶学性质研究

为了探索白灵侧耳Pleurotus eryngii var.tuoliensis漆酶性质,以白灵侧耳菌株00485为试验材料,从发酵液中分离纯化得到胞外漆酶并对其酶学性质进行测定。纯化流程依次为DEAE-Cellulose阴离子交换层析,CM-Cellulose阳离子交换层析,SP-Sepharose强阳离子交换层析以及Superdex 75凝胶过滤层析,获得胞外白灵侧耳漆酶(Pn Lac)。SDS-PAGE检测结果表明Pn Lac为65k Da的单一蛋白。Pn Lac经过胰蛋白酶水解得到3种肽段,经过NBCI-BLAST后发现它们与糙皮侧耳、环柄韧伞、刺芹侧耳等的漆酶具有同源性。底物为2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)时该种漆酶的最适反应温度和p H分别为50℃和3.0,Ca2+和Hg2+能够抑制它的活性,相反地Cu2+和Mn2+能够提高它的活性,米氏常数Km和Vmax分别是0.17mmol/L和1.76OD/min/U。     

白灵侧耳栽培种质对高温胁迫的反应研究

本文以“中农一号”、“华杂13”、“中农翅鲍”和“CCMSSC 00488”4个白灵侧耳菌株为材料,通过测定高温胁迫下菌丝体内硫代巴比妥酸反应物（thiobarbituric acid-reactive substances,TBARS）和蛋白质羰基（protein carbonyl,PCO）含量,观察适温培养和高温胁迫后恢复培养时菌落的生长速率、生长势和菌丝形态特征,研究不同白灵侧耳栽培种质对高温胁迫的反应。研究表明,白灵侧耳不同栽培种质对高温胁迫的反应在氧化损伤程度、菌落形态和菌丝形态特征上都有变化,不同材料的高温伤害程度有着较显著的差异。4个材料高温胁迫条件下TBARS和PCO含量均显著升高,但是升高程度不同,按照TBARS和PCO含量多少排序,依次为CCMSSC 00488>华杂13>中农翅鲍>中农一号。高温胁迫处理后恢复生长需要的时间依次为：CCMSSC 00488>华杂13>中农翅鲍、中农一号;菌落生长势：中农一号>中农翅鲍=华杂13>CCMSSC 00488;菌丝形态特征：菌丝表面增长率、菌丝顶端细胞表面积、菌丝直径和菌丝体分支频率均显著降低,但降低程度以中农一号为最大。菌丝体分支频率与菌落生长势相对应。     

Cultivation of different strains of king oyster mushroom (Pleurotus eryngii) on saw dust and rice straw in Bangladesh

Pleurotus eryngii is a popular mushroom due to its excellent consistency of cap and stem, culinary qualities and longer shelf life. In Bangladesh, where Pleurotus mushrooms are very popular, P. eryngii may take position among the consumers, but currently this mushroom is not cultivated in large scale there. In this study, 3 strains of P. eryngii such as Pe-1 (native to Bangladesh), Pe-2 (germplasm collected from China) and Pe-3 (germplasm collected from Japan) were cultivated on saw dust and rice straw and their growth and yield parameters were investigated. Pe-1 on saw dust showed the highest biological yield and efficiency (73.5%) than other strains. Also, the mycelium run rate and number of fruiting bodies were higher in Pe-1 than other two strains. The quality of mushroom strains was near about similar. On saw dust, the yield and efficiency were better than those cultivated on rice straw, however, on straw; the mushroom fruiting bodies were larger in size. This study shows the prospects of P. eryngii cultivation in Bangladesh and suggests further study in controlled environment for higher yield and production.     

Bioconversion of agricultural lignocellulosic wastes through the cultivation of the edible mushrooms Agrocybe aegerita,Volvariella volvacea and Pleurotus spp.

Ten selected wild and commercial strains of Pleurotus ostreatus,Pleurotus eryngii,Pleurotus pulmonarius, Agrocybe aegerita andVolvariella volvacea were cultivated on three agricultural wastes, i.e. wheat straw (WS), cotton waste (CW) and peanut shells (PS). All species demonstrated significantly higher colonization rates on WS and CW than on PS. WS supported faster growth of A. aegerita and Pleurotus spp., whereas V. volvacea performed better on CW. Comparison of growth rates on composted and non-composted WS and CW substrates revealed that in the latter case faster colonization was achieved, particularly for Pleurotus spp. However, one commercial strain of V. volvacea presented higher growth rates when the composted CW medium was used. Furthermore, earliness in the fructification of P. ostreatus, P. pulmonarius and V. volvacea strains was promoted in CW substrates, while WS favoured earliness of P. eryngii and A. aegerita. Similarly, high sporophore yields were obtained by P. ostreatus and P. pulmonarius on both wastes, whereas WS enhanced yield and basidioma size of P. eryngii and A. aegerita strains and CW production of V. volvacea. The substrates cellulose:lignin ratios were found to be positively correlated to mycelial growth rates and to mushroom yield of P. ostreatus and P. pulmonarius; in addition, positive correlation was also detected for carbon:nitrogen ratio and mushroom yield in P. eryngii and A. aegerita and between cellulose content and mushroom yield for V. volvacea strains.     

First environmental isolation of Cryptococcus neoformans var. neoformans and var. gatti from the Gharbia Governorate,Egypt

Flowers from two Eucalyptus camaldulensis trees in the Qutur area and one tree from the Tanta area yielded three isolates of Cryptococcus neoformans var. gattii. Pigeon and sparrow droppings were also investigated for the occurrence of C. neoformans within the study area. Ninety five isolates of the neoformans variety of C. neoformans were recovered from 550 samples of avian droppings. This revised version was published online in October 2005 with corrections to the Cover Date.     

Evaluation of morphological and molecular variation in Plantago asiatica var. densiuscula, with special reference to the systematic treatment of Plantago asiatica var. yakusimensis

Morphological and molecular variations in Plantago asiatica L. var. densiuscula Pilg. were analyzed to evaluate the genetic basis for recognizing the dwarf variety P. asiatica var. yakusimensis (Masam.) Ohwi. Considerable variation in the leaf size of P. asiatica var. densiuscula was observed, and no morphological discontinuities were found between the dwarf types of P. asiatica var. densiuscula and P. asiatica var. yakusimensis. Morphological analysis of plants grown under standardized conditions revealed that both environmental plasticity and genetic differentiation contributed to the dwarfisms. Molecular phylogenetic analysis of rDNA internal transcribed spacer (ITS) regions and the SUC1 locus encoding a sucrose transporter revealed that P. asiatica var. yakusimensis was genetically unique although the differentiation level was low. From the above results, we concluded that P. asiatica var. yakusimensis should be reduced to a form of P. asiatica var. densiuscula. Furthermore, the geographic distribution of the SUC1 genotype suggested multiple origins of dwarves, and possible hypotheses for the origins of dwarves are discussed.     

Comparative study of leaf carbon gain in saplings ofThujopsis dolabrata var.hondai andQuercus mongolica var.grosseserrata in a cool-temperate deciduous forest

Seasonal courses of leaf CO₂ gas exchange in a growing season were examined in saplings ofThujopsis dolabrata var.hondai andQuercus mongolica var.grosseserrata in a cool temperate deciduous forest. Between the two tree species there were no large differences in the light compensation point of leaf photosynthesis, except for the season of new leaf expansion. However, light-saturated rates of net photosynthesis were obviously high inT. dolabrata var.hondai. EvergreenT. dolabrata var.hondai saplings had large photosynthetic production in two seasons, before the emergence of new foliage and after foliage fall of the overstory deciduous trees, because of the significantly high solar radiant energy penetrating under the forest canopy during the seasons. Saplings of deciduousQ. mongolica var.grosseserrata were heavily shaded throughout the growing season by foliage of the overstory trees, which resulted in a low daily surplus production. The annual surplus production of leaves in the growing season was estimated to be 2300 mmol CO₂ m⁻² inT. dolabrata var.hondai and −100 mmol CO₂ m⁻², slightly negative, inQ. mongolica var.grosseserrata. These results supported the high survivability ofT. dolabrata var.hondai saplings and the high mortality ofQ. mongolica var.grosseserrata in the deciduous forest.     

Mitochondrial DNA analysis of Exophiala jeanselmei var. lecanii-corni and Exophiala castellanii

A Japanese clinical isolate (KU-A-0094) which was identified by de Hoog et al. as Exophiala jeanselmei var. lecanii-corni with difficulty, was compared with 5 strains including the type cultures of E. jeanselmei var. lecanii-corni, var. jeanselmei and E. castellanii using RFLP (restriction fragment length polymorphism) patterns of mtDNA (mitochondrial DNA). RFLP patterns of KUA-0094 were identical with those of E. jeanselmei var. lecanii-corni and different from those of E. castellanii with restriction enzymes of HaeIII, MspI and hindIII. Therefore, de Hoog et al.'s identification of KU-A-0094 was confirmed. Additionally, mtDNA-RFLP patterns of E. jeanselmei var. lecanii-corni and E. jeanselmei var. jeanselmei were also different from each other. Consequently E. jeanselmei var. lecanii-corni seem to be a species in its own right rather than a variant of E. jeanselmei. This revised version was published online in June 2006 with corrections to the Cover Date.     

Waste-reducing cultivation of Pleurotus ostreatus and Pleurotus pulmonarius on coffee pulp: changes in the production of some lignocellulolytic enzymes

Fruiting body production for one strain of Pleurotus ostreatus and three strains of P. pulmonarius was evaluated on coffee pulp pasteurized at 80 °C for 1 h. Based upon three harvests per strain, the single P. ostreatus line was found to display a 40-day culture cycle, whereas the three P. pulmonarius strains completed their cycles after more than 50 days of incubation. These time periods were notably shorter than those observed in previous studies using other growth substrates. Nevertheless, yields expressed as biological efficiencies were not significantly different among strains, fluctuating between 125 and 138%. Extracellular enzymatic activity was also monitored for P. ostreatus and P. pulmonarius (one strain only). To do this, samples of mycelium-bearing substrate were taken every 4 days throughout the incubation period. Care was taken to represent all developmental stages, including primordial and fruiting bodies. Samples were either lyophilized and then analysed or, in some cases, analysed immediately without lyophilization. Hydrolase activity (i.e. endoglucanase (CMC) and cellobiohydrolase (CBH)) was found to depend on developmental stage, showing peak production during fruiting body formation. On the other hand, oxidase activity-(i.e. laccase (LAC) and Mn-peroxidase (MnP)) was associated with phenol degradation. Nevertheless, in the case of oxidases developmental timing differences were also observed. Specifically, LAC activity was detected as early as 8 days after inoculation in non-lyophilized samples, whereas MnP appeared near the end of the incubation period. No LAC activity was observed in lyophilized samples. This study concludes that coffee pulp might be successfully employed in the cultivation of mushrooms, not only because important extracellular enzymes are produced by mushrooms when grown upon this substrate, but also because the abbreviated cultivation cycle associated with this medium favours commercial processes. Commercialization might be further improved if strains specifically adapted to this novel substrate are selected.     

Recycling spent larval food of Corcyra cephalonica Stainton for preparing spawn and sporophore of Pleurotus sajor-caju (Fr.) Singer

Large scale production of the rice moth, Corcyra cephalonica Stainton in pearl millet grain medium leads to a huge accumulation of spent larval medium in commercial insectaries. We attempted bioconversion of spent larval medium of C. cephalonica (CLM) for cultivation of the mushroom Pleurotus sajor-caju (Fr.) Singer, to increase the usage of these residues. Maximum efficiency limits of CLM for spawn run, sporophore cropping and as bed substrate were assessed with varying combinations of sorghum and rice straw. Sorghum grains and rice straw were the best substrates for spawn run and sporophore yield respectively. Having been crushed, macerated, heated and sterilized, CLM could also become a suitable substrate along with sorghum or rice straw. Sorghum and CLM at 16.7% + 83.3% and 33.3% + 66.7% combinations were very effective in supporting mycelial growth and quicker colonization of fungus, and mother spawn yield. The spawn that was obtained from these combinations yielded higher sporophore as well. The fungus did not rapidly colonize on other combinations (50% + 50%, 66.7% + 33.3% and 83.3% + 16.7%), and was completely unable to grow on CLM 100%. Combination of rice straw and CLM at 75% + 25% and 50% + 50% as bed substrate contributed higher sporophore yield. Analysis of the substrates indicated variation in their chemical and mineral composition, but they were good sources of N, P and Ca. The prospects of exploring CLM for the mushroom cultivation are discussed.     

Micropropagation of Cypripedium macranthos var. rebunense through Protocorm-like Bodies Derived from Mature Seeds

Cypripedium macranthos var. rebunense is the most famous terrestrial orchid in Japan, since the variety has large beautiful yellowish-white flowers and is threatened with extinction. Establishment of an efficient method for micropropagation is urgently needed. When imbibed mature seeds of the orchid, that had been pre-chilled at 4°C for 3 months, were sown onto Hyponex-peptone medium that contained both α-naphthaleneacetic acid (NAA) and cytokinin, protocorm-like bodies (PLBs) were formed from germinated seeds. Although the growth of PLBs was very slow, plantlets were easily regenerated from the PLBs on hormone-free medium. The PLBs were subcultured eight times along 2 years without loss of ability to regenerate plantlets, and one aggregate of PLBs (ca. 5 mm in diameter) produced ca. 10 plants within a year. A reduction of commercial value through a large-scale micropropagation by this method will be able to prevent illegal collection from the wild populations.     

Behaviour of the yeast Kluyveromyces marxianus var. marxianus during its autolysis

The lactic yeast Kluyveromyces marxianus var.marxianus (formerly K. fragilis) autolyzates at faster rate than Saccharomyces cerevisiae. During K. marxianus autolysis, quite similar release kinetics were observed for intracellular space markers (potassium ions, nucleotides), cell-wall components (polysaccharides, N-acetyl-D-Glucosamine) and non specific products (amino nitrogen). By Scanning Electronic Microscopy examination, no cell burst was observed, but a variation in cell shape (from ellipsoidal to cylindrical), as well as a 43% decrease in the internal volume were observed. The mechanism proposed for S. cerevisiae autolysis appeared also likely for K. marxianus.Abbreviations NacGlc N-acetyl-D-glucosamine - x total biomass (dry cellular weight) concentration     

Production of <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis> strain PS-2001 biomass in submerged culture

Mushrooms or fruiting bodies of many basidiomycetes are commonly produced in solid-state fermentation, generally after 20-60 days of growth. However, it is also possible to produce biomass from these fungi, in submerged fermentation in shorter time. This work was aimed at evaluating biomass production with the basidiomycete Pleurotus sajor-caju, in a submerged process and to determine the proportion of chemical components of this biomass. Initially, an optimization of the culture medium was done to produce a faster growth of microbial mass by changing the concentrations of ammonium sulfate, soy protein and yeast extract. Using the optimized culture medium, values of approximately 5.5 g L(-1) of biomass in a medium with 10 g L(-1) of glucose were attained. When the optimized culture medium was tested in a 5-L stirred tank bioreactor, using 10 g L(-1) of glucose or sucrose as carbon source, values of 8.18 and 5.94 g L(-1) of biomass concentration were obtained, respectively. In the medium with glucose, high yields (0.82 g g(-1)) and productivity of 0.085 g L(-1) h(-1) were obtained. The exopolysaccharide content (1.58 g dry matter L(-1)) in the culture was higher in the fermentation with sucrose. The nutritional composition of the biomass obtained in the submerged fermentation was similar to that of the fruiting body in terms of quantities of total carbohydrates, ash and calories, but total fat and protein were higher.     

Structural investigation of a polysaccharide (Fr. II) isolated from the aqueous extract of an edible mushroom, Pleurotus sajor-caju

A water-soluble polysaccharide, (Fr. II) isolated from aqueous extract of an edible mushroom Pleurotus sajor-caju, was found to consist of D-glucose, D-galactose, and D-mannose in a molar proportion of 1:1:1. Compositional analysis, methylation analysis, periodate oxidation study, partial hydrolysis, and NMR experiments ((1)H, (13)C, 2D-COSY, TOCSY, NOESY, HSQC, and HMBC) revealed the presence of the following repeating unit in the polysaccharide: [formula: see text]     

青稞HvBAS1基因的克隆及表达分析

细胞色素P450单加氧酶(CYP450)是参与植物代谢的最大酶家族,其中CYP734A亚家族成员广泛参与植物激素油菜素类固醇(BRs)的失活。该研究以青稞农家品种‘肚里黄’幼苗为实验材料,通过人工合成激素24 表油菜素内酯(24 eBL)和BRs合成抑制剂油菜素唑(BRZ)处理,分析BRs对青藏高原特色作物青稞(Hordeum vulgare L. var. nudum Hook. f.)的影响;采用RT PCR技术从青稞中克隆HvBAS1基因,并运用实时定量PCR检测其表达特征,为深入分析HvBAS1基因的功能奠定研究基础。结果显示：(1)24 eBL能够显著促进青稞幼苗的生长,而BRZ处理后幼苗长势明显减缓。(2)从青稞中成功克隆到2个与拟南芥BRs失活基因BAS1高度同源的CYP734A亚家族基因,即HvBAS1 1和HvBAS1 2;HvBAS1 1开放阅读框全长1 629 bp,编码542个氨基酸;HvBAS1 2长1 689 bp,编码562个氨基酸,二者氨基酸序列相似性为76.42%;亚细胞定位预测结果显示二者均存在于内质网。(3)实时定量PCR检测发现,青稞HvBAS1 1与HvBAS1 2的表达模式完全不同,其中HvBAS1 1在青稞根中的表达量高于叶中,而HvBAS1 2在叶中的表达量高于根,且随着苗龄的增大,HvBAS1 1在根中的表达呈先升高后降低的趋势,HvBAS1 2在叶片中呈先降低后升高的变化趋势;BRZ处理青稞幼苗后,其HvBAS1 1与HvBAS1 2基因的表达较对照均显著下调,且HvBAS1 2下调更为明显。研究表明,HvBAS1 1和HvBAS1 2很可能都参与了青稞内源BRs的失活,但二者的功能存在差异。