Continuous cropping obstacle (CCO) is becoming a major restrictive factor limiting the sustainable development of morel industry. The species-specific autotoxicity of extracellular self-DNA (esDNA) may be one of the primary drivers underlying the occurrence of CCO. In this study, the effects of short fragments (≤250 bp) of esDNA or extracellular DNA (exDNA) on mycelial growth of cultivable Morchella eximia and M. sextelata were assayed. These effects were quantified using a response index (RI). The results indicated the dose-dependent, strain-specific, and conspecific autotoxicity of esDNA in cultivable morels. At ecologically relevant DNA concentrations, the strain-specific and conspecific growth inhibitory effects of esDNA in tested Morchella strains were consistently negative (RI < 0). Additionally, our study found that the growth-inhibitory effects of exDNA from M. sextelata on M. eximia strains were weaker than those observed in the reverse scenario. Taken together, our study suggests, for the first time, the conspecific autotoxicity of esDNA in cultivable Morchella under laboratory conditions, providing novel insights into the potential mechanisms of CCO and highlighting its prospective applications in morel production.

p-coumaric acid (p-CA) is one of the main allelochemicals of cultivable Morchella mushrooms. However, its toxicity mechanism has not been elucidated. Therefore, we used physiological and comparative transcriptomic analyses to reveal its toxicity mechanism. The results suggest that the mycelial growth and sclerotial production of M. importuna were promoted under treatment with a low dosage of p-CA (10 μg/mL). The treatment induced moderate reactive oxygen species (ROS) accumulation, with an upregulation of genes associated with antioxidant regulation, energy supply and damage repair. In contrast, oxidative stress induced under treatment with a high dosage of p-CA (50 μg/mL) led to strain ageing. The contents of ROS were significantly increased, along with decreased peroxidase and catalase activity. Moreover, the genes associated with H2O2 synthesis were upregulated, while those responsible for H2O2 decomposition, non-enzymatic antioxidant components and damage repair were downregulated. Meanwhile, the carbohydrate and lipid metabolic pathways, and the signal transduction and cell division pathways, were impaired. Taken together, moderate stress induced under a low concentration of p-CA promotes the mycelial growth and sclerotial metamorphosis of M. importuna. This study provides new insights into the potential mechanisms of continuous cropping obstacles in the cultivation of morel mushrooms, which is of great importance for the practical aspects of mushroom cultivation.

ABSTRACT True morels are precious edible and medicinal mushrooms. The sustainable development of morel cultivation necessitates urgent breeding programmes to develop improved varieties or strains. Some soil bacteria, including Pedobacter spp., are recognised as morel's beneficial microbes. In this paper, the potential beneficial bacterium Pedobacter sp. DDGJ with minor chitinolytic activity was isolated and confirmed for improving morel's mycelial growth and identified using a combination of morphological characteristics, 16 S rRNA gene sequencing, and comparative genomics. Then, it was introduced into the hyphal cells of three cultivated Morchella strains (M. sextelata 13, M. eximia SM, and M. importuna Y2) through a confrontation culture method. The successful establishment of artificial endosymbiosis was confirmed by PCR assay, green fluorescent protein gene (gfp) localisation, and the cryo‐preparation system conjunction with high‐resolution field emission scanning electron microscopy. Laboratory assay indicated that artificial endosymbiosis of the bacterium significantly enhanced morel's growth potential, resistance to allelochemicals of 4‐coumaric acid and antagonistic capability against the pathogenic fungus Fusarium oxysporum and soil‐dominant fungus Chaetomium globosum. Moreover, the outdoor planting experiment showed significant increase in productivity among the endosymbiotic morel strains. The study provides an additional tool to improve Morchella strains with high yield and strong stress resistance.

Natural acylsucrose, often found in the glandular trichomes of Solanaceae plants, has potential applications in many industries, including food, cosmetics, and pharmaceuticals. In this study, we engineered an Escherichia coli strain to complete the biosynthesis of acylsucroses through whole-cell transformation. Using acylsucrose acyltransferases and CoA ligases, acylsucroses, including monoacylsucrose S1:5 ("S" represents an acylsucrose backbone, the number before the colon indicates the number of acyl chains, and the number after the colon indicates the sum of carbons in all acyl chains), diacylsucrose S2:10, triacylsucrose S3:14, and triacylsucrose S3:15 were synthesized from the substrate sucrose and short branched-chain fatty acids by the engineered E. coli EcoSE07, of which S3:15 was the primary product. Several strategies were applied to improve acylsucrose production, including codon optimization, constitutive promoter replacement, and serial resting cell assays. The use of fed-batch fermentation with an engineered E. coli strain of EcoSE22 containing a constitutive promoter further improved the production of acylsucroses. Serial resting cell assays with an optical density of 50 at 600 nm significantly increased the production of acylsucroses S3:15 and S2:10. These findings will facilitate the synthesis of natural acylsucroses through whole-cell transformations and provide the potential for future industrial applications.

Continuous cropping obstacle has been becoming the bottleneck for the stable development of morel cultivation. The allelopathic effect of soil allelochemicals may play an instrumental role in the morel soil sickness. In this study, the allelochemicals were identified by gas chromatography-mass spectrometry (GC-MS) combined with in vitro bioassay. A total of 61 chemical substances were identified through the GC-MS analysis of 12 replanting and control soil samples, comprising 10 phenolic acids, 36 acids, 3 aldehydes, etc. Among which, 15 compounds with values of variable importance for the projection (VIP) greater than 1 in the orthogonal partial least squares-discriminant analysis (OPLS-DA) were selected as the differential metabolites between soil samples of continuous cropping and control. The bioassay showed that 4-coumaric acid and vanillic acid exhibited inhibitory effect on mycelial growth of three cultivated Morchella mushrooms (M. sextelata, M. eximia and M. importuna) under soil native concentrations. Analysis of potential biosynthetic pathways of phenolic acids found that the 3 cultivable Morchella mushrooms are unable to synthesize phenolic acid allelochemicals. Therefore, although they were detected in trace amounts in static culture broth of M. sextelata, the two phenolic acids can only be defined as morel allelochemicals rather than autotoxins. Taken together, we found two morel allelochemicals that may derive from morel related microbes and plants, which will be helpful for further fundamental study and application in morel artificial cultivation.

The prominent problem of continuous cropping obstacle has been frustrating the morel farming. To deepen the understanding on morel continuous cropping obstacle, the allelopathic effects of phenolic acid extracts from morel continuous cropping soils on growth and development of Morchella sextelata, M. eximia, M. importuna, pathogenic fungus Fusarium sp. and soil-dominant fungus Chaetomium sp. were investigated. These effects were expressed as response index (RI). Under actual content of phenolic acids (6.150 μg/g fresh mixed continuous cropping soil), the mycelial growth of all tested morel strains was inhibited (RI < 0), while the allelopathic effect of control phenolic acids (4.252 μg/g fresh mixed control soil) was between promotion and inhibition, which suggested that the phenolic acid extracts from morel continuous cropping soils may exhibit certain extent of autotoxicity for the existence of morel-specific allelochemicals. In addition, the aggravated pigmentation and reduced occurrence of sclerotium in three Morchella fungi at growth inhibitory concentrations of phenolic acids indicated the induction of morel strain aging. Meanwhile, most concentrations of phenolic acids showed stimulatory effects on sporulation of Fusarium sp. and Chaetomium sp. (RI > 0), manifesting the enrichment of soil-borne pathogenic fungi and dominance of certain fungal population in soil ecosystem. Collectively, the allelopathic effects of phenolic acid extracts play an instrumental role in morel continuous cropping obstacle. The study will be beneficial for healthy development of morel artificial cultivation.

Morels are one of the most highly prized edible and medicinal mushrooms worldwide. Therefore, historically, there has been a large international interest in their cultivation. Numerous ecological, physiological, genetic, taxonomic, and mycochemical studies have been previously developed. At the beginning of this century, China finally achieved artificial cultivation and started a high-scale commercial development in 2012. Due to its international interest, its cultivation scale and area expanded rapidly in this country. However, along with the massive industrial scale, a number of challenges, including the maintenance of steady economic profits, arise. In order to contribute to the solution of these challenges, formal research studying selection, species recognition, strain aging, mating type structure, life cycle, nutrient metabolism, growth and development, and multi-omics has recently been boosted. This paper focuses on discussing current morel cultivation technologies, the industrial status of cultivation in China, and the relevance of basic biological research, including, e.g., the study of strain characteristics, species breeding, mating type structure, and microbial interactions. The main challenges related to the morel cultivation industry on a large scale are also analyzed. It is expected that this review will promote a steady global development of the morel industry based on permanent and robust basic scientific knowledge.

目的:烟草酰基糖酰基转移酶(Nicotiana tabacum acylsugar acyltransferase,NtASAT1)可催化短支链脂肪酸与蔗糖形成蔗糖单酯.利用大肠杆菌原核表达系统分析NtASAT1的表达,并对其进行纯化及功能验证.方法:首先利用生物信息学软件对烟草NtASAT1的理化性质、二级结构及同源性进行分析;之后从烟草腺毛的cDNA中克隆NtASAT1基因,并构建表达载体,研究其在BI21(DE3)中的表达情况;最后利用镍柱对NtASAT1进行纯化,将纯化后得到的目标蛋白进行酶反应,通过液相色谱-质谱法(liquid chromatography-mass spectrometry,LC-MS)检测产物并验证其功能活性.结果:C端截短93个氨基酸之后的NtASAT1能够在BL21(DE3)中表达.表达后的蛋白质大部分以不可溶状态存在于细胞中,不同的诱导剂浓度、诱导时间及诱导温度对于蛋白质可溶性表达影响不明显.利用镍柱对其纯化得到目标蛋白,加入底物进行酶反应,通过LC-MS检测到产物蔗糖单酯的存在.结论:通过克隆及纯化得到重组蛋白trNtASAT1,加入底物进行酶反应后可检测到产物,证明纯化后的NtASAT1具有一定功能,为ASAT类酶的纯化及应用奠定基础.

Morels, which belong to the Ascomycete genus Morchella, are highly valued edible fungi treasured by gourmet chefs worldwide. Some species are saprotrophic and others are able to form facultative mycorrhizal-like associations with plant roots without establishing true ectomycorrhizal symbioses. In general, it is considered that the formation of asexual spores, or mitospores, is an important step in the life cycle of morels. However, ultrastructure characterization and physiological attributes of morel mitospores have received little attention. In this contribution, the mitospores of M. sextelata were successfully induced under laboratory conditions and their ultrastructure, occurrence, germination, physiological characteristics and mating type gene structure were studied. Mitospore production was closely related to aeration, nutrition and humidity conditions. The average germination rate of mitospores on different media and under various induction stimuli was very low, with an average of 1/100,000. Based on the ultrastructure characterization, low germination rate, growth rate decline, rapid aging and mating genotyping, it was concluded that the mitospores of M. sextelata had lost their conventional function as conidia and might act more as mate sperm-like (gamete) structures. Thus, this study contributed to a deeper understanding of the life cycle of the economically and ecologically important morel fungal group.

Strain aging has been mainly contributing to the "uncertainty" of Morchella farming. The situation calls for urgent quantitative assessment of strain aging in cultivated Morchella mushrooms. In this paper, systemic senescence of the productive strains of M. eximia, M. importuna, and M. sextelata was achieved through successive subculturing to provide subcultures with different degree of aging for further studies. Then the quantitative assessment of morel strain aging was conducted by activity assay of amylase and xylanase using dinitrosalicylic acid (DNS) method. The results suggested that both activity of amylase and xylanase decreased along with the rise of subculture times. Meanwhile, the correlation between xylanase activity and time of subculturing in the tested morel strains was higher than that of amylase assay. Consequently, assay of amylase and xylanase activity by DNS method can be used in the quantitative assessment of morel strain aging, and assay of xylanase activity is the better alternative. The work will improve the settlement of "uncertainty" in the morel industry and thus be beneficial for stable development of morel farming.

Verpa spp. are potentially important economic fungi within Morchellaceae. However, fundamental research on their mating systems, the key aspects of their life cycle, remains scarce. Fungal sexual reproduction is chiefly governed by mating-type genes, where the configuration of these genes plays a pivotal role in facilitating the reproductive process. For this study, de novo assembly methodologies based on genomic data from Verpa spp. were employed to extract precise information on the mating-type genes, which were then precisely identified in silico and by amplifying their single-ascospore populations using MAT-specific primers. The results suggest that the MAT loci of the three tested strains of V. bohemica encompassed both the MAT1-1-1 and MAT1-2-1 genes, implying homothallism. On the other hand, amongst the three V. conica isolates, only the MAT1-1-1 or MAT1-2-1 genes were present in their MAT loci, suggesting that V. conica is heterothallic. Moreover, bioinformatic analysis reveals that the three tested V. bohemica strains and one V. conica No. 21110 strain include a MAT1-1-10 gene in their MAT loci, while the other two V. conica strains contained MAT1-1-11, exhibiting high amino acid identities with those from corresponding Morchella species. In addition, MEME analysis shows that a total of 17 conserved protein motifs are present among the MAT1-1-10 encoded protein, while the MAT1-1-11 protein contained 10. Finally, the mating type genes were successfully amplified in corresponding single-ascospore populations of V. bohemica and V. conica, further confirming their life-cycle type. This is the first report on the mating-type genes and mating systems of Verpa spp., and the presented results are expected to benefit further exploitation of these potentially important economic fungi.

该研究以酿酒酵母(Sacchromyces cerevisiae)IMX581为出发菌株,利用Crispr-Cas9基因编辑的方法敲除其蔗糖消耗途径中的蔗糖转化酶基因Suc2和麦芽糖酶基因MAL32、MAL12、MAL22,构建工程菌株IMX581△Suc2和IMX581△Suc2△MAL32△MAL12△MAL22,并对其生长特性及蔗糖消耗情况进行分析.结果表明,当初始OD600 nm值为0.1时,菌株IMX581△Suc2在以蔗糖为唯一碳源的培养基上仍然可以生长,但培养56 h时,生物量是出发菌株IMX581的52％,而菌株IMX581△Suc2△MAL32△MAL12△MAL22几乎不生长.当初始OD600 nm值为0.1、5.0及10.0时,菌株IMX581△Suc2和IMX581△Suc2△MAL32△MAL12△MAL22培养72 h后,蔗糖的消耗率较出发菌株IMX581均显著下降,分别为28.5％、35.5％、38.5％和7.0％、18.4％、22.5％.因此,成功构建了显著降低蔗糖消耗的菌株IMX581△Suc2△MAL32△MAL12△MAL22,为研究酿酒酵母对蔗糖的利用及调控提供了参考.

基于浓香型白酒挥发性化合物的主要特征和基本信息(感官阈值、风味描述、嗅闻时间强度、香气活力值等),综述了浓香型白酒重要呈香物质种类、香气感官描述及其关联性挥发性化合物,以及挥发性化合物的地域特征等,认为:含量低但种类多、感官阈值范围广、香气特征多样及交互作用复杂为浓香型白酒微量成分的主要特征;对浓香型白酒中115种挥发性化合物的感官阈值及其香气贡献度进行梳理,归纳出其中38种重要呈香物质,主要为酸类、酯类、芳香族及醇类化合物;挥发性化合物的组成和含量与浓香型白酒地域特征和香气感官存在明显关联性,如吡嗪类化合物是浓香型白酒中呈现焙烤香的物质基础,乙酸乙酯、2,3,5-三甲基吡嗪、2-戊酮等22种化合物是区分川派和江淮派浓香型白酒的特征挥发性化合物.针对"风味轮"构建及浓香型白酒感官评价中存在的不足,指出未来应就白酒感官描述术语标准体系构建、香气感官特征与挥发性化合物组成的关联性、白酒微量成分之间的交互作用等进行深入研究,促进浓香型白酒关键风味成分筛选及溯源、异味物质控制、系统感官评价方法建立及基于地域环境特征的个性化生态酿造理念形成,为白酒产业高质量发展奠定基础.

羊肚菌是世界知名的食药兼用真菌.自2012年以来,我国羊肚菌人工栽培的规模不断扩大,然而生产不稳定一直困扰着羊肚菌产业的发展.造成羊肚菌生产不稳定的重要原因是菌株栽培适宜性评价技术不完善、栽培菌株质量不稳定等.与分类学属于担子菌门的多数食用菌相比,属于子囊菌门的羊肚菌在物种多样性、交配型基因丢失、快速老化等方面表现突出,因而关于其栽培菌株相关性状的检测要求更高.基于长期理论研究和生产实践经验,提出了包括菌株身份(Identity)识别、交配型(Mating Type)基因检测和菌株活力(Vitality)测定的羊肚菌生产菌株栽培适宜性IMV评价系统,叙述了该评价系统的技术必要性和具体操作方法.两个产季的大田栽培实际应用情况表明,该评价系统可以实现对羊肚菌生产菌株的科学、系统的筛选与评估,以确保适宜菌株用于人工栽培,增加羊肚菌人工栽培的稳定性,进而推动羊肚菌产业的持续和稳定发展.

Morel mushrooms are iconic ascomycetes that are economically valuable and have attracted the attention of a large number of scientific researchers around the world. However, the tendency of morel fungi to undergo heterokaryosis through plasmogamy is not well understood. In this study, three populations of stipe tissue isolates were isolated from ascocarps of Mel-21 (Morchella sp.). Mating type gene analysis showed that a large proportion of the mating type idiomorphs were missing in the population. According to the mating type structure, one population could be divided into three categories: isolates containing the only Mat1-1, the only Mat1-2, and both isolated containing both Mat1-1 and Mat1-2 idiomorphs. While random amplified polymorphism DNA (RAPD) molecular markers showed clear polymorphisms among the tissue isolates from the same ascocarp, and there was no consistency between this polymorphism and the mating type structure. The characteristic band of RAPD was further developed into a more specific and stable sequence-characterized amplified region (SCAR) marker to further detect the isolate population, and the results were consistent with the RAPD polymorphism. This study showed that the stipe tissue of Mel-21 morel ascocarp had obvious genetic diversity and was more than two homogenous karyotypes. We speculated that this might be due to the frequent fusion of the hyphae germinated by the genetically diverse ascospores in nature, which eventually led to the heterokaryotic state in the stipe of the ascocarps. This heterokaryotic state should be related to the competition at the nucleus level and/or the heterogeneous distribution of nuclei in the tissue. This genetic polymorphism might be related to the character diversity, ecological environment diversity and rapid species evolution of Morchella.

采用宏转录组学技术,对浓香型白酒酒醅中具有生理活性的细菌和真菌群落在窖池不同空间位置上的组成及差异表达基因进行研究.结果表明:细菌群落共检测到87个门、78个纲、165个目、396个科、1612个属和7234个种,真菌群落共检测到8个门、26个纲、58个目、127个科、223个属和394个种,且酒醅上、中、下层之间的活性微生物数目差异较小;各层酒醅中的细菌和真菌群落组成无明显差异,均以厚壁菌门和子囊菌门为主要优势细菌门和真菌门,以芽孢杆菌纲和酵母纲为主要优势细菌纲和真菌纲,以乳杆菌属为主要优势细菌属,以酵母菌属(上层和下层)或Scheffersomyces(中层)为主要优势真菌属;下层与中层酒醅、中层与上层酒醅之间的差异表达基因数量较多,分别为90个和67个,且主要富集在RNA降解和糖酵解/糖异生通路上,而各层酒醅之间的差异表达基因的功能大部分为代谢活动;整体上,中层酒醅中的活性微生物菌群对RNA降解的影响最低,下层酒醅中的活性微生物菌群在代谢活动上的贡献最大.

利用宏转录组学技术解析浓香型白酒主发酵期过程中(3、7、25 d)酒醅活性生物群落的多样性、演替及差异转录基因.结果表明,酒醅活性生物群落由6个界、124个门、195个纲、2 824个属及部分未知和不可鉴定的种属组成,其中可鉴定生物主要由细菌和真菌组成.随着发酵时间延长,真菌相对含量由42.8％降至0.01％,细菌相对含量由17.7％增至56.3％,其中厚壁菌门和子囊菌门分别是驱动细菌和真菌组成变化的主要菌门.3个发酵节点的酒醅生物差异表达基因数分别为6 895(3 dvs7d)、2 640(7dvs25d)和6 124(3dvs25d).发酵过程中,下调差异基因数量明显多于上调差异基因数量.发酵3～7 d,酒醅微生物上调的差异基因显著富集到与糖和醇类化合物代谢的功能条目,而在整个主发酵期,下调的差异基因主要富集到与细胞组分或与其相关的生物过程条目中.本研究有助于进一步阐明浓香型酒醅复杂生态体系中活性微生物群落结构、演替和功能,为深入揭示浓香型白酒固态酿造机理提供基础数据和理论支撑.

以牛粪堆肥为天然菌源、葡萄糖为发酵底物,考查微波辐射预处理菌源对生物暗发酵制氢的影响,并采用Illumina NovaSeq高通量测序技术分析微波辐射预处理前后菌源中细菌和古生菌的群落结构变化,揭示预处理引起的菌源微生物群落结构变化对暗发酵制氢的影响机制.结果表明,与对照组相比,微波辐射预处理后菌源的累积产H2量和丁酸含量分别提高了27.9％和24.1％,菌源中微生物多样性下降,主要产氢微生物芽孢杆菌的相对丰度增加,产甲烷耗氢菌的相对丰度减小;产氢微生物的富集及耗氢菌活性的抑制可有效提高暗发酵制氢系统的产H2量.