羊肚菌菌种继代培养代数与菌种退化关系

为探究羊肚菌继代培养代数同菌种退化的关系,对梯棱羊肚菌菌丝体进行继代培养,并对各代菌丝显微形态、菌落形态、菌丝生长速度和胞外酶活性进行观察和测定,以揭示母种的频繁继代与菌种退化的关系.研究结果表明:随着继代培养代数的增加,菌丝生长速度、菌丝显微形态、菌落形态以及酶活性总体呈现下降趋势,要保持菌种的优良性状及活性,继代代数少于6代为宜.其中,继代1代(G1代)在22℃条件下,菌丝体的生长速度呈稳定上升趋势,菌丝浓密度均一,主干菌丝与侧生菌丝间的分叉约为45°,可以形成大量的菌核,且酶活力最强.     

林生地霉血液分离株的形态学观察

目的 进一步了解林生地霉的形态学特征和产孢方式。方法受试菌株复苏后,转种于沙堡培养基,37℃和27℃温箱培养3周,每天观察菌落生长情况,并对其丝状型和酵母样型两种菌落进行光镜观察。在培养1周时,挑取菌落常规制片后行扫描电镜和透射电镜观察。结果 培养第2～3d,菌落开始生长,初为白色绒毛样、粉末样,1～2周后菌落均转变为乳酪样,其中37℃下培养的菌落转变较早。菌丝相时,光镜下可见丰富的分支、分隔菌丝,成链的关节孢子和圆形、椭圆形小分生孢子。酵母相时,光镜下可见丰富的芽生孢子,少量的厚膜孢子、外生或内生的关节孢子,菌丝稀少。扫描电镜下可见菌丝分支末端或侧面着生球形、棒状的分生孢子,末端凹陷。透射电镜下可见分生孢子与菌丝分离时全壁断裂,另一端正在出芽。结论 林生地霉包括丝状型和酵母样型两种菌落形态和链状排列的矩形关节孢子、圆形或椭圆形的小分生孢子、厚膜孢子以及分支、分隔菌丝为其形态学特征,有芽殖、外生和内生节孢子三种产孢类型。     

裂褶菌培养基形态学观察和DNA序列分析

目的通过观察裂褶菌在5种培养基上的生长状态、扫描电镜及DNA序列分析,了解该菌形态学及分子生物学等方面的特征。方法菌落转种于沙氏培养基(SDA),麦芽浸膏琼脂(MEA),马铃薯葡萄糖琼脂(PDA),玉米粉琼脂(CMA)和察氏琼脂(CZA)平皿培养基,27℃和37℃培养2周,观察菌落生长情况,进行扫描电镜检测及DNA序列分析。结果菌落在SDA,MEA和PDA上生长状态较好,呈蓬松白色羊毛状;尿素酶试验阳性,放线菌酮耐受试验阴性。光镜下见分支分隔菌丝、侧生的钉状突起及类水母体变异子实体。扫描电镜见菌丝分隔处闭锁联合、侧生钉状突起和泪滴状球形分泌物。经26S rDNAD1/D2区序列分析证实该菌株为裂褶菌。结论裂褶菌只有丝状型一种菌落形态;分支分隔菌丝及分隔处闭锁联合,侧生钉状突起和泪滴状球形分泌为其形态学特征;孢子由类水母状子实体产生。     

1株短帚霉临床分离株的形态学和rDNA序列的鉴定

目的研究短帚霉的形态学特征,产孢方式和分子生物学特性。为临床快速、准确鉴定这类真菌奠定一定基础。方法受试菌株常温复苏24h后,转种于沙堡弱培养基上,27℃和37℃温箱培养4周,每天光镜观察菌落的生长情况。在培养10d时,挑取菌落常规制片后行扫描电镜观察。并且对其rDNA进行序列分析。结果27℃培养3—4d后,菌落开始生长,初为白色膜样菌落,生长迅速,1周时形成直径达2cm的白色毡样菌落,以后菌落逐渐成灰褐色粉末状,边缘淡黄色,背面黄褐色。37℃培养2—3d后菌落开始生长呈灰褐色粉末状。光镜下可见丰富的分枝、分隔菌丝,透明、细长,分生孢子由环孢子梗产生一串,球形或柠檬形,壁厚、表面光滑或粗糙有刺,常成链状排列,自动卷曲或成团,有帚状枝。扫描电镜可见环痕梗外壁破裂,梗与孢子间产生横隔,遗留一圈环痕。向基性的连续产孢,孢子球形,菌丝或孢子表面有饰纹。rDNA序列分析证实此菌为短帚霉。结论根据培养形态,光镜和扫描电镜下表现以及DNA序列分析可以对短帚霉进行准确的鉴定。     

一株绿僵菌的分离研究

利用马铃薯葡萄糖琼脂培养基,对一株绿僵菌进行了分离培养。对不同生长发育天数的菌落特征即颜色、大小、孢子堆的形成等特点进行了描述。在光学显微镜下观察了菌丝的形态,成熟孢子及分生孢子梗的形状、颜色及连接状况。利用扫描电镜观察了分生孢子和孢子梗的超微形态。经培养特征和形态学鉴定,确定该昆虫病原真菌是金龟子绿僵菌。     

壳聚糖酶产生菌KJ0312的鉴定

通过对产壳聚糖酶菌KJ0312群体形态包括菌落大小、颜色、质地等特征的观察,和个体形态包括菌丝、分生孢子梗、分生孢子等特征的研究,以及部分18sDNA序列的鉴定,初步确定产壳聚糖酶菌KJ0312的分类地位属于淡紫拟青霉.     

蛹虫草子囊孢子萌发及其后代群体培养性状观察

从野生和人工栽培的蛹虫草子实体共分离了6个子囊孢子群体,对子囊孢子形态、萌发过程、培养性状及子实体产生等进行了观察。结果表明,蛹虫草子囊孢子为线形、多细胞,169.78~364.57×1.72~2.04μm;每个子囊孢子有56~114个细胞,每个细胞大小为1.77~4.53×1.72~2.04μm。子囊孢子弹射后13h开始萌发,30h后开始形成分生孢子。多数子囊孢子的每个细胞都能萌发,但少数子囊孢子的部分细胞不萌发。子囊孢子群体的培养性状表现多样性,以I型所占比列较大。野生群体的菌落颜色以杏橙色和淡柠檬黄色为主,而人工栽培的群体以橙铬色所占比例最大。不同个体的菌落生长速率有差异。野生群体产生扇形突变的比例高于人工栽培群体。在人工栽培的CM群体中出现了气生菌丝较多、白色、生长缓慢的个体。菌落为I型,橙铬色或杏橙色,生长速率正常,无突变的单子囊孢子菌株产生子实体的可能性较大,子实体产量较高,但大部分单子囊孢子菌株不能产生发育良好的子实体。     

6株食根结线虫真菌厚垣普奇尼亚菌的形态学比较

采用玻片和平板菌落培养方法,对6株不同来源的食根结线虫真菌——厚垣普奇尼亚菌进行了形态学观察和比较。结果表明:不同菌株在形态和培养性状上存在明显差异。各菌株在CMA培养基上的生长速率不同,QNMP0214和NRRL13094生长最快,气生菌丝发达;QNZFF0601-3菌落背面乳白色;QNAV97-5分生孢子梗细长。6个菌株均能够产生菌丝膨大和厚垣孢子,其中QNAV97-6和QNMP0214厚垣孢子着生方式除间生外还有侧生,NRRL13094厚垣孢子大量间生成串,QNZFF0601-3和QNMP0214厚垣孢子稀少。QNAV97-2、MP0214、NRRL13094和QNAV97-5能产生晶体。     

马尔尼菲青霉菌的鉴定

为了提高马尔尼菲青霉菌的分离率和鉴定的正确率,通过培养检查特征性的菌丝和观察菌落形态,通过形态学观察其在骨髓和外周血以及PAS染色的形态。马尔尼菲青霉菌为双相菌,霉菌型菌落可发现特征性的扫帚状菌丝,沙保罗培养3 d后产生水溶性玫瑰色素;骨髓和外周血细胞内外均可发现孢子;PAS染色可见菌体呈圆形、椭圆形或腊肠状,大小不一,约为2~8μm,胞壁染红色且清楚连续,在腊肠状的细胞内可见一明显的横隔,胞质不易着色。鉴定结论:特征性的扫帚状菌丝;骨髓和外周血细胞内外均可发现孢子;水溶性玫瑰色素;PAS染色对其有鉴定价值。     

不同培养基对冬虫夏草菌丝生长的影响

不同培养基对3个来源不同的冬虫夏草菌菌丝生长影响的研究结果表明,在不同培养基上,各菌株之间在菌丝萌发时间、40d菌落直径和菌落形态上表现出一定的差异。同时筛选到的最优培养基比已报道培养基的菌落生长速度快2～3倍,且菌丝为白色、粗壮,菌丝质量较优。     

Cellular automata simulations of fungal growth on solid substrates

Growth of filamentous fungi on the surface of cereal grains is a critical aspect of solid substrate fermentation (SSF). Numerous mathematical models have been developed to describe various aspects of fungal growth in SSF. These models consider hyphal geometry and nutrient availability as determinants of colony morphology and fungal physiological state. This work describes the use of cellular automata (CA) as an alternative method of modeling fungal growth. CA models reliant on a very limited set of rules or "knowledge base" display a rich array of behaviors that mimic fungal growth. By incorporating probablistic growth rules into CA models, colony characteristics such as biomass accumulation rate, colony radial growth rate, mycelial density and fungal differentiation are readily generated.     

Understanding the morphology of fungi

Filamentous fungi comprise an industrially very important collection of microorganisms, since they are used for the production of a wide variety of products ranging from primary metabolites to secondary metabolites and further on to industrial enzymes (such as proteases, lipases and antibiotics). It is known that fungal morphology is often considered as one of the key parameters in industrial production. For the production of fungal metabolite products, the desired morphology varies from one product to another. Many parameters affect the morphology of fungi during the process of fermentation, among them speed of agitation, specific growth rate, dissolved oxygen, number of spores or conidia per liter of fermentation broth are important and should be considered when higher yield is desired in the process. It is, therefore, of considerable importance to understand the mechanism underlying the morphology of the cell, its growth and product formation by filamentous fungi. Such knowledge may be used in the optimization of the microbial process. Several literatures with various fungi to study their morphology, relating enzyme or product production to the character of the fungi in the study is reviewed. It is also considered that how the process parameters affects the morphology. The aim of this communication is to review the relevant literature to understand the morphology of filamentous fungi.     

Autophagy in filamentous fungi

Autophagy is a ubiquitous, non-selective degradation process in eukaryotic cells that is conserved from yeast to man. Autophagy research has increased significantly in the last ten years, as autophagy has been connected with cancer, neurodegenerative disease and various human developmental processes. Autophagy also appears to play an important role in filamentous fungi, impacting growth, morphology and development. In this review, an autophagy model developed for the yeast Saccharomyces cerevisiae is used as an intellectual framework to discuss autophagy in filamentous fungi. Studies imply that, similar to yeast, fungal autophagy is characterized by the presence of autophagosomes and controlled by Tor kinase. In addition, fungal autophagy is apparently involved in protection against cell death and has significant effects on cellular growth and development. However, the only putative autophagy proteins characterized in filamentous fungi are Atg1 and Atg8. We discuss various strategies used to study and monitor fungal autophagy as well as the possible relationship between autophagy, physiology, and morphological development.     

Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production

Chemostat cultivation of Aspergillus niger and other filamentous fungi is often hindered by the spontaneous appearance of morphologic mutants. Using the Variomixing bioreactor and applying different chemostat conditions we tried to optimize morphologic stability in both ammonium- and glucose-limited cultures. In most cultivations mutants with fluffy (aconidial) morphology became dominant. From an ammonium-limited culture, a fluffy mutant was isolated and genetically characterized using the parasexual cycle. The mutant contained a single morphological mutation, causing an increased colony radial growth rate. The fluffy mutant was subjected to transformation and finally conidiospores from a forced heterokaryon were shown to be a proper inoculum for fluffy strain cultivation.     

An X-ray microtomography-based method for detailed analysis of the three-dimensional morphology of fungal pellets

Filamentous fungi are widely used in the production of biotechnological compounds. Since their morphology is strongly linked to productivity, it is a key parameter in industrial biotechnology. However, identifying the morphological properties of filamentous fungi is challenging. Owing to a lack of appropriate methods, the detailed three-dimensional morphology of filamentous pellets remains unexplored. In the present study, we used state-of-the-art X-ray microtomography (µCT) to develop a new method for detailed characterization of fungal pellets. µCT measurements were performed using freeze-dried pellets obtained from submerged cultivations. Three-dimensional images were generated and analyzed to locate and quantify hyphal material, tips, and branches. As a result, morphological properties including hyphal length, tip number, branch number, hyphal growth unit, porosity, and hyphal average diameter were ascertained. To validate the potential of the new method, two fungal pellets were studied—one from Aspergillus niger and the other from Penicillium chrysogenum. We show here that µCT analysis is a promising tool to study the three-dimensional structure of pellet-forming filamentous microorganisms in utmost detail. The knowledge gained can be used to understand and thus optimize pellet structures by means of appropriate process or genetic control in biotechnological applications.     

Use of pH auxostats to grow filamentous fungi in continuous flow culture at maximum specific growth rate

Abstract The pH auxostat employs a pH-derived feedback control of biomass concentration and allows continuous flow cultures of organisms to be grown at their maximum specific growth rate. We examine three modifications of the pH auxostat system for the growth of filamentous fungi and discuss the suitability of each method according to the biomass concentration desired, the medium used and the equipment available. Fusarium graminearum and Geotrichum candidum were used for this work and it was possible to maintain steady state pH auxostats of these filamentous fungi for up to 20 days. pH auxostats have not previously been described for filamentous fungi.     

丝状真菌形态控制及其在发酵过程优化中的应用

丝状真菌广泛用于发酵产业,在液体深层发酵过程中,其生长形态与产物种类及产量间存在重要关联,成为发酵过程调控的热点。采用数学模型解释丝状真菌的形态发育与调控机制,是近年来工程学界颇为关注的研究手段。本文结合自己的工作着重解释丝状真菌各种生长形态的生长机理及形态发育的数学描述方式,以及如何采用数学模型描述丝状真菌发酵过程中产物、形态及环境的关联,最终实现形态的控制,完成生物发酵过程优化。具体包括:1)丝状真菌的生长机制;2)形态发育数学模型在发酵过程优化中的应用;3)控制丝状真菌形态的策略。     

The ham-2 locus, encoding a putative transmembrane protein, is required for hyphal fusion in Neurospora crassa.

Somatic cell fusion is common during organogenesis in multicellular eukaryotes, although the molecular mechanism of cell fusion is poorly understood. In filamentous fungi, somatic cell fusion occurs during vegetative growth. Filamentous fungi grow as multinucleate hyphal tubes that undergo frequent hyphal fusion (anastomosis) during colony expansion, resulting in the formation of a hyphal network. The molecular mechanism of the hyphal fusion process and the role of networked hyphae in the growth and development of these organisms are unexplored questions. We use the filamentous fungus Neurospora crassa as a model to study the molecular mechanism of hyphal fusion. In this study, we identified a deletion mutant that was restricted in its ability to undergo both self-hyphal fusion and fusion with a different individual to form a heterokaryon. This deletion mutant displayed pleiotropic defects, including shortened aerial hyphae, altered conidiation pattern, female sterility, slow growth rate, lack of hyphal fusion, and suppression of vegetative incompatibility. Complementation with a single open reading frame (ORF) within the deletion region in this mutant restored near wild-type growth rates, female fertility, aerial hyphae formation, and hyphal fusion, but not vegetative incompatibility and wild-type conidiation pattern. This ORF, which we named ham-2 (for hyphal anastomosis), encodes a putative transmembrane protein that is highly conserved, but of unknown function among eukaryotes.     

Probing the growth dynamics of Neurospora crassa with microfluidic structures

Despite occupying physically and chemically heterogeneous natural environments, the growth dynamics of filamentous fungi is typically studied on the surface of homogeneous laboratory media. Fungal exploration and exploitation of complex natural environments requires optimal survival and growth strategies at the colony, hyphal, and intra hyphal level, with hyphal space-searching strategies playing a central role. We describe a new methodology for the characterisation and analysis of hyphal space-searching strategies, which uses purposefully designed three-dimensional microfluidics structures mimicking some of the characteristics of natural environments of the fungi. We also demonstrate this new methodology by running a comparative examination of two Neurospora crassa strains, i.e., the wild type of N. crassa -- a commonly used model organism for the study of filamentous fungi -- and the N. crassa ro-1 mutant strain -- which is deficient in hyphal and mycelial growth. Continuous live imaging showed that both strains responded actively to the geometrically confined microstructured environments without any detectable temporal delay or spatial adjustment. While both strains navigated the test structures exhibiting similar geometry-induced space-searching mechanisms, they presented fundamentally different growth patterns that could not be observed on geometrically unconfined, flat agar surfaces.     

Macroautophagy-mediated degradation of whole nuclei in the filamentous fungus Aspergillus oryzae

Filamentous fungi consist of continuum of multinucleate cells called hyphae, and proliferate by means of hyphal tip growth. Accordingly, research interest has been focusing on hyphal tip cells, but little is known about basal cells in colony interior that do not directly contribute to proliferation. Here, we show that autophagy mediates degradation of basal cell components in the filamentous fungus Aspergillus oryzae. In basal cells, enhanced green fluorescent protein (EGFP)-labeled peroxisomes, mitochondria, and even nuclei were taken up into vacuoles in an autophagy-dependent manner. During this process, crescents of autophagosome precursors matured into ring-like autophagosomes to encircle apparently whole nuclei. The ring-like autophagosomes then disappeared, followed by dispersal of the nuclear material throughout the vacuoles, suggesting the autophagy-mediated degradation of whole nuclei. We also demonstrated that colony growth in a nutrient-depleted medium was significantly inhibited in the absence of functional autophagy. This is a first report describing autophagy-mediated degradation of whole nuclei, as well as suggesting a novel strategy of filamentous fungi to degrade components of existing hyphae for use as nutrients to support mycelial growth in order to counteract starvation.