调查双孢蘑菇产业发展现状,分析双孢蘑菇工厂化生产技术标准化现状,结合上海市金山区双孢蘑菇工厂化生产实际及发展要求探讨构建双孢蘑菇工厂化生产技术标准体系,以推动双孢蘑菇全产业链标准化建设,提升产品质量及竞争力,为保障农产品质量安全、增加绿色优质农产品供给和推动农业高质量发展提供有力支撑.

The cultivation of Agaricus bisporus was investigated with two commercial strains, A15 and W192. Nitrogen and lignocellulose were analyzed in absolute amounts based on mass balance to accurately compare the degradation efficiency of the compost, and the correlation between the degradation efficiency and extracellular enzyme activity of the mycelium was analyzed. Lignocellulose utilization efficiency positively correlated with mushroom yield. For the same strain, the compost with high lignocellulose content resulted in high utilization efficiency, which increased the yield of A. bisporus. For the same compost, the lignocellulose utilization efficiency of A15 was higher than that of W192. The activities of manganese-dependent peroxidase and β-glucosidase indicated that W192 may have a higher demand for lignin and cellulose. Therefore, a higher yield of W192 was obtained with high-lignocellulose compost. The metabolism of cellulose and hemicellulose in the mycelial growth stage seemed to be conducive to high mushroom yield.

Stropharia rugosoannulata uses straw as a growth substrate during artificial cultivation and has been widely promoted in China. However, its fruiting body formation and development processes have not been elucidated. In this study, the developmental transcriptomes were analyzed at three stages: the mycelium (G-S), primordium (P-S) and fruiting body (M-F) stages. A total of 9690 differentially expressed genes (DEGs) were identified in the different developmental stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that these DEGs were involved mainly in hydrolase activity, structural molecule activity and oxidoreductase activity as well as xenobiotic biodegradation and metabolism and energy metabolism pathways. We further found that the higher expression of most carbohydrate enzyme (i.e., GH, CE, CBM, AA and PL) genes in the hyphal (i.e., G-S) stage was related mainly to substrate degradation, while the upregulation of glycosyltransferase (GT) gene expression in the P-S and M-F stages may be related to cell wall synthesis. In addition, we found that CO2-sensing-related genes (i.e., CA-2, CA-3, PKA-1 and PKA-2) were upregulated in the P-S and M-F stages, heat shock protein genes (HSP60 and HSP90) were significantly downregulated in the P-S stage and upregulated in the M-F stage and the transcription factors (i.e., steA, MYB, nosA, HAP1, and GATA-4/5/6) involved in growth and development were significantly upregulated in the P-S stage. These results suggest that environmental factors (i.e., CO2 and temperature) and transcription factors may play a key role in primordium formation. In short, this study provides new insights into the study of stimulating primordia formation affecting the development of fruiting bodies of S. rugosoannulata.

为筛选适合双孢蘑菇(Agaricus bisporus)三次发酵料工厂化栽培的优良菌株,首先测定20个菌株的菌丝生长速度和菌丝生物量,再通过二次发酵料栽培观察不同菌株催蕾期菌丝生长状态,记录原基形成时间,采收后观察子实体颜色,记录第一潮菇采摘时间和采收持续时间,统计子实体产量;分别随机选取10个子实体,测量菌盖直径、菌盖厚度、菌柄直径、菌柄长度;采摘第二潮菇,统计子实体产量,计算总产量.采用三次发酵料工厂化栽培901商业菌株和K6菌株,901商业菌株为对照,按照等级分别统计三潮菇产量和计算总产量.结果表明:菌株U3菌丝生长速度快,为4.85 mm·d-1,且与其他菌株相比,组间存在显著性差异;8302菌丝生物量较大,为2.48 g·100 mL-1,其次是K6,为2.33 g·100 mL-1,两者组间差异不显著;二次发酵料栽培结果表明,菌株MG、XXX、901、FG、W192、914、福蘑38、8305和K6综合表现较好,菌丝长势较好,覆土后第一潮菇采摘时间为17～19 d,第一潮菇采收持续时间为8～10 d,其中菌株K6的子实体形态与商业化菌株XXX和A15类似,菌盖直径和菌盖厚度较大,综合评价,K6菌株子实体总产量较高,出菇时间较早、采菇时间较集中,菇型较好,适合作为双抱蘑菇工厂化生产菌株;三次发酵料工厂化栽培结果表明,K6菌株第一潮A级菇产量稍低于901商业菌株,但组间无显著性差异,而B级菇产量高于901商业菌株,组间存在显著性差异;其中商品菇(A+B)为26.36 kg·m-2,高于901商业菌株的25.78 kg·m-2,比901提高6.18％;K6菌株的总产量达38.44 kg·m-2,略低于901商业菌株的41.32 kg·m-2,但组间无显著性差异.本研究证实K6菌株是适合三次发酵料工厂化栽培的双孢蘑菇新菌株.

将40RH冷藏集装箱改建成方舱式可移动低压双孢蘑菇培养料发酵隧道,该发酵隧道由隧道主体、气候系统、通风格栅地板、控制系统四大部分组成.经过双孢蘑菇培养料的三次发酵试验,结果料温稳定,发菌状况良好,三次发酵培养料的关键理化指标与国际标准化隧道的三次发酵培养料无显著差异.该发酵隧道建造成本低、风险可控,可以用于小批量双孢蘑菇培养料的试验与生产,是国内大型发酵隧道小型化的初步探索,为双孢蘑菇培养料发酵领域的新实践.

In this study, changes in metabolites during the fermentation of Agaricus bisporus compost under the Shanghai Lianzhong secondary fermentation method and Jiangsu Yuguan tertiary fermentation method were analysed by applying gas chromatography–mass spectrometry (GC–MS) to understand the differences in metabolites under different fermentation methods and find metabolic markers at different fermentation stages in different fermentation methods. The results showed that 1002 compounds were identified. Based on the differential metabolites from pathways of significant enrichment, it was found that L-aspartic acid and 5-aminobenzolevulinic acid could be used as potential metabolic markers to evaluate the phase 2 fermentation method of Shanghai Lianzhong and the phase 3 fermentation method of Jiangsu Yuguan, respectively. This study provides a reference for the preparation of quality-stable fermentation materials and further understanding of the cultivation of A. bisporus with fermentation materials.

抗氧化酶和热激蛋白是双孢蘑菇Agaricus bisporus抵御逆境胁迫的重要蛋白,高温胁迫下菌丝会通过二者基因的差异表达来减少对自身的损伤.通过对双孢蘑菇菌丝进行40℃热胁迫处理0-120min后发现,随着热胁迫时间延长,菌丝生长速度降低、气生菌丝增多和菌丝分叉明显.转录组分析抗氧化酶和热激蛋白基因差异表达发现,在热胁迫30-60min时抗氧化酶基因gpx、ppo3和cat3与热激蛋白基因hsp、hsp70-1和hsp70-17上调表达明显,而在90-120min时抗氧化酶基因ppo1、ppo2和cat2与热激蛋白基因hsp16、hsp70-14、hsp70-3和hsp70-16上调表达明显.跟踪抗氧化酶活性发现,热胁迫能激活过氧化氢酶(CAT)和过氧化物酶(POD),使酶活性提高2-3倍;同时热胁迫降低了超氧化物歧化酶(SOD)酶活性,而对多酚氧化酶(PPO)影响不明显.此外,研究还发现热胁迫能使双孢蘑菇积累更多的超氧阴离子氧化自由基(O2-),从而对菌丝造成损伤.因此,双孢蘑菇在热胁迫过程中可以通过启动不同的抗氧化酶和热激蛋白基因表达来抵御高温胁迫对菌丝造成的损伤,其中CAT和POD可能起到主要清除氧化自由基的作用,对双孢蘑菇耐高温基因的初步研究为选育耐高温品种奠定基础.

以传统菇房发菌料为对照,从菌丝生长及培养料降解角度研究了隧道发菌对双孢蘑菇(Agaricus bisporus)栽培的影响.结果表明:隧道发菌料的菌丝生长速度比菇房发菌料快13.6％,基因拷贝数是菇房发菌料的3.7倍;在菌丝生长阶段,隧道发菌料中的蛋白酶、漆酶、锰过氧化物酶、滤纸酶、酸性木聚糖酶活性显著提高,是菇房发菌料的1.2～3.0倍,隧道发菌料中半纤维素、纤维素、木质素利用率高于菇房发菌料,与酶活性结果一致;在子实体生长阶段,隧道和菇房发菌料的胞外酶活性及氮、木质纤维素的利用率均没有显著差异.

为探讨双孢蘑菇(Agaricus bisporus)培养料制作过程中预湿阶段不同堆置时间对麦秸秆温度、结构、持水量及成分的影响,将麦秸秆方捆浸泡90 s至饱和,横排堆置于场地上,分别堆置0(对照)、24、48、72、96、120 h.每组随机取3捆测量温度,采用扫描电子显微镜观察麦秸秆的内外表面结构,采用傅里叶变换红外光谱仪检测外表面的官能团信息,并测定持水量以及可溶性糖、半纤维素、纤维素和木质素含量.结果 表明:麦秸秆堆置0～72 h温度由16.6℃(0 h)升高至64.5℃(72 h);堆置24～48 h蜡质晶体明显减少,堆置48 h后麦秸秆内表面结构开始打开;堆置24～120 h Si-O伸缩振动峰和多糖类的C-O伸缩振动峰强度降低,表明麦秸秆外表面蜡质层、多糖类物质变少;麦秸秆持水量由堆置0h的565.2％增加至72 h的701.8％,在堆置24 h后可溶性糖含量迅速下降,在堆置24～72 h期间木质纤维素组分没有变化;堆置96 h时,麦秸秆方捆温度达到69.3℃,与堆置0h相比麦秸秆中半纤维素含量降低了16.9％;堆置120 h,麦秸秆方捆的温度降低至65.5℃,麦秸秆结构破坏明显,木质纤维素骨架暴露,与堆置0h相比纤维素和木质素含量分别下降了11.7％和13.6％.

本研究以双孢蘑菇Agaricus bisporus工厂化菌株A15和筛选得到的耐高温菌株A15-TH为研究对象,比较了高温胁迫对两个菌株菌丝生长的影响,并从氧化损伤修复及基础碳代谢-糖酵解途径两个角度探索双孢蘑菇对高温胁迫的响应及耐热机理.高温胁迫下,对照菌株A15的菌丝生长速度降低,菌丝分叉增加;而耐高温菌株A15-TH菌丝生长速度高于A15,菌丝形态优于对照菌株,表现出对高温具有一定的耐受性.对两个菌株高温胁迫下氧化损伤及抗氧化酶系统进行研究发现,高温胁迫30-90min导致对照菌株A15的三磷酸腺苷(ATP)含量下降54.4％-59.6％,线粒体复合物Ⅰ、Ⅱ、Ⅲ活性升高,超氧阴离子(O2-)含量增加了34.9％-71.3％;此外高温胁迫降低了超氧化物歧化酶(SOD)的活性,影响了O2-的清除效率.耐高温菌株在受到高温胁迫后的氧化损伤及氧化修复效果与对照菌株不同,一方面体现在正常状态下维持较低的细胞能量代谢和较高的ROS合成量;另一方面抗氧化系统中sod1、sod2、cat1与对照菌株相比有不同程度的上调,SOD和过氧化氢酶(CAT)活性增强,可以更有效地清除过量的活性氧,减轻高温对菌丝的氧化损伤.尤其在高温胁迫120min时,A15的线粒体功能及抗氧化系统受到严重损伤,线粒体复合物Ⅰ、Ⅱ、Ⅲ活性和CAT活性大幅度下降,但是A15-TH线粒体复合体Ⅰ、Ⅲ活性分别增加至正常状态下的1.4倍和8.9倍,CAT活性比对照菌株高128％,维持了正常的线粒体功能及对活性氧的有效清除.进一步研究发现高温胁迫下,双孢蘑菇菌丝的己糖激酶和丙酮酸激酶活性增加,糖酵解途径加快;耐高温菌株A15-TH在正常状态下和高温胁迫下,己糖激酶和丙酮酸激酶的活性均高于对照菌株A15,具有更活跃的碳代谢.

以麦秸秆配方为对照,测定不同配方培养料在不同阶段的理化指标、质量损失以及不同配方栽培的双孢蘑菇产量、成品菇率,筛选适合三次发酵技术的稻秸秆工厂化栽培双孢蘑菇(Agaricus bisporus)的配方.结果 表明:从预湿至三次发酵结束,供试配方的pH、含水量、灰分、C/N和纤维素、半纤维素、木质素含量变化趋势相近.从菇蕾期开始,与对照相比,配方Ⅰ(37.5％麦秸秆、32％鸡粪、17.5％稻秸秆、7.5％棉籽壳、5％石膏、0.5％花生粕)和配方Ⅱ(36％鸡粪、30.5％麦秸秆、21％稻秸秆、7％棉籽壳、5％石膏、0.5％花生粕)的pH、含水量、C/N和纤维素、半纤维素含量递减速度较慢,灰分含量递增速度较慢;在供试配方中,配方Ⅱ一次发酵培养料质量损失较低、产量和成品菇率较高,可用于工厂化栽培双孢蘑菇.

The cultivation of Agaricus bisporus with compost made from wheat (Triticum aestivum L.), rice (Oryza sativa L.), and reed (Phragmites australis Trin.) straw was investigated. Straw degradation was analyzed at the microscopic level, and the corresponding changes in the breakdown of different lignocellulose components during different phases of composting and mushroom production helped in understanding the yield-limiting factors of using different straws to grow mushrooms. The wheat straw compost resulted in the highest mushroom production and had the highest bioconversion efficiency. The rice straw was limited by the softer texture, which resulted in low-porosity and overdecomposed compost in the composting process and decreased the amount of available lignocellulose during mycelial growth. Although reed straw had the largest carbon resources, its utilization rate was the lowest. The hard structure, low water holding capacity, and high porosity increased the recalcitrance of reed straw to degradation and prolonged the composting time, which resulted in large N and C losses and an increased C/N ratio. Moreover, reed straw failed to transform into “ready-to-consume C” in composting. Therefore, a high C/N ratio and deficiency of available nutrition decreased the utilization efficiency of the lignocellulosic components by A. bisporus during mycelial colonization and mushroom production. The investigation revealed that degradability by and availability to microbiota and A. bisporus seemed to be the overriding factors for optimizing the composting process with different straw types. • The physical structure of compost has a significant influence on the composting process. • Degradability and availability are key factors in compost quality evaluation. • Lignocellulose utilization efficiency positively correlated with mushroom yield.

为了探讨施肥对'飞寒樱'苗木生长的影响,设计复合肥和微生物菌剂6种不同配方施肥处理,对'飞寒樱'苗木进行施肥,调查分析'飞寒樱'苗木叶片大小、叶绿素含量、地径和新稍生长量等指标,结果表明:施肥对'飞寒樱'苗木叶片大小、叶绿素含量、新稍和地径生长均有促进作用,单独使用微生物菌剂对'飞寒樱'苗木新稍和地径生长的促进作用不显著,而单独使用复合肥及一定剂量的微生物菌剂与复合肥混合使用对'飞寒樱'苗木叶片大小、叶绿素含量、新稍和地径生长均有明显的促进作用.通过比较分析筛选出的最佳施肥处理为:复合肥150 g+微生物菌剂100 g,其次是复合肥150 g+微生物菌剂50 g.

为了探究不同浓度的钠盐对双孢蘑菇A15菌株菌丝生长特性及工厂化生产产量的影响,分别测定了醋酸钠、柠檬酸钠处理后双孢蘑菇的菌丝生长速度、菌丝生物量以及工厂化栽培的产量.结果表明:在PDA培养基中添加0.2 g/L醋酸钠或0.5 g/L柠檬酸钠均可显著提高菌丝生长速度;在液体培养基中添加0.1 g/L醋酸钠或0.2 g/L柠檬酸钠均可提高菌丝的生物量,缩短培养周期.栽培试验表明:在培养料中喷施0.5 g/L柠檬酸钠能够增加双孢蘑菇产量,而喷施醋酸钠对双孢蘑菇产量无显著影响;覆土后喷施0.1 g/L醋酸钠和1.0 g/L柠檬酸钠使双孢蘑菇产量分别提高17.4％和21.6％.可见,适量的醋酸钠和柠檬酸钠溶液能够促进双孢蘑菇菌丝生长,提高生物量和工厂化生产产量.

本试验采集了双孢蘑菇工厂制备的二次发酵料,通过配制不同类型的选择培养基,采用涂布分离的方法,于温度为55℃、湿度为60％～70％的恒温培养箱中培养,从二次发酵料浸出液中得到高温菌单菌落,再进行纯化,然后提取纯化后的高温菌基因组DNA,采用NCBI物种相似度比对进行鉴定.结果表明,在马丁氏培养基上得到棕黑腐质霉(Humicola fuscoatra)和短柄帚霉(Scopulariopsis brevicaulis),相似度对比均达到99.4％以上.

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.

Hypsizygus marmoreus is an important commercial edible fungus, but the lack of basic studies on this fungus has hindered further development of its commercial value. In this study, we found that the treatment of damaged vegetative mycelia with 1 mM L-ascorbic acid (ASA) significantly increased the antioxidant enzyme activities (GPX, GR, CAT and SOD) and antioxidant contents (GSH and ASA) and reduced the ROS levels (H2O2 and O-2(-)) in mechanically damaged mycelia. Additionally, this treatment increased mycelial biomass. At the reproductive stage, our results demonstrated that the treatment of damaged H. marmoreus mycelia with 2.24 mM ASA significantly increased the antioxidant enzyme activities (GPX, GR, GST, TRXR and CAT), endogenous ASA contents and GSH/GSSG ratios in different developmental stages and significantly decreased the MDA and H2O2 contents. Furthermore, this study showed that the expression levels of the antioxidant enzyme genes were consistent with the enzyme activities. Damaged mycelia treated with ASA regenerated 2-3 d earlier than the control group and showed significantly enhanced fruiting body production. These results suggested that exogenous ASA regulated mycelia intracellular ASA content to increase mycelial antioxidant abilities, induce the regeneration of damaged mycelia and regulate the development of fruiting bodies in H. marmoreus. (C) 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

为了探明漆酶在斑玉蕈生长发育过程中的功能,对斑玉蕈转录测序预测的13个漆酶基因序列进行分析、鉴定和构建分子系统发育树;检测了不同生长发育时期漆酶的活性和漆酶基因表达水平.研究结果显示:13个基因片段中有10个是漆酶基因.不同的漆酶同工酶之间进化关系存在明显差异,大多数漆酶与木腐菌(金针菇Flammulina filiformis和侧耳属Pleurotus)进化关系较近.对斑玉蕈不同生长发育时期的酶活检测结果显示,从斑玉蕈的菌丝恢复期到钉头期,漆酶活性逐渐升高,而在子实体形成后期酶活逐渐降低.对培养40d、60d和80d的菌丝样品以及不同生长发育时期的样品进RT-qPCR检测,结果显示在菌丝营养生长时期,大多数漆酶基因在第40-60天表达量持续增加1-3倍,而在第60-80天时表达量出现降低的情况.而在生殖生长时期,大多数漆酶基因在转色期或者原基期相对表达量达到最大值,并在子实体期出现降低,这与漆酶活性的检测具有一致性.Icc3、Icc7、Icc8和Icc9在斑玉蕈生殖生长过程中相对表达量出现了10-100倍的上调.这说明从菌丝培养到菌丝扭结形成子实体和子实体发育的过程中,不同的漆酶可能发挥着不同的作用,表达量较高的漆酶基因可能对基质降解和子实体形成起主要作用.

为了验证艾加3号生物肥在农业生产中的增产增收作用,以玉米为试验对象,开展了田间小区试验.试验结果表明:艾加3号生物肥可以使玉米提早成熟,增产增收效果显著.