Synchrony of Nuclear Replication in Individual Hyphae of Aspergillus nidulans

The synchrony of nuclear replication in individual, multinucleate hyphae of Aspergillus nidulans has been investigated. Samples were taken from cultures of germinating conidiospores, and the relative frequency of hyphae containing two to eight nuclei was determined. Because the conidiospores are mononucleate, complete synchrony will yield populations of hyphae containing only 2ⁿ nuclei, n being the number of doublings after germination. The appearance of hyphae with total numbers of nuclei other than 2ⁿ will indicate lack of synchrony. The relative frequency of hyphae not having 2ⁿ nuclei will depend on the degree of synchrony in the individual hyphae; numerical aspects of this relation are discussed. In two different strains, replication of the nuclei in any one hypha was highly synchronized when the dry weight doubling time was 1.4 to 1.8 hr. As the doubling time was made longer by changing the nitrogen or carbon source, synchrony was progressively lost. At the slowest growth rate tested, the interval between the division of the fastest and the slowest nucleus equaled 48% of the dry weight doubling time. The active replication of some nuclei in a hypha where other nuclei were resting suggested that nuclear duplication in this eukaryotic organism may be controlled by specific initiators.     

Nutrient requirements in germination of conidiospores of Aspergillus niger V. Tieghen

Germination of conidiospores of A. niger was stimulated by the addition of some carbohydrates and nitrogenous compounds to the growth medium. Glucose at 0.5 and 1.0 mg/ml concentrations was the best sugar for germination. Sucrose and fructose were the second best. Glutamic acid and valine were the most effective nitrogenous compounds for the spore germination of the fungus. Glucose, sucrose, and glutamic acid initiated germination within the first 6 h of incubation. When cellobiose, galactose and valine were used germination started after 9 h. With leucine, cysteine and arginine, however, not only the percentage of germination reduced, but also the latent period increased, significantly. A period of 15 h was needed before germination had been started.     

Sporulation et mycosporines chez un champignon imparfait: Ascochyta fabae

Sporalation and mycosporins in the Deuteromycete Ascochyta fabae .
To investigate the sporogenic activity of mycosporins, a strain of Ascochyta fabae Speg., in which conidiogenesis and mycosporin synthesis are easily induced, has been selected. In darkness, few mature pycnidia and little mycosporin are produced. Dry weight and mycosporin production were followed during mycelial growth and the conidiogenic phase, in both illuminated and non-illuminated cultures: Mycosporin production appears closely linked with the morphogenic differentiation; its synthesis starts when the exponential growth is over. There is a good correlation between conidiogenesis and mycosporin production.
Irrespective of illumination, there is no increase in the rate of sporulation when maycosporins are added at different concentration levels. This cannot be explained by impermeability of the hyphae to mycosporins since uptake starts after a lag phase of 7 days and then proceeds until 15 to 20 days; at the same time the mycelium of A. fabae keeps its sporulation competence until 16 days after transfer to complete darkness. Mycosporin accumulated into conidiospores – 3 to 5% of dry weight – is not metabolized during the germination; on the contrary, a slight synthesis occurs after 20 h of germination.
Mycosporins thus seem to be secondary metabolites appearing as products from a type of metabolism that occurs at a very low level during mycelial growth and increases its activity during reproductive morphogenesis in numerous species of fungi.     

Thiamine binding and metabolism in germinating seeds of selected cereals and legumes.

The basic characteristics of thiamine metabolism in germinating seeds of maize (Zea mays), oat (Avena sativa), faba bean (Vicia faba) and garden pea (Pisum sativum) are presented with a special emphasis of a possible thiamine storage function of seed thiamine-binding proteins (TBPs). Seeds were germinated for 6 d in the dark. Thiamine-binding activity in seeds decreased during germination by 50% in cereals and by 30% in legumes. The degradation of TBPs was also detected by polyacrylamide gel electrophoresis. The total thiamine content decreased rapidly to 20-40% of the initial value in cereal seeds during first 3 d of germination while in legume seeds thiamine content started changing from the fourth day and dropped by 50% at the sixth day. A composite pattern was found for the changes in thiamine pyrophosphate (TPP) contribution to total thiamine during seed germination. A peak of the coenzyme percentage was usually detected at the second day of germination. Another gain of TPP was often seen toward the sixth day of germination. The activity of thiamine pyrophosphokinase (EC 2.7.6.2) was high in resting legume seeds and did not significantly change during germination. In contrast, the low activity of this thiamine-activating enzyme in cereal seeds progressively increased during germination. Thiamine phosphate synthase (EC 2.5.1.3) was also detected in seeds and was shown to contribute significantly to the balance of thiamine compounds during seed germination.     

Changes of the cytoplasmic ultrastructure during development of sclerotia in Claviceps purpurea (Fr.) Tul.

The development of sclerotia of Claviceps purpurea was investigated by light and electron microscopy. During the first days after infection sterigma and conidiospores are formed. The spores show a moderately developed vacuolar system, they are thick walled and contain about 20% lipid (related to the cell volume) embedded in glycogen. The sterigma are cylindrical unicellular hyphae with electron dense cytoplasm and isolated strongly contrasted lipid droplets. In maturing sclerotia the hyphae become septated with increasingly thick cell walls and a large lipid content. The lipid forms small droplets in young cells, while in the mature sclerotium it occurs in the form of very large drops, occupying the major part of the cell. Simultaneously the composition of the lipid is changed. The mature cells have several nuclei. They are partially connected by osmiophilic substances, forming a network of intercellular spaces.Abbreviations HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - DMSO Dimethylsulfoxide     

Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae,as revealed by high resolution two-dimensional polyacrylamide gel electrophoresis

On artificial polyethylene membranes providing a thigmotropic signal, uredospores of the broad bean rust fungus Uromyces viciae-fabae differentiated a series of infection structures which in nature are necessary to invade the host tissue through the stomata. Within 24 h germ tubes, appressoria, substomatal vesicles, infection hyphae and haustorial mother cells were developed successively. Alterations in protein metabolism during infection structure differentiation of this obligate plant pathogen were analyzed in the absence of the host plant by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver staining. The norm pattern representing the 2-DE protein patterns of the whole developmental sequence of infection structures of U. viciae-fabae showed 733 spots. During infection structure differentiation 55 proteins were newly formed, altered in quantity, or disappeared. Major alterations in the protein pattern occurred during uredospore germination and when infection hyphae were formed. Uredospore germination was characterized by a decrease of acidic proteins and an increase mainly of proteins with isoelectric points ranging from weakly acidic to basic.Abbreviations 2-DE two-dimensional polyacrylamide gel electrophoresis - DAPI 4,6-diamino-phenylindol - kDa kilo Dalton - pl isoelectric point - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Nuclear inheritance of the biosynthesis of cyclopenin and cyclopenol in Penicillium cyclopium

Balanced heterokarions were grown from Penicillium cyclopium aux-glu 1, a glutamic acid auxotroph producing benzodiazepine alkaloids of the cyclopenin-cyclopenol group, and P. viridicatum aux-met 1, a methionine auxotroph forming these alkaloids in traces only. In contrast to the hyphae of the parent strains, the hyphae of the heterokarions were dark orange-brown and grew well on media without the auxotrophic factors. In surface cultures they synthesized the benzodiazepine alkaloids cyclopenin and cyclopenol in amounts similar to those formed by the hyphae of P. cyclopium aux-glu 1. From the monokariotic conidiospores of the heterokarions homokariotic daughter strains were obtained which were similar to the parent strains in every respect. Hence no exchange of features of cyclopenin-cyclopenol biosynthesis took place between the parent strains at the stage of the heterokarion. This result indicates that the formation of cyclopenin and cyclopenol in P. cyclopium aux-glu 1 and the nearly complete lack of biosynthesis of these compounds in P. viridicatum aux-met 1 is encoded within the nucleus and is not influenced by plasmic genetic material.     

Metabolic products of microorganisms

1. The total nitrogen content of spores of Penicillium notatum increased during swelling and reached a maximum before germ-tubes were formed. It subsequently decreased during germ-tube formation until a constant value in hyphae was reached.
2. An increase in the protein content of the spores was found during swelling, followed by a decrease when germ-tubes were formed. After germination, the protein content increased again to a constant level in hyphae.
3. The content of total lipids steadily decreased during swelling of spores. It reached a minimum value in germinating spores, followed by a net accumulation during hyphal growth. Similar changes occurred in free lipids, phospholipids and the acetone-soluble lipids but not in bound lipids.
4. After an initial decrease during swelling, no further change took place in the neutral carbohydrates content of the spores at the time of germ-tube formation. An accumulation of neutral carbohydrates occurred during late hyphal extension.
5. The nucleic acid content increased sharply during swelling, and reached the highest value in swollen spores just prior to the initiation of germ-tube formation. Afterwards, its content steadily decreased during germ-tube formation and hyphae elongation.

     

Mode of Infection of Phyllosticta maculata on Banana as Revealed by Scanning Electron Microscopy

The initial infection stages of Phyllosticta maculata on banana were studied using scanning electron microscopy. Conidial germination on the banana leaf surface commenced within 3 h postinoculation to produce a long and slender germ tube. The hyphae developed secondary branches and mostly grew randomly across the leaf surface. Appressoria were formed at the apex of the germ tubes within 18 h postinoculation and were variable in shape. A layer of an extracellular matrix surrounded the appressoria at the pathogen–host interface. On the fruit surface, conidia germinated to produce predominantly swollen germ tubes which functioned as lateral appressoria together with some slender ones. These germ tubes were formed within 3 h postinoculation. There was no stomatal penetration apparent on the leaf; instead, direct penetration through the cuticle with and without the formation of appressoria was observed. Cuticular degradation on the leaf surface was evident with a circular, darkened area around the point of penetration by hyphae or appressoria. The significant role of pycnidia and conidia in the epidemiology of the disease was further demonstrated in naturally infected leaf samples.     

Morphological quantification of filamentous fungal development using membrane immobilization and automatic image analysis

Mycelial morphology is a critically important process property in industrial fermentations of filamentous micro-organisms, as particular phenotypes are associated with maximum productivity. However, the accurate quantification of complex morphologies still represents a significant challenge in elucidating this relationship. A system has been developed for high-resolution characterisation of filamentous fungal growth on a solid substrate, using membrane immobilization and fully-automatic plug-ins developed for the public domain, Java-based, image-processing software, ImageJ. The system has been used to quantify the microscopic development of Aspergillus oryzae on malt agar, by measuring spore projected area and circularity, the total length of a hyphal element, the number of tips per element, and the hyphal growth unit. Two different stages of growth are described, from the swelling of a population of conidiospores up to fully developed, branched hyphae 24 h after inoculation. Spore swelling expressed as an increase in mean equivalent spore diameter was found to be approximately linear with time. Widespread germination of spores was observed by 8 h after inoculation. From approximately 12 h, the number of tips was found to increase exponentially. The specific growth rate of a population of hyphae was calculated as approximately 0.24–0.27 h⁻¹. A wide variation in growth kinetics was found within the population. The robustness of the image-analysis system was verified by testing the effect of small variations in the input data.     

粉虱拟青霉对黑刺粉虱的侵染过程

本文通过扫描电镜和透射电镜 ,观察了粉虱拟青霉对黑刺粉虱的侵染过程。用粉虱拟青霉分生孢子接种于黑刺粉虱幼虫后 ,12h孢子萌芽 ,36h菌丝穿透寄主表皮进入体腔 ,在虫体内进行生长繁殖 ,逐渐充满虫体内部 ,5d后 ,菌丝穿透寄主表皮长出体外 ,并继续生长 ,逐渐将整个虫体包裹 ,形成一个半球形的子痤 ,颜色也由白色变为栗褐色或棕红色。     

A sensitive method for examining whole-cell biochemical composition in single cells of filamentous fungi using synchrotron FTIR spectromicroscopy

Cell function is related to cell composition. The asexual state of filamentous fungi (molds and mildews) has two main life cycle stages: vegetative hyphae for substrate colonization and nutrient acquisition, and asexual spores for survival and dispersal. Hyphal composition changes over a few tens of microns during growth and maturation; spores are different from hyphae. Most biochemical analyses are restricted to studying a few components at high spatial resolution (e.g. histochemistry) or many compounds at low spatial resolution (e.g. GC-MS). Synchrotron FTIR spectromicroscopy can be used to study fungal cell biology by fingerprinting varieties of carbohydrates, proteins, and lipids at about 6 microm spatial resolution. FTIR can distinguish fungal species and changes during hyphal growth, and reveals that even fungi grown under optimal vs mildly stressed conditions exhibit dramatic biochemical changes without obvious morphological effects. Here we compare hypha and spore composition of two fungi, Neurospora and Rhizopus. There are clear biochemical changes when Neurospora hyphae commit to spore development, during spore maturation and following germination, many of which are consistent with results from molecular genetics, but have not been shown before at high spatial resolution. Rhizopus spores develop within a fluid-containing sporangium that becomes dry at maturity. Rhizopus spores had similar protein content and significantly more carbohydrate than the sporangial fluid, both of which are novel findings.     

Cyclic adenosine 3′,5′-monophosphate and germination of sporangiospores from the fungus Mucor

Cyclic adenosine 3,5-monophosphate (cAMP) metabolism was examined in germinating sporangiospores of Mucor genevensis and Mucor mucedo. Exogenous cAMP prevented normal hyphal development from sporangiospores. Internal pools of cAMP fluctuated profoundly during development. Spherical growth of the spores was characterized by large pools of cAMP whereas germ tube emergence and hyphal elongation were characterized by small pools of cAMP. These observations suggest a possible role for cAMP in sporangiospore germination. Adenylate cyclase activities fluctuated significantly during germination with maximum values attained during spherical growth. In contrast, cAMP phosphodiesterase activities remained constant throughout germination. Internal cAMP levels may therefore be regulated by adjustment of adenylate cyclase activities. The binding of cAMP by soluble cell proteins was measured. cAMP-binding activity changed greatly during germination. Dormant and spherically growing spores possessed the highest activities. Developing hyphae contained the lowest activities. Use of the photoaffinity label, 8-azido-[^32P]cAMP, in conjunction with sodium dodecyl sulfate-polyacrylamide-gel electrophoresis allowed the identification of a small population of morphogenetic-stage-specific proteins which bind cAMP and may be of regulatory significance to development.     

Histochemistry and storage of organic compounds during basidiosporogenesis in the ectomycorrhizal fungus <Emphasis Type="Italic">Pisolithus microcarpus</Emphasis>

Knowledge on the distribution and storage of different organic compounds during basidiosporogenesis in P. microcarpus is paramount to a better understanding of basidiospore recalcitrance to germination. The objective of this work was to detect the presence and distribution of phenolics, reducing sugars, starch, glycogen, total polysaccharides, RNA, and proteins during P. microcarpus basidiosporogenesis. Starch and reducing sugars were not detected in the fungal basidiocarps, while other polysaccharides predominated in the extracellular matrix at the base of the basidiocarp containing unconsolidated peridioles. Phenolics were also detected in this region. Glycogen was present inside the hyphae, basidia, and basidiospores and constitutes an important storage compound in the fungal basidiocarps. In mature basidiospores, RNA accumulation occurred at discrete locations in the cytoplasmatic periphery, while polysaccharides and proteins were shown to predominate in the cell wall. The presence of glycogen, RNA, and proteins inside the basidiospores strongly indicates provision for future germination and suggests that other factors may also influence basidiospore recalcitrance.     

Infection process of Colletotrichum dematium on mulberry leaves: An unusual method of sporulation

Abstract

The pre-penetration and infection process of Colletotrichum dematium on mulberry leaf was investigated by scanning electron microscope. Conidia produced on germination appressoria directly or at the end of short germ tubes. Appressoria were formed mostly over cuticle, but sometimes over stomata also. At 72 h post-inoculation, an extensive network of sub-cuticular runner hyphae (RH) was produced. The RH were traceable by the cuticular bulgings on leaf surface. The RH emerged to leaf surface through ruptured cuticle to form secondary infection hyphae (SIH). The SIH re-entered the leaf tissue by sending penetration branches through stomata. Conidia were formed singly on short conidiophores from the RH and SIH, at short intervals. The conidia developed on RH were exposed to leaf surface through ruptured cuticle. Some times conidia were released through stomata also. The RH and SIH had thick knots from which hyphal branches and conidia were developed. Definite acervuli were not developed.     

尖角突脐孢菌在稗草和水稻上的萌发和侵染行为比较研究

本试验结合曲利苯兰和荧光素钠两种染色方法的优点,从显微角度研究了尖角突脐孢菌(Exserohilum monoceras)两个菌株X-27和HN-14在稗草和水稻上萌发和侵染行为的差异。结果表明,在寄主稗草上,接种4h后尖角突脐孢菌孢子开始从一端或两端萌发形成初生芽管;10h后芽管顶端膨大形成附着胞,附着在寄主表面,两端萌发的孢子约90%一端败育,仅一端形成成熟附着胞;在接种后24h内成熟附着胞形成率与接种时间成正相关,24h后趋于稳定;16h后在成熟附着胞下方受侵染的细胞内指状吸器开始形成,随后发育为掌状吸器;接种36h后菌丝在组织表面扩展形成网状,同时稗草叶片上显现叶斑病症状,部分菌丝能在细胞间蔓延扩展。在非寄主植物水稻上,同样观察到孢子萌发产生芽管,但是萌发起始时间滞后大约4h,初生菌丝分枝明显减少,且未能观察到附着胞和吸器的产生。     

A proteomic analysis of powdery mildew (Blumeria graminis f.sp. hordei) conidiospores

Conidiospores are the asexual propagation units of many plant-pathogenic fungi. In this article, we report an annotated proteome map of ungerminated conidiospores of the ascomycete barley powdery mildew pathogen, Blumeria graminis f.sp. hordei . Using a combination of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, we have identified the proteins in 180 spots, which probably represent at least 123 distinct fungal gene products. Most of the identified proteins have a predicted function in carbohydrate, lipid or protein metabolism, indicating that the spore is equipped for the catabolism of storage compounds as well as for protein biosynthesis and folding on germination.     

磷和镉对根内球囊霉Glomus intraradices孢子萌发、菌丝生长和外生菌丝内聚磷酸累积的影响

丛枝菌根真菌(AMF)能够显著提高植物对重金属的抗性,菌丝内聚磷酸(Polyphosphate,PolyP)可能参与了这种抗性的形成。试验以Glomus intraradices孢子为试材,对灭菌条件进行了优化,并进一步研究了不同P和Cd2+水平对孢子萌发、菌丝生长、分支和外生菌丝中聚磷酸含量的影响。结果表明,孢子在1%氯胺T+0.02%链霉素+0.01%庆大霉素+1/100(V/V)吐温-20中灭菌5min的效果最好。孢子萌发率、菌丝分支和菌丝长度随着Cd2+浓度的增加不断降低;当Cd2+浓度达到0.1mmol/L时,孢子萌发率降低为0%,表明Glomus intraradices的孢子萌发对Cd2+的耐受极限为0.1mmol/L;1mmol/L的P促进菌丝分支增加,却降低了萌发率,但对菌丝生长没有影响;在培养23d以后,三者基本不再变化。外生菌丝内的聚磷酸含量随着P的升高而增加;在Cd2+胁迫作用下,聚磷酸的含量降低而菌丝密度随着聚磷酸的升高而升高,表明聚磷酸在减弱重金属毒性方面起了重要作用。     

pH值对Gigaspora margarita孢子萌发、菌丝生长与聚磷酸盐含量的影响

土壤pH值是影响AM真菌的生理与生态过程的重要因子之一,本试验在培养基上接种Gigasporamargarita的孢子,研究了pH值分别为5.2、6.0和6.8时孢子萌发率、菌丝生长和菌丝中聚磷酸盐(polyP)的含量。结果表明,不同pH条件下的孢子萌发率没有明显差异,培养12d后的萌发率为70%左右;随着pH的升高,菌丝的长度逐渐增加,表明低pH对菌丝的生长有一定的抑制效应;培养12d后,孢子中polyP含量低于菌丝中polyP含量,pH6.0和pH6.8的条件下菌丝中polyP含量明显高于pH5.2的含量,表明低pH也能降低菌丝中的聚磷酸盐含量。认为低pH对菌丝生长和polyP含量的抑制可能是其限制AM真菌功能发挥的重要机制之一。