The filamentous fungal pellet and forces driving its formation

Filamentous fungi play an important role not only in the bio-manufacturing of value-added products, but also in bioenergy and environmental research. The bioprocess manipulation of filamentous fungi is more difficult than that of other microbial species because of their different pellet morphologies and the presence of tangled mycelia under different cultivation conditions. Fungal pellets, which have the advantages of harvest ease, low fermentation broth viscosity and high yield of some proteins, have been used for a long time. Many attempts have been made to establish the relationship between pellet and product yield using quantitative approaches. Fungal pellet formation is attributed to the combination of electrostatic interactions, hydrophobicity and specific interactions from spore wall components. Electrostatic interactions result from van der Waals forces and negative charge repulsion from carboxyl groups in the spore wall structure. Electrostatic interactions are also affected by counter-ions (cations) and the physiologic conditions of spores that modify the carboxyl groups. Fungal aggregates are promoted by the hydrophobicity generated by hydrophobins, which form a hydrophobic coat that covers the spore. The specific interactions of spore wall components contribute to spore aggregation through salt bridging. A model of spore aggregation was proposed based on these forces. Additionally, some challenges were addressed, including the limitations of research techniques, the quantitative determination of forces and the complex information of biological systems, to clarify the mechanism of fungal pellet formation.     

Evaluation of inoculum performance for enhancing gamma-linolenic acid production from Mucor rouxii

Aims: To facilitate a cost‐effective preparation of spore inoculum with good capacity for gamma‐linolenic acid (GLA) production from Mucor rouxii. Methods and Results: Sporangiospore production, mycelial growth ability and fatty acid composition of M. rouxii were determined. Compared with fungal cultivation on solid semi‐synthetic media, high spore production was achieved from M. rouxii grown on rice grains, particularly polished rice (30·7 g kg^?1 initial substrate). Variations in the fatty acid profiles were found in the spores grown on different types of solid media, whereas the spores obtained at different ages from cultivated polished rice showed a similar fatty acid profile. Using the inocula from different spore‐forming media and culture ages, and low temperature storage, not much change in the vegetative growth of submerged cultures or fatty acid composition of mycelia was observed. Conclusion: The spores generated on polished rice exhibited a high performance for GLA production. Age of spore and timing of spore storage at low temperature did not affect on fatty acid profile of the mycelial cultures. Significance and Impact of the Study: The simple, low cost method of inoculum preparation can be applied for large‐scale production of GLA‐rich oils, which reduce a time constraint and variation in fatty acid composition.     

草坪草离蠕孢叶枯病菌生物学特性的研究

对草坪离蠕孢叶枯病病原菌进行分离鉴定,并对其生物学特性进行了研究。结果表明:该病害由禾草离蠕孢(Bipolaris sorokiniana)引起。该病原菌的菌丝生长及产孢的最适温度为30℃,孢子萌发最适温度为25℃,菌丝的致死温度为65℃,而孢子的致死温度则为55℃;该菌对酸碱度的适应能力较强,中性偏酸性的条件对菌丝的生长有利,而pH值为8.0时最易产孢;各碳源对菌丝的生长均有促进作用,但不同碳源对产孢量的影响很大,单糖和双糖利于产孢,多糖对产孢的影响不大。氮源对菌丝生长和产孢量非常重要,无机氮效果较好,硝态氮好于氨态氮,有机氮效果最差。花粉、叶面物质和草坪草汁液可促进孢子萌发。     

粉被虫草N~6-(2-羟乙基)腺苷高产菌株的原生质体诱变育种

在粉被虫草（CordycepspruinosaPetch）无性型─-粉被马利娅霉（MariannaeaPruinosaLiang）原生质体形成和再生的前期研究基础上,报道了通过原生质体诱变培育高产N6－（2－羟乙基）腺苷菌株的研究结果。粉被马利娅霉Cp－14单孢子株经15W紫外灯照射后,获得一高产突变株MpM－135。经多代移植后,此突变株比出发菌株Cp－14的N6－（2－羟乙基）腺苷含量提高了14．8％。实验还表明,粉被虫草无性型菌丝提取物对枯草杆菌（Bacillussubtilis）抗紫外辐射效果与菌丝所含N6－（2－羟乙基）腺苷含量有正相关性。     

Regulation of trehalose metabolism by Streptomyces griseus spores.

Spores of Streptomyces griseus contain trehalose and trehalase, but trehalose is not readily hydrolyzed until spore germination is initiated. Trehalase in crude extracts of spores, germinated spores, and mycelia of S. griseus had a pH optimum of approximately 6.2, had a Km value for trehalose of approximately 11 mM, and was most active in buffers having ionic strengths of 50 to 200 mM. Inhibitors or activators or trehalase activity were not detected in extracts of spores or mycelia. Several lines of evidence indicated that trehalose and trehalase are both located in the spore cytoplasm. Spores retained their trehalose and most of their trehalase activity following brief exposure to dilute acid. Protoplasts formed by enzymatic removal of the spore walls in buffer containing high concentrations of solutes also retained their trehalose and trehalase activity. Protoplasts formed in buffer containing lower levels of solutes contained low levels of trehalose. The mechanism by which trehalose metabolism is regulated in S. griseus spores is unresolved. A low level of hydration of the cytoplasm of the dormant spores and an increased level of hydration during germination may account for the apparent inactivity of trehalase in dormant spores and the rapid hydrolysis of trehalose upon initiation of germination.     

Ester antibiotic accumulation by Streptomyces hygroscopicus

In an attempt to maximize the ester antibiotic production by Streptomyces hygroscopicus D1.5, its efficacy was found to be enhanced by manipulation of the nutrient and physical environment. The two stage fermentation using seed inoculum (10% v/v) resulted in better production while fermentation continued for 5 days in pH 7.0 at 30 degrees C. Enhanced yield was also observed in whole cell immobilization. Under entrapment, maximum yield was achieved at 7th and 9th day of fermentation for mycelia and spore. In addition, the beads could be reused up to the 3rd cycle.     

灵芝子实体、菌丝体及孢子粉中多糖成分差异比较研究

为探讨灵芝子实体、菌丝体和孢子粉3种材料中多糖成分的差异,分别运用苯酚硫酸法进行多糖含量测定,运用离子色谱分析其酸水解后单糖组成,并运用HPLC分析各多糖图谱及经α-淀粉酶和β-1,3-葡聚糖酶处理后HPLC图谱的变化,结果发现,灵芝菌丝体中多糖含量最高,达到3.81%,孢子粉多糖含量为1.8%,灵芝子实体中多糖含量最低,仅为0.59%;水解后的单糖组成及摩尔比也有差异,子实体的单糖主要为葡萄糖和半乳糖,菌丝体和孢子粉的单糖主要为葡萄糖;HPLC图谱显示3种多糖出峰位置和分子量也不同,酶解效果表明多糖结构也相差较大。各样品多糖对小鼠巨噬细胞RAW264.7释放NO的产量的影响上,菌丝体与子实体多糖都表现出了很好的活性,而孢子粉多糖却呈现出较低活性。实验结果表明灵芝子实体、菌丝体和孢子粉3种材料的多糖成分差异大,在医药保健品使用中应区分使用。     

Tryptophan supplementation and pH adjustment for optimizing the sporulation of <Emphasis Type="Italic">Coniothyrium minitans</Emphasis>

To determine the effect of tryptophan and pH on sporulation of Coniothyrium minitans, the fungus was cultivated using a two-stage, agar plate method in which addition of tryptophan and pH were controlled at the sporulation stage. The spore yield was enhanced by 4 times with 0.1 g tryptophan/l addition after 72 h. The optimal pH values were 4 for mycelia growth and 5.8–6 for sporulation. Mycelia grown at pH 6 had a higher productivity of spore production than did those grown at pH 4.     

适合飞虱虫疠霉菌丝生产的液体培养基组分及发酵条件

蚜虫、飞虱、叶蝉等是威胁农业生产的刺吸式口器害虫,长期以来依赖化学农药防治,严重影响农产品的安全性和农业生态环境.探讨这类害虫的生物防治途径,减轻农产品化学农药的残留污染,一直受到国内外学者的关注.虫霉(En-tomophthorales)的许多种类具有优异的诱病杀虫能力,尤其通过主动弹射孢子而在害虫种群中高强度流行的特征,可在短期内迅速压低虫口密度,是极其重要的杀虫微生物资源[1～3].     

Phosphorus and carbon availability regulate structural composition and complexity of AM fungal mycelium

The regulation of the structural composition and complexity of the mycelium of arbuscular mycorrhizal (AM) fungi is not well understood due to their obligate biotrophic nature. The aim of this study was to investigate the structure of extraradical mycelium at high and low availability of carbon (C) to the roots and phosphorus (P) to the fungus. We used monoxenic cultures of the AM fungus Rhizophagus irregularis (formerly Glomus intraradices) with transformed carrot roots as the host in a cultivation system including a root-free compartment into which the extraradical mycelium could grow. We found that high C availability increased hyphal length and spore production and anastomosis formation within individual mycelia. High P availability increased the formation of branched absorbing structures and reduced spore production and the overall length of runner hyphae. The complexity of the mycelium, as indicated by its fractal dimensions, increased with both high C and P availability. The results indicate that low P availability induces a growth pattern that reflects foraging for both P and C. Low C availability to AM roots could still support the explorative development of the mycelium when P availability was low. These findings help us to better understand the development of AM fungi in ecosystems with high P input and/or when plants are subjected to shading, grazing or any management practice that reduces the photosynthetic ability of the plant.     

月腺大戟根总黄酮对尖孢镰刀菌抑制作用的研究

采用生长速率法和孢子萌发法分别测定月腺大戟根总黄酮对尖孢镰刀菌菌丝生长和分生孢子萌发的抑制率,显微观察总黄酮处理尖孢镰刀菌后菌丝体形态结构的变化,并测定菌丝体相对电导率,菌丝体内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性。结果表明:月腺大戟根总黄酮对尖孢镰刀菌菌丝生长和分生孢子萌发均有显著的抑制作用,抑制率随总黄酮浓度增加而增高,20 mg/L时抑制率达100%。总黄酮处理后的尖孢镰刀菌菌丝较细,分支减少,透明度差,液泡数量增多且形成较大的液泡;菌丝体细胞膜透性增加,SOD、CAT活性呈先上升后下降的趋势。以上实验结果可为植物病害生物防治及开发植物源农药方面提供理论依据和实验技术。     

Production of conidia of Alternaria cassiae with alginate pellets

Formulations of alginate-encapsulated mycelia are used to generate spores for mycoherbicidal application to weed-infested fields and for bulk production of spore-based products. Spore yield of such formulations is a primary determinant of product efficacy. A number of parameters of the alginate process were studied to develop an optimal alginate formulation for field application of Alternaria cassiae, a mycoherbicide for sicklepod (Cassia obtusifolia). The composition of the fermentation medium and of the filler used in formulation and the fermentation time were important variables. The addition of nutrients to the mycelial homogenate after fermentation increased sporulation but the amount and ratio of nutrients in the fermentation medium had a greater influence on spore yield from pellets. Optimal sporulation resulted from mycelia produced during a 60- to 70-h fermentation in 2.4% dehydrated potato dextrose broth and 14% V-8 vegetable juice and entrapped in pellets containing corn cob grits as the filler.     

Colony development in Streptomyces carpinensis: a streptomycete with substrate mycelium spores

In this paper we present an ultrastructural study of spore formation in aerial vs. substrate mycelia of Streptomyces carpinensis. Both mycelia initiated spore formation at nearly the same time of colony development but exhibited different patterns of spatial localization of sporulation: spore formation took place throughout the aerial mycelium whereas in the substrate mycelium was confined to a narrow zone at the bottom of the colony. The ultrastructural changes leading to spore formation, however, were quite similar in both mycelia, differing only with respect to the outer components of the sporal wall. Spores formed in the aerial mycelium were covered by a thin sheath whereas the spores formed in the substrate mycelium were covered by an amorphous electron-dense material.     

Genetic analysis of spore killing in the filamentous ascomycete Podospora anserina

In the present study, we analysed different Podospora anserina strains for their ability to induce spore killing and identified three new killer strains. Test crosses of killer strains with different sensitive strains revealed different second division segregation ratios suggesting an influence of the sensitive strain on the crossing-over frequency. In crosses of killer strain O with a sensitive strain, the frequency of two-spored asci was found to vary extremely from perithecium to perithecium. Furthermore, crosses of strain O with sensitive strain Us5 led to a significant proportion of asci containing an unexpected high number of surviving spores as the result of gene conversion. Finally, for the first time, we present data demonstrating that in a number of ascospores the killer and the corresponding sensitive allele is located in one individual nucleus. Mycelia derived from such ascospores display a "sensitive killer" phenotype. Crosses of these mycelia with a killer strain as well as with a sensitive strain result in spore killing. Strikingly, heterokaryotic spores containing the recombined "sensitive/killer" allele and a nucleus with a killer allele give rise to mycelia protected against spore killing during selfing.     

具抗菌活性放线菌菌株JSM 20.1的分离和鉴定

从药用植物银杏(Ginkgo biloba)根际土壤中分离到1株放线菌菌株JSM20.1,其发酵产物具有广谱抗菌活性。该菌株在多数培养基上生长良好,具有链霉菌属的典型形态特征。菌丝多分枝,不断裂;基内菌丝黄白色至棕褐色,气生菌丝黄白色至棕红色;孢子链直且长,偶有波曲,孢子柱状、光滑、不运动。在察氏、马铃薯浸汁、葡萄糖-天门冬酰胺、酵母膏-麦芽膏(ISP2)、燕麦(ISP3)、无机盐淀粉(ISP4)和甘油-天门冬酰胺(ISP5)琼脂上均产生可溶性色素,而在营养琼脂上不产生可溶性色素。生长温度范围为7～40℃,最适生长温度28℃;生长pH范围为5.0～10.0,最适pH7.0;能在含0～3%(质量与体积比)NaCl的培养基上生长,而在无NaCl的ISP2培养基生长最佳。根据其形态学特征、生理生化特征、细胞化学分类特征和基于16SrRNA基因序列的系统发育分析结果,菌株JSM20.1被鉴定为链霉菌属的有效发表种Streptomyces cyaneofuscatus的1个菌株。     

Comparison of rapid DNA extraction methods applied to PCR identification of medicinal mushroom Ganoderma spp

Four different DNA extraction methods were used to extract genomic DNA of the medicinal mushroom Lingzhi from its developing stage materials, such as mycelium, dry fruiting body, or sliced and spore powder or sporoderm-broken spore powder. The DNA samples were analyzed using agarose gel electrophoresis, UV spectrophotometer, and PCR amplification. According to the average yields and purity of DNA, high salt concentrations and low pH methods were the best for DNA extraction. The mycelia and sporoderm-broken spore powder yielded higher and purer DNA. The method developed could effectively eliminate the influence of the secondary metabolites to DNA extraction. The DNA samples extracted from the developed method could be successfully used for PCR applications.     

Basidiospore dispersal in the old-growth forest fungus Phlebia centrifuga (Basidiomycetes)

Dispersal of the old‐growth forest hngus Phlebia centrifuga was studied under natural conditions in an old‐growth forest stand in south Sweden using spore trapping with single spore mycelia as bait. Sampling was done not only in the forest but also 500 and 1000 m outside it. Within 100 m of the basidomata source, spore dispersal was very efficient, and it was estimated that 385 spores settled per m² during 24 h. The minimum total number of spores calculated to have fallen within the 100 m circle was 1.21 × 70⁷. The spore fall decreased significantly with distance from the basidiomata. Almost all of the mycelia were dikaryotised at lm distance, compared with less than half at 10 and 100 m. Dikaryotisation was observed in a small but significant number of mycelia at 500 and 1000 m. Mating success between different single spore mycelia differed significantly and we speculate that effective population size may be affected by mating choice in this species. What factors restrict this species to old‐growth forests? In the light of the dikaryotisation observed at 1000 m from the source, it seems unlikely that this fungus is restricted by spore dispersal distance, which implies good prospects for its successful establishment in restored habitats, i.e. first‐generation forests with much coarse woody debris.     

Formulating Atoxigenic Aspergillus flavus for Field Release

A procedure was developed to encapsulate mycelia of an atoxigenic strain of Aspergillus flavus in alginate pellets for seeding into agricultural fields in order to reduce aflatoxin contamination via competitive exclusion. Kaolin, a clay filler commonly employed in alginate formulations, was detrimental to pellet performance as measured by spore yield. Corn cob grits, a by-product of the corn industry, was found to be an excellent replacement for kaolin. Of nine nutritive adjuvants tested, wheat gluten improved pellet performance the most, although gluten concentrations above 5% were difficult to process. The best formulation tested consisted of 1% sodium alginate, 5% corn cob grits and 5% wheat gluten. On a 'per gram' basis, this alginate formulation yielded more spores than either A. flavus sclerotia or colonized wheat seed. Pesticides were also tested as adjuvants with potential use for protecting pellets under field conditions. Only one (chloramphenicol) of four tested pesticides (the others were dichloran, rose Bengal and cyfluthrin) reduced pellet sporulation. Formulations with or without pesticide adjuvants retained similar spore yield potential during a 2-year storage at 8 C. However, spore production in stored products lagged behind that of fresh products. At 75% relative humidity (RH), pellet storage stability decreased with increasing temperature from 27 to 42 C. Pellet spore yield at 32 C decreased as RH decreased from 100 to 90%. Sporulation occurred at 90% RH but not at 88% RH. Spore yield varied widely in four field tests, and the cumulative spore yield was inversely correlated (r2= -0.798, P 0.01) with rainfall. The results suggest that alginate pellets may be effective formulations for delivery of atoxigenic A. flavus strains to furrow-irrigated cotton in desert environments, where aflatoxin contamination of cottonseed is most severe.     

Biodiesel fuel production from lipids of filamentous fungi

The main stages in the production of biodiesel fuel from lipids of filamentous fungi belonging to the order Mucorales are described. Fungi of the family Cunninghamellaceae have been screened; the lipogenic activity of the examined strains has been assessed; and a producer generating up to 50% of lipids, represented by triacylglycerols, has been found. The substitution effect of a source of carbon and nitrogen with less expensive components (in particular, various industrial wastes) has been studied, as well as their influence on the quantity and major characteristics of the final product. An ecologically friendly method for extracting lipids from fungal mycelia, utilizing supercritical technologies, has been used. A correlation between the lipid content in the spore inoculum and the maximal lipid content in biomass has been discovered; this correlation is proposed for optimizing the biotechnology and increasing the yield of final products.     

Isolating Mortierella alpina strains of high yield of arachidonic acid

AIMS: To develop a fast isolation method for arachidonic acid-producing fungi of high yield. METHODS AND RESULTS: Relation between the staining degree of mycelia of Mortierella alpina stained with triphenyltetrazolium chloride (TTC) and arachidonic acid content in the fungal lipids was investigated. Results showed that staining degree of mycelia stained with TTC increased when arachidonic acid content in mycelia lipids increased. This finding was used to isolate strains of high arachidonic acid yield. Arachidonic acid producing fungi were selectively isolated from soil at a low temperature of 4 degrees C and the mycelia of these isolates were stained with TTC. CONCLUSIONS: The strain M. alpina M6 that had the highest staining degree had the highest arachidonic acid content (72.3%). The yield of arachidonic acid in this strain reached 4.82 g l(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: A fast and effective method to isolate strains of high arachidonic acid yield was established according to the finding that staining degree of mycelia of M. alpina was positively correlated with arachidonic acid content in mycelia lipid.