侧耳属食用菌高温胁迫条件优化研究

侧耳属真菌是我国最大规模栽培的食用菌,主要是糙皮侧耳Pleurotus ostreatus和白黄侧耳P. cornucopiae。高温是影响侧耳产量和质量的重要因子之一,确定高温胁迫条件是开展高温胁迫机制研究的方法学基础。以糙皮侧耳和白黄侧耳的11个菌株为材料,以硫代巴比妥酸反应产物（thiobarbituric acid reactive substances,TBARS）含量为主要生理指标,结合菌丝形态和生长等微生物学参数,开展了双核体菌丝高温胁迫条件的研究,包括胁迫的适宜温度、时间和菌龄等。结果表明,所有供试材料,高温胁迫后菌丝体内TBARS含量在28-40℃、0-48h范围内与温度和时间呈正相关,与之后的恢复生长率呈负相关,高温胁迫后菌丝体内TBARS含量和之后的恢复生长能力均与自身菌丝体生长和子实体形成的适宜温度无关。TBARS含量可作为菌丝高温胁迫生理研究条件确定的指标。与植物的最适温度加10℃不同,基于TBARS含量的糙皮侧耳和白黄侧耳高温胁迫研究条件应为Difco^TM Potato Dextrose Agar培养基,菌丝培养3d,最适温度加12℃,高温胁迫处理48h。     

卵孢侧耳野生菌株人工栽培及营养成分分析

采自西藏日喀则地区的两份野生蘑菇标本及其分离菌株,通过形态学及基于ITS序列的分子系统发育分析鉴定为卵孢侧耳Pleurotus placentodes。该物种在2016年首次报道于中国。人工驯化栽培试验表明卵孢侧耳可以在人工条件下出菇,生长周期80-95d;初步的营养成分分析表明,卵孢侧耳蛋白质、粗多糖、氨基酸含量比香菇、糙皮侧耳（平菇）、金针菇、柱状田头菇（茶树菇）等都高,尤其含有较高的组氨酸和丰富的矿物质,如铁、钙、锌等。卵孢侧耳可作为新的食用菌进行开发利用。     

锰离子Mn(Ⅱ)对糙皮侧耳木质素降解酶活性和原基形成的影响

木质素降解酶是糙皮侧耳Pleurotus ostreatus分解木质素的关键酶。本文以糙皮侧耳栽培菌株New831为试验材料,通过固体培养法和液体培养法测定了不同浓度锰离子Mn(II)对其菌丝生长、偶氮染料橙黄II降解率、木质素降解酶活性和原基形成的影响。结果表明,添加Mn(II)可促进糙皮侧耳菌丝生长,GP-Orange液体培养时的最适Mn(II)浓度为50μmol/L,GP-Urea-Orange固体培养时的最适Mn(II)浓度为70μmol/L;添加Mn(II)可加速橙黄II的降解,Mn(II)浓度为70μmol/L时,橙黄II降解率最高、脱色圈最大;漆酶和锰过氧化物酶活性均随Mn(II)浓度增加呈现"先升后降"的趋势,而木质素过氧化物酶活性则随Mn(II)浓度的增加而降低;添加Mn(II)可诱导原基形成,Mn(II)浓度为70μmol/L时,原基数量最多。此外,平板培养结果揭示木质素降解酶活性与菌丝生物量之间无相关性,其主要是在营养限制条件下表达。     

不同栽培基质对糙皮侧耳不同发酵方式下产漆酶活性的影响

为了比较糙皮侧耳栽培种在不同常规栽培基质上的漆酶活性,分析更适合糙皮侧耳生长的栽培基质,以1株糙皮侧耳Pleurotus ostreatus栽培菌株为研究材料,研究在固态和液态发酵条件下添加木屑、玉米芯和棉籽壳这3种栽培基质后,对其产漆酶活性的影响。结果表明,不同栽培基质对糙皮侧耳漆酶活性具有极显著的影响（P<0.001）;不同发酵方法对糙皮侧耳漆酶活性也具有极显著的影响（P<0.001）,仅第2天差异不显著。固体发酵与液体发酵条件下,糙皮侧耳在棉籽壳培养基上所检测到的漆酶活性均高于在木屑或者玉米芯培养基上,表明棉籽壳对提高糙皮侧耳漆酶活性的诱导能力更强。此外,糙皮侧耳在棉籽壳培养基上能够快速分泌漆酶,表明棉籽壳对缩短糙皮侧耳漆酶分泌时间的诱导能力更强。     

磷酸氢二铵对糙皮侧耳菌丝生长及基质降解酶活性的影响

氮源种类和浓度均会对糙皮侧耳Pleurotus ostreatus菌丝长势、生长速率以及代谢产生影响,而氮匮乏与氮过量积累都会对糙皮侧耳的生长产生不利影响.为了研究氮源对糙皮侧耳菌丝生长和基质降解酶活性的影响,本研究选取磷酸氢二铵[(NH4)2HP04]作为唯一氮源,通过在培养基中添加不同浓度的磷酸氢二铵检测糙皮侧耳菌...     

Effect of mannan oligosaccharide elicitor and ferulic acid on enhancement of laccases production in liquid cultures of basidiomycetes

The effects of mannan oligosaccharides preparation (MO), as elicitor, and ferulic acid inducer for enhancement in laccases production in liquid cultures of three strains of basidiomycetes, Pycnoporus sanguineus, Coriolopsis polyzona and Pleurotus ostreatus was studied using a full factorial 3² experimental design. MO, either individually or combined with ferulic acid, enhanced laccases levels in the three different strains of the white-rot fungi. The enhancement of laccases production was species specific with the highest increase in liquid cultures of P. sanguineus (88-fold) followed by P. ostreatus (3-fold) and C. polyzona (2-fold). Separate additions of 75 mg/l of MO to the cultures of P. sanguineus and P. ostreatus caused the increase while a combined addition of 150 mg/l of MO and 1 mM ferulic acid resulted in the optimal production of laccases in the cultures of C. polyzona.     

Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg²⁺ but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.     

Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.     

Several pseudomonads,associated with the cultivated mushrooms Agaricus bisporus or Pleurotus sp., are hemolytic

Pseudomonas tolaasii, causing brown blotch disease on cultivated mushrooms, and yielding a white line precipitate towards P. “reactans”, has been shown to induce lysis of erythrocytes. Some Finnish strains isolated from diseased mushroom fruit bodies, although harboring the typical features of P. tolaasii, proved to be distinct, and have been allocated to a nov. sp. P. costantinii. We examined in these study whether all brown blotch causing agents were hemolytic. The induction of erythrocytes lysis seemed to be a rather common feature of mushroom associated-pseudomonads, especially for strains involved in the production of a white-line-in agar.     

不同诱导培养基对糙皮侧耳液体发酵产漆酶活性的影响

利用1株糙皮侧耳Pleurotus ostreatus栽培菌株为材料,研究添加碱性木质素或者配合简单碳源或氮源后对其液体发酵产漆酶活性的影响。结果表明,不同诱导培养基对糙皮侧耳漆酶活性具有极显著的影响（P<0.001）,而且不同诱导培养基对糙皮侧耳菌丝生物量也产生了极显著的影响（P<0.001）。此外,只利用碱性木质素或者是再添加碳源葡萄糖均有利于糙皮侧耳产漆酶,既包括产漆酶酶活性的提升,同时也包括产漆酶时间的提前,但只利用碱性木质素诱导不利于菌丝生物量的积累;而富含简单碳/氮源的诱导培养基,无论是否含碱性木质素,均有利于菌丝生物量的积累,其中,富含简单碳/氮源的培养基中再添加碱性木质素后的菌丝生物量和漆酶活性均高于不添加碱性木质素时的菌丝生物量和漆酶活性。相比而言,含碱性木质素的培养基中测得的漆酶活性大部分时间下都要高于不含木质素的简单碳/氮源培养基,含碱性木质素的培养基对糙皮侧耳菌株产漆酶的诱导作用更强。     

Interaction of ostreolysin, a cytolytic protein from the edible mushroom Pleurotus ostreatus, with lipid membranes and modulation by lysophospholipids.

Ostreolysin is a 16-kDa cytolytic protein specifically expressed in primordia and fruiting bodies of the edible mushroom Pleurotus ostreatus. To understand its interaction with lipid membranes, we compared its effects on mammalian cells, on vesicles prepared with either pure lipids or total lipid extracts, and on dispersions of lysophospholipids or fatty acids. At nanomolar concentrations, the protein lysed human, bovine and sheep erythrocytes by a colloid-osmotic mechanism, compatible with the formation of pores of 4 nm diameter, and was cytotoxic to mammalian tumor cells. A search for lipid inhibitors of hemolysis revealed a strong effect of lysophospholipids and fatty acids, occurring below their critical micellar concentration. This effect was distinct from the capacity of ostreolysin to bind to and permeabilize lipid membranes. In fact, permeabilization of vesicles occurred only when they were prepared with lipids extracted from erythrocytes, and not with lipids extracted from P. ostreatus or pure lipid mixtures, even if lysophospholipids or fatty acids were included. Interaction with lipid vesicles, and their permeabilization, correlated with an increase in the intrinsic fluorescence and alpha-helical content of the protein, and with aggregation, which were not detected with lysophospholipids. It appears that either an unknown lipid acceptor or a specific lipid complex is required for binding, aggregation and pore formation. The inhibitory effect of lysophospholipids may reflect a regulatory role for these components on the physiological action of ostreolysin and related proteins during fruiting.     

Conversion of Japanese red cedar (Cryptomeria japonica) into a feed for ruminants by white-rot basidiomycetes

The potential of five white-rot fungi to convert Japanese red cedar (Cryptomeria japonica) into feed for ruminants was determined. Pleurotus ostreatus (ATCC 66376), Pholiota nameko (IFO 30373), Dichomitus squalens (CBS 432.34), Lentinula edodes (IFO 6654) and Ceriporiopsis subvermispora (ATCC 90467) were inoculated into chips of cedar sapwood. L. edodes was cultured at 25 °C, and the other fungi at 28 °C, for 4, 8, 12, or 20 weeks. The in vitro organic matter (OM) digestibility (IVOMD) in cedar wood cultured without fungus were between 0.047 and 0.068, while it was elevated to 0.446 by culturing with C. subvermispora and to 0.281 by culturing with L. edodes for 20 weeks. In contrast, the IVOMD were 0.200, or lower, in cedar wood cultured with P. ostreatus, P. nameko, or D. squalens. The in vitro gas production (IVGP) in cedar wood cultured with P. ostreatus, P. nameko, or D. squalens was 37 ml/g OM, or lower, while that in cedar not inoculated with fungus was between 4 and 17 ml/g OM. In contrast, the IVGP in cedar wood cultured with C. subvermispora for 20 weeks increased to 107 ml/g OM, and that in cedar wood cultured with L. edodes increased to 58 ml/g OM. Lignin degradability in cedar wood cultured with C. subvermispora and L. edodes for 20 weeks were 0.578 and 0.288, respectively. These changes in IVOMD and IVGP demonstrate that a selective white-rot fungus, C. subvermispora has the ability to convert cedar wood into feed for ruminants, although further increase is required before the cultured cedar wood would have widespread feed potential.     

菌核多孔菌培养特性及驯化栽培

为了充分开发利用菌核多孔菌Polyporus tuberaster资源,对采自吉林省露水河东升林场的野生菌核多孔菌进行分离纯化获取纯菌株作为实验材料,采用十字划线法研究固体培养条件下不同碳源、氮源、pH和温度对其菌丝生长的影响,从以上4个单因素实验中选出3个最优水平进行正交实验。结果表明,菌核多孔菌P. tuberaster的最适培养条件为：碳源糊精粉（20mg/mL）、氮源酵母浸粉（2mg/mL）、pH 5.0、温度25℃。在最适培养条件下采用试剂盒测定液体培养条件下菌株的产酶活力,结果表明,菌核多孔菌产漆酶能力较强,酶活力最高可达386.96U/L;木质素过氧化物酶活力仅为5.23U/L;未检测到锰过氧化物酶。出菇实验中,二级种选用麦粒菌种,500mL罐头瓶装干料160g,每瓶接种蚕豆大小菌块3-4块,25℃培养,菌丝满瓶时间为20d;栽培种配方为阔叶树木屑78%、麦麸20%、石灰粉1%、石膏1%、含水量65%左右,17cm×33cm×0.05cm栽培袋装干料520g,每袋接种蚕豆大小二级种3-4块,菌丝发菌时间为26d;在92%-95%空气湿度、一定散射光和20-21℃低温刺激下培养13d后原基分化形成菇蕾,此后加大空气湿度至97%-98%并保持温度24-25℃培养32d后子实体成熟。     

Yield and size of oyster mushroom grown on rice/wheat straw basal substrate supplemented with cotton seed hull

Oyster mushroom (Pleurotus ostreatus) was cultivated on rice straw basal substrate, wheat straw basal substrate, cotton seed hull basal substrate, and wheat straw or rice straw supplemented with different proportions (15%, 30%, and 45% in rice straw substrate, 20%, 30%, and 40% in wheat straw substrate) of cotton seed hull to find a cost effective substrate. The effect of autoclaved sterilized and non-sterilized substrate on growth and yield of oyster mushroom was also examined. Results indicated that for both sterilized substrate and non-sterilized substrate, oyster mushroom on rice straw and wheat basal substrate have faster mycelial growth rate, comparatively poor surface mycelial density, shorter total colonization period and days from bag opening to primordia formation, lower yield and biological efficiency, lower mushroom weight, longer stipe length and smaller cap diameter than that on cotton seed hull basal substrate. The addition of cotton seed hull to rice straw and wheat straw substrate slowed spawn running, primordial development and fruit body formation. However, increasing the amount of cotton seed hull can increase the uniformity and white of mycelium, yield and biological efficiency, and increase mushroom weight, enlarge cap diameter and shorten stipe length. Compared to the sterilized substrate, the non-sterilized substrate had comparatively higher mycelial growth rate, shorter total colonization period and days from bag opening to primordia formation. However, the non-sterilized substrate did not gave significantly higher mushroom yield and biological efficiency than the sterilized substrate, but some undesirable characteristics, i.e. smaller mushroom cap diameter and relatively long stipe length.     

Development and growth of fruit bodies and crops of the button mushroom, Agaricus bisporus

We studied the appearance of fruit body primordia, the growth of individual fruit bodies and the development of the consecutive flushes of the crop. Relative growth, measured as cap expansion, was not constant. It started extremely rapidly, and slowed down to an exponential rate with diameter doubling of 1.7 d until fruit bodies showed maturation by veil breaking. Initially many outgrowing primordia were arrested, indicating nutritional competition. After reaching 10 mm diameter, no growth arrest occurred; all growing individuals, whether relatively large or small, showed an exponential increase of both cap diameter and biomass, until veil breaking. Biomass doubled in 0.8 d. Exponential growth indicates the absence of competition. Apparently there exist differential nutritional requirements for early growth and for later, continuing growth. Flushing was studied applying different picking sizes. An ordinary flushing pattern occurred at an immature picking size of 8 mm diameter (picking mushrooms once a day with a diameter above 8 mm). The smallest picking size yielded the highest number of mushrooms picked, confirming the competition and arrested growth of outgrowing primordia: competition seems less if outgrowing primordia are removed early. The flush duration (i.e. between the first and last picking moments) was not affected by picking size. At small picking size, the subsequent flushes were not fully separated in time but overlapped. Within 2 d after picking the first individuals of the first flush, primordia for the second flush started outgrowth. Our work supports the view that the acquisition of nutrients by the mycelium is demand rather than supply driven. For formation and early outgrowth of primordia, indications were found for an alternation of local and global control, at least in the casing layer. All these data combined, we postulate that flushing is the consequence of the depletion of some unknown specific nutrition required by outgrowing primordia.     

Detection, quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry

This study presents a combined method to analyze extracellular fungal laccases using a new anti-laccase antibody together with the identification of tryptic laccase peptides by mass spectrometry (nanoLC–ESI–MS/MS). The polyclonal anti-laccase antibody LccCbr2 was raised against peptides designed from the copper binding region II of fungal laccases using in silico data obtained from GenBank database. As a consequence, detection requires denaturation of the enzymes due to the stable conformation of the copper binding region II. The specificity of the antibody was shown with denatured laccase Lcc1 of Coprinopsis cinerea and laccase of Hypholoma fasciculare. LccCbr2 detected amounts as low as 5 ng of highly purified laccase, indicating a possible use of the antibody for quantification of laccase proteins. Denatured extracellular laccases from culture supernatants of the basidiomycetes C. cinerea, H. fasciculare, Lentinula edodes, Mycena sp., Piriformospora indica, Pleurotus cornucopiae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Trametes versicolor and furthermore the ascomycete Verpa conica were detected with apparent molecular masses between 60 and 70 kDa by LccCbr2. The identity of extracellular laccases from C. cinerea, H. fasciculare, P. ostreatus, P. cinnabarinus and T. versicolor were verified by tryptic peptides using nanoLC–ESI–MS/MS.     

糙皮侧耳原基期差异表达基因分析

为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期cDNA为检测子（tester）、双核菌丝期cDNA为驱赶子（driver）,采用抑制性消减杂交法（suppression subtractive hybridization,SSH）构建了糙皮侧耳SSH cDNA文库。菌液PCR验证SSH cDNA文库插入cDNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列（expressed sequence tag,EST）,重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH cDNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。     

信息素信号通路基因在斑玉蕈生长发育过程中的差异表达

为了更清楚地了解斑玉蕈菌丝成熟、原基形成和子实体发育的过程,本研究对不同菌丝培养时期的栽培瓶进行出菇实验,并对其不同培养时期和生长发育关键时期的信息素通路基因进行差异表达分析,以期揭示信息素信号通路基因参与调节斑玉蕈菌丝的生长、子实体形成和发育的作用。研究结果表明：斑玉蕈菌丝培养40-80d过程中,子实体产量呈上升的趋势,说明菌丝的成熟程度对产量会产生重要影响。对斑玉蕈基因组中的信息素信号通路基因进行分析鉴定共获得了8个关键基因。信息素通路基因差异表达分析表明：在菌丝培养40-80d过程中,大部分信息素信号通路基因在第60天时表达量最高,其中ste20、cdc24和ste12上调了4-20倍,而在第80天出现下降。从菌丝恢复到扭结形成原基和子实体发育的过程中,大多数基因在原基时期表达量最高,其中ste20、cdc24、ste11和ste12表达量上调最为显著,在子实体成熟期这些基因表达量下降。因此,这说明在菌丝营养生长过程中,在第60天菌丝细胞增殖生长最为旺盛,而在第80天菌丝细胞基本停止生长,菌丝也逐渐达到成熟。同时,在菌丝生殖生长过程中,斑玉蕈持续地上调信息素通路基因表达使菌丝细胞不断地分裂增殖,从而使新生的菌丝扭结形成原基,其中ste3、ste20、cdc24、ste11和ste12基因可能对斑玉蕈菌丝细胞的分裂增殖和诱导子实体形成起到关键的作用。     

两个糙皮侧耳菌株的比较基因组学研究

随着糙皮侧耳基因组测序的完成,基因组数据的深入挖掘和利用成为研究重点。本研究基于较高质量的糙皮侧耳CCMSSC00389-1和CCMSSC03989-1基因组图谱,开展了种内比较基因组学分析。CCMSSC00389-1和CCMSSC03989-1的11条染色体序列表现出良好的共线性,且分别含有89.92%和91.68%的保守基因。对菌株特有基因的GO富集分析表明,两菌株各自分化出一些独特的调控方式或途径。CCMSSC00389-1和CCMSSC03989-1之间共存在931 542个单核苷酸多态性位点,231 654个插入/缺失和9 221个结构变异。对与遗传变异重叠基因的GO富集分析表明,碳水化合物降解、物质运输/催化和调控/蛋白活性相关的基因分别容易发生SNP、In/Del和SV变异。CCMSSC00389-1中50.326kb缺失序列的断点两侧含有双链断裂修复（DSB）信号,推测DSB介导50.326kb序列的缺失。