Spore ontogeny of the arbuscular mycorrhizal fungus<Emphasis Type="Italic"> Archaeospora trappei</Emphasis> (Ames &; Linderman) Morton &; Redecker (<Emphasis Type="Italic">Archaeosporaceae</Emphasis>)

Arbuscular mycorrhizal (AM) fungi in the genus Archaeospora (family Archaeosporaceae) contain both monomorphic and dimorphic species. The synanamorphism is often hard to discern without ontogenetic observations. Here, the spore ontogeny of Ar. trappei is reported from single species pot culture studies. The sporogenous hypha swelled up to a terminal sporiferous saccule and produced a lateral spore primordium on its neck. The saccule expanded fully before the spore primordium emerged. The saccule transferred its contents into the expanding spore and collapsed while wall differentiation continued inside the spore. The spore wall of Ar. trappei differentiated sequentially, in discrete steps, as in Acaulosporaceae members. In contrast, Ar. trappei produced a simplified spore wall in which the components differed in chemical and physical characteristics from those of the Acaulosporaceae members. Ontogenetic studies confirmed Ar. trappei to be monomorphic and producing acaulosporoid spores. The fungus is a new record to New Zealand.     

Taxonomic revision transferring species in Kuklospora to Acaulospora (Glomeromycota) and a description of Acaulospora colliculosa sp. nov. from field collected spores

In a phylogenetic study of arbuscular mycorrhizal fungal species in Acaulospora (Acaulosporaceae, Glomeromycota) we discovered that species classified in genus Kuklospora, a supposed sister clade of Acaulospora, did not partition as a monophyletic clade. Species in these two genera can be distinguished only by the position of the spore relative to a precursor structure, the sporiferous saccule, as either within (entrophosporoid) or laterally (acaulosporoid) on the saccule subtending hypha. Subsequent spore differentiation follows identical patterns and organization. Molecular phylogeny reconstructed from nrLSU gene sequences, together with developmental data, support the hypothesis that the entrophosporoid mode of spore formation evolved many times and thus represents a convergent trait of little phylogenetic significance. Therefore genus Kuklospora is rejected as a valid monophyletic group and it is integrated taxonomically into genus Acaulospora. Thus Acaulospora colombiana and Acaulospora kentinensis are erected as new combinations (formerly Kuklospora colombiana and Kuklospora kentinensis). Mode of spore formation is demoted from a genus-specific character to one that is included with other traits to define Acaulospora species. In addition we describe a new AM fungal species, Acaulospora colliculosa (Acaulosporaceae), that originated from a tallgrass prairie in North America. Field-collected spores of A. colliculosa are small (<100 μm diam), hyaline or subhyaline to pale yellow and form via entrophosporoid development based on structure and organization of cicatrices and attached hyphae. Each spore consists of a bilayered spore wall and two bilayered inner walls. A germination orb likely forms after the completion of spore development to initiate germination, but this structure was not observed. A character distinguishing A. colliculosa from other Acaulospora species is hyaline to subhyaline hemispherical protuberances on the surface of the outer spore wall layer. A phylogeny reconstructed from partial nrLSU gene sequences unambiguously placed A. colliculosa in the Acaulospora clade.     

Otospora bareai, a new fungal species in the Glomeromycetes from a dolomitic shrub land in Sierra de Baza National Park (Granada, Spain)

A new fungal species of the Glomeromycetes was isolated from the rhizosphere of Pterocephalus spathulatus and Thymus granatensis, two rare endemic plants growing on dolomite in the Sierra de Baza (Granada, southern Spain). The fungus was propagated in pot cultures of Sorghum vulgare and Trifolium pratense for 4 y and it is described here on the basis of the spores found in nature and formed in pot cultures. Its brown spores (140-210 microm diam) form laterally on a persistent, brown stalk (=neck) of a sporiferous saccule. They have two walls without ornamentation: a brown, three- to four-layered outer wall and a hyaline two- to three-layered inner wall. The unique combination of spore formation and spore wall structure does not fit with any of the known fungal genera. Spore formation is similar to that of Acaulospora spp. and Archaeospora trappei, but Acaulospora spp. has three spore walls with a characteristic "beaded" wall, and the outer wall of Ar. trappei is simple, thin, hyaline and only bilayered. Spore wall structure of the new fungus is similar to that of Entrophospora infrequens, however this fungus forms its spores internally, inside the hyphal stalk of the sporiferous saccule. Molecular analyses of the small subunit of the ribosomal gene phylogenetically place the new fungus next to Diversispora spurca, which forms one-walled glomoid spores (i.e. terminally on hyphae). Based on these analyses we place the new fungus into a new genus in the family Diversisporaceae under the epithet Otospora bareai.     

Acaulospora alpina, a new arbuscular mycorrhizal fungal species characteristic for high mountainous and alpine regions of the Swiss Alps

Acaulospora alpina sp. nov. forms small (65-85 microm diam), dark yellow to orange-brown spores laterally on the neck of hyaline to subhyaline sporiferous saccules. The spores have a three-layered outer spore wall, a bi-layered middle wall and a three-layered inner wall. The surface of the second layer of the outer spore wall is ornamented, having regular, circular pits (1.5-2 microm diam) that are as deep as wide and truncated conical. A "beaded" wall layer as found in most other Acaulospora spp. is lacking. The spore morphology of A. alpina resembles that of A. paulinae but can be differentiated easily by the unique ornamentation with the characteristic pits and by the spore color. A key is presented summarizing the morphological differences among Acaulospora species with an ornamented outer spore wall. Partial DNA sequences of the ITS1, 5.8S subunit and ITS2 regions of ribosomal DNA show that A. alpina and A. paulinae are not closely related. Acaulospora lacunosa, which has similar color but has generally bigger spores, also has distinct rDNA sequences. Acaulospora alpina is a characteristic member of the arbuscular mycorrhizal fungal communities in soils with pH 3.5-6.5 in grasslands of the Swiss Alps at altitudes between 1800 and 2700 m above sea level. It is less frequent at 1300-1800 m above sea level, and it so far has not been found in the Alps below 1300 m or in the lowlands of Switzerland.     

Mycelium development and architecture, and spore production of Scutellospora reticulata in monoxenic culture with Ri T-DNA transformed carrot roots

Mycelium development and architecture and spore production were studied in Scutellospora reticulata from single-spore isolates grown with Ri T-DNA transformed carrot root-organ culture in monoxenic system. Culture establishment, anastomosis occurrence and auxiliary cell development also were examined. Seventy percent of the pregerminated disinfected spores colonized the transformed carrot roots. After 8 mo, the average spore production was 56 (24-130) per 30 cm(3) of medium. Of the spores produced, 75% germinated and produced new generations in monoxenic culture. The mycelium network was formed by thick light-brown hyphae, which exhibit two major architecture patterns related to either root colonization or resource exploitation, and lower-order hyphae, bearing auxiliary cells, branched absorbing structures (BAS), hyphal swellings (HS) and forming anastomoses. BAS were formed abundantly in extramatrical mycelium and frequently had HS resembling vesicles, a feature not previously reported in the Gigasporaceae, to the best of our knowledge. Few anastomosis were observed within the mycelium and most often corresponded to a healing mechanism that form hypha bridges to reconnect broken hyphae or overcoming obstructed areas within a hypha. Numerous auxiliary cells were produced during culture development and their role was inferred.     

Regulation of hyphal growth and sporulation of the insect pathogenic fungus Entomophthora thripidum in vitro

Entomophthora thripidum is an obligate biotrophic insect pathogenic fungus that grows as protoplasts within the hemocoel of thrips. Prior to penetration through the insect cuticle and spore formation at the insect surface the protoplasts switch to hyphal growth. In vitro, the differentiation to hyphal growth was a prerequisite for the subsequent formation of infectious spores and was detected 10-20 days after inoculation. E. thripidum secreted a factor that autoinduced the differentiation to hyphal growth. The discovery of this activity inducing hyphal growth made possible the reliable production of spores, the infection of host insects and the consecutive re-isolation of the fungus from the infected insects.     

Acaulosporoid glomeromycotan spores with a germination shield from the 400-million-year-old Rhynie chert

Scutellosporites devonicus from the Early Devonian Rhynie chert is the only fossil glomeromycotan spore taxon known to produce a germination shield. This paper describes a second type of glomeromycotan spore with a germination shield from the Rhynie chert. In contrast to S. devonicus, however, these spores are acaulosporoid and develop laterally in the neck of the sporiferous saccule. Germination shield morphology varies, from plate-like with single or double lobes to tongue-shaped structures usually with infolded margins that are distally fringed or palmate. Spore walls are complex and appear to be constructed of at least three wall groups, the outermost of which includes the remains of the saccule. The complement of features displayed by the fossils suggests a relationship with the extant genera Ambispora, Otospora, Acaulospora or Archaeospora, but which of these is the closest extant relative cannot be determined. The acaulosporoid spores from the Rhynie chert document that this spore type was in existence already ∼400 mya, and thus contribute to a more complete understanding of the evolutionary history of the Glomeromycota. This discovery pushes back the evolutionary origin of all main glomeromycotan groups, revealing that they had evolved before rooted land plants had emerged.     

广东省三种植物群落土壤AMF孢子比较

应用样方法,以三叶草(Trifolium repens)作盆栽扩繁,利用湿筛倾析法和蔗糖离心法分离土壤孢子,研究了广东省常绿阔叶林、人工林(台湾相思林、湿地松林、刚果按林)和木薯地等3种植物群落11个土样的丛枝菌根菌(Arbuscular mycorrhizal fungi,AMF)孢子组成及分布状况.根据孢子形状、颜色、大小、孢壁饰纹及连孢菌丝等形态,将所分离的孢子划分为20个形态型(morphotypes),分属于3科5属.其中球囊霉属(Glomus)有10种形态型,无梗囊霉属(Acaulospora)5种,巨孢囊霉属(Gigaspora)和盾巨孢囊霉属(Scutellospora)各2种,内养囊霉属(Entrophospora)1种.在20个形态型中,以球囊霉属最丰富,分布最广.其中4种类型Glomus dolichosporum(TypeI)、Glomus eburneum(TypeJ)、Glomus versiforme(TypeM)和Acaulospora scrobiculata(TypeD)在常绿阔叶林、人工林和木薯地中均有出现,其余类型仅见个别土样.     

安徽茶区茶树丛枝菌根真菌多样性

调查了安徽茶区茶树丛枝菌根真菌(AMF)资源分布情况,为菌根生物技术在茶产业中的应用提供了具有应用价值的菌种资源.采用醋酸-墨水染色法观察茶树丛枝菌根(AM)的侵染率、侵染级数、侵染强度和菌根类型;采用湿筛法获得AMF孢子,进行形态学鉴定.结果表明: 在安徽茶区,AMF能够侵入茶树根系形成典型的共生体,侵染率在36%～95%,侵染级数均在3级以上,侵染强度大;从安徽茶树根际土中共鉴定出8属36种AMF,其中缩管柄囊霉是优势种,网状球囊霉、刺无梗囊霉、孔窝无梗囊霉、詹氏无梗囊霉、双网无梗囊霉和凹坑无梗囊霉是常见种,褐色管柄囊霉、疣突管柄囊霉、毛氏无梗囊霉、近明管柄囊霉、瑞氏无梗囊霉、空洞无梗囊霉、晕环球囊霉、细齿无梗囊霉、地管柄囊霉、幼套球囊霉、蜜色无梗囊霉、稀有内养囊霉是稀有种,其余17种为少见种;不同采样地茶树根际AMF群落相似性系数(0.14～0.55)较低,多属于低和中等水平;相关性分析表明,孢子密度与侵染率呈显著正相关,种的丰度与侵染率和总球囊霉素相关土壤蛋白呈显著正相关.安徽茶区茶树根系存在典型的AM结构,其根际AMF多样性丰富,为开发茶树专用AMF肥料提供了丰富的菌种资源.     

Redescription of Glomus caledonium based on correspondence of spore morphological characters in type specimens and a living reference culture

Glomus caledonium accession UK301 from Rothamsted, England was designated a living reference culture of the species based on close correspondence of spore morphological characters with those of preserved holotype (Farlow Herbarium) and paratype (Oregon State University) specimens. Morphological characters were defined and interpreted according to their origin and sequence in spore differentiation. Three discrete stages were discriminated by simultaneous appearance of new layers in the spore wall and in the wall of the subtending hypha. An outer mucilaginous layer nonreactive in Melzer's reagent and a more rigid hyaline layer were present initially in the most juvenile spore wall, followed by de novo formation of a granular layer and and a yellow-brown laminate layer. Spore wall differentiation terminated with the innermost sublayer of the laminate layer occluding the hyphal pore. A germ tube, when present, originated in the lumen of the subtending hypha near the occluding sublayer. A preserved voucher of an isolate from France and a living culture from Denmark possessed corresponding subcellular characters to those of isolate UK301, thus establishing definitive morphological criteria to group different geographic isolates of the species.     

BASIDIOSPOROGENESIS IN BOLETUS RUBINELLUS. I. STERIGMAL INITIATION AND EARLY SPORE DEVELOPMENT

Sterigmal initiation in Boletus rubinellus resembled hyphal tip growth. Four stages in early basidiospore development have been delineated based on gross morphology, and changes in wall layers and cytoplasm. Changes in wall layers and cytoplasm during spore development were stage-specific. During Stage 1 the spore wall consisted of two layers identical to those of the sterigmal wall with occasional pellicle remnants on the outer surface. The onset of wall differentiation began in Stage 2, and during Stage 3 wall layers characteristic of the mature spore developed. At Stage 4 there was a pronounced gradient in wall thickness from the apex to the base of the spore. Small vesicles (30–60 nm diam) were uniformly distributed in the cytoplasm of spherically enlarging spores (Stage 2), but during spore elongation (Stages 3 and 4) numerous larger vesicles as well as small vesicles aggregated at the spore apex. A variety of cytoplasmic organelles entered the spore during Stage 3; however, migration of storage materials and the nucleus to the spore did not occur until late basidiospore development. The hilar appendix body developed in the earliest spore primordium and persisted until Stage 3. Development of wall layers and their differential thickening, distribution of vesicles, and probable function of the hilar appendix body are discussed with reference to the control of spore shape. Systematic implications of the data are considered.     

Composition and Ultrastructure of Streptomyces venezuelae

Streptomyces venezuelae is a filamentous bacterium with branching vegetative hyphae embedded in the substrate and aerial hyphae bearing spores. The exterior of the spore is inlaid with myriads of tiny rods which can be removed with xylene. The spore wall is approximately 30 nanometers thick. Occasionally, it can be seen that the plasma membrane and the membranous bodies within a spore are connected. The spore's germ plasm is not separated from the cytoplasm by a nuclear envelope. The cell walls of the vegetative hyphae, which are about 15 nanometers thick, are structurally and chemically similar to those of gram-positive bacteria. The numerous internal membranous bodies, some of which arise from the plasma membrane of the vegetative hypha, may be vesicular, whirled, or convoluted. Membranous bodies are usually prominent at the hyphal apices and are associated with septum formation. The germ plasm is an elongate, contorted, centrally placed area of lower electron density than the hyphal cytoplasm. The spores differ from the vegetative hyphae, not only in fine structure, but also in the arginine and leucine contents of their total cellular proteins.     

Status of nuclear division in arbuscular mycorrhizal fungi during in vitro development

Summary The number of nuclei in spores and along hyphae of an arbuscular mycorrhizal fungiGigaspora margarita was measured in digital images of fluorescence arising from mithramycin stained cultures. Typical dormant spores (250 m diameter) contained 2000 nuclei. Eight hundred nuclei were mobilized during the first 3 days of germination. The number of nuclei in the spores nearly returned to the initial number after 22 days of hyphal growth. The average relative DNA content in the nuclei of dormant spores and in the nuclei of spores incubated for 22 days was comparable, as judged from fluorescence intensity. Hyphal elongation occurred with 460 nuclei per cm under a special set of in vitro conditions that promote extensive hyphal growth of arbuscular mycorrhizal fungi. We found an average total of 26000 hyphal nuclei per germinating spore after 22 days. The specific DNA polymerase inhibitor aphidicolin did not inhibit spore germination but it rapidly reduced the rate of hyphal growth and arrested growth after 4 days. No nuclei were produced de novo during this time. These results demonstrate thatG. margarita replicates nuclear DNA and undergoes nuclear division when grown in vitro even in the absence of a plant host.     

CabC, an EF-hand calcium-binding protein, is involved in Ca2+-mediated regulation of spore germination and aerial hypha formation in Streptomyces coelicolor

Ca(2+) was reported to regulate spore germination and aerial hypha formation in streptomycetes; the underlying mechanism of this regulation is not known. cabC, a gene encoding an EF-hand calcium-binding protein, was disrupted or overexpressed in Streptomyces coelicolor M145. On R5- agar, the disruption of cabC resulted in denser aerial hyphae with more short branches, swollen hyphal tips, and early-germinating spores on the spore chain, while cabC overexpression significantly delayed development. Manipulation of the Ca(2+) concentration in R5- agar could reverse the phenotypes of cabC disruption or overexpression mutants and mimic mutant phenotypes with M145, suggesting that the mutant phenotypes were due to changes in the intracellular Ca(2+) concentration. CabC expression was strongly activated in aerial hyphae, as determined by Western blotting against CabC and confocal laser scanning microscopy detection of CabC::enhanced green fluorescent protein (EGFP). CabC::EGFP fusion proteins were evenly distributed in substrate mycelia, aerial mycelia, and spores. Taken together, these results demonstrate that CabC is involved in Ca(2+)-mediated regulation of spore germination and aerial hypha formation in S. coelicolor. CabC most likely acts as a Ca(2+) buffer and exerts its regulatory effects by controlling the intracellular Ca(2+) concentration.     

红盖鳞毛蕨孢子囊早期发育与质体分化的研究

利用光学显微镜和透射电子显微镜观察了红盖鳞毛蕨(Dryopteris erythrosora(Eaton)O.Ktze.)孢子囊的发育及在此期间质体的分化过程。研究表明:(1)红盖鳞毛蕨孢子囊的发育类型属于薄囊蕨型;(2)绒毡层为混合型,即内层绒毡层为原生质团型,外层绒毡层为腺质型;(3)孢子囊原始细胞中的质体通过3条路径分化,其一,原始细胞中含淀粉粒的质体通过分裂分配到下方细胞,继而进入孢子囊柄;其二,原始细胞分裂产生的新生质体被分配到上方细胞,进而被分配到除顶细胞外的原基细胞中,顶细胞将含淀粉粒的质体通过分裂分配到外套层原始细胞中;其三,顶细胞也将具淀粉粒的质体通过分裂分配到内部细胞,使分裂产生的孢原细胞和绒毡层原始细胞具新生质体;造孢细胞和孢子母细胞的质体具淀粉粒,孢子母细胞还具油体,新生孢子中具造粉体和油体;两层绒毡层具新生质体,随着退化外层绒毡层出现造粉体,内层绒毡层出现油体;(4)红盖鳞毛蕨与少数被子植物小孢子发育阶段质体分化模式类似,由前质体分化为造粉体再到油体。研究结果为蕨类植物质体在孢子囊发育过程不同组织细胞中的差异分化提供了新观察资料,为蕨类植物发育生物学和系统演化研究提供科学依据。     

Ascocarp and spore ontogeny in two species of Sphaerophorus (Caliciales)

Two different ascocarp ontogenies have been demonstrated in Sphaerophorus (Caliciales). A globose primordium arises in both species investigated. In S. globosus , the primordium wall encloses the whole structure during the hymenium development, although a weak differentiation of the basal part takes place, while in S. tener the wall is clearly differentiated into a watchglass-like basal part and a cap-like apical part not in contact with the basal part. In S. globosus , the cover disintegrates during the mazaedial development. In S. tener , the cover persists until the mazaedium is well developed, and is then shed, most often in one piece, thereby exposing the spores. The mechanism of the shedding and the possible biological consequences of the specialized ascocarp ontogeny in S. tener is discussed. The spore ontogeny of S. tener has also been investigated. No ornamentation is deposited on the spore surface until the spores are liberated from the asci. The spore ontogeny thus resembles that of S. murrayii , but only a small amount of mazaedial material is deposited on the spore surface. The classification of the species is discussed and it is concluded that the genus Sphaerophorus as presently circumscribed is very heterogeneous.     

Effect of trehalose on the viability of sporangiospores of the mucorous fungus <Emphasis Type="Italic">Blakeslea trispora</Emphasis>

Sporangiospores of Blakeslea trispora are in a state of exogenous dormancy, and water is the key factor controlling their germination. A wide range of carbohydrates, ammonium salts, and yeast extract had a weak stimulating effect (less than 50%) on spore germination, whereas amino acids could significantly inhibit this process. Cultivation of B. trispora on sporogenous sucrose- and trehalose-containing media (S and T spores, respectively) resulted in significant changes in spore formation, as well as in the chemical composition of spores and their viability. In the presence of trehalose, the amount of spores increased twofold; spore viability during storage increased as well. All changes in the carbohydrate composition of the cytosol and in the composition of the spore membrane lipids indicated that the dormancy of T spores was deeper than that of S spores, which has a favorable effect on their viability.     

Ammonia promotes accumulation of intracellular cAMP in differentiating amoebae of Dictyostelium discoideum

We used sporogenous mutants of Dictyostelium discoideum to investigate the mechanism(s) by which exogenous NH4Cl and high ambient pH promote spore formation during in vitro differentiation. The level of NH4Cl required to optimize spore formation is correlated inversely with pH, indicating that NH3 rather than NH4+ is the active species. The spore-promoting activity of high ambient pH (without exogenous NH4Cl) was eliminated by the addition of an NH3-scavenging cocktail, suggesting that high pH promotes spore differentiation by increasing the ratio of NH3:NH4+ secreted into the medium by developing cells. High ammonia levels and high pH stimulated precocious accumulation of intracellular cAMP in both sporogenous and wild-type cells. In both treatments, peak cAMP levels equaled or exceeded control levels and were maintained for longer periods than in control cells. In contrast, ammonia strongly inhibited accumulation of extracellular cAMP without increasing the rate of extracellular cAMP hydrolysis, indicating that ammonia promotes accumulation of intracellular cAMP by inhibiting cAMP secretion. These results are consistent with previous observations that factors that raise intracellular cAMP levels increase spore formation. Lowering intracellular cAMP levels with caffeine or progesterone inhibited spore formation, but simultaneous exposure to these drugs and optimal concentrations of NH4Cl restored both cAMP accumulation and spore formation to normal levels. These data suggest that ammonia, which is a natural Dictyostelium morphogen, favors spore formation by promoting accumulation or maintenance of high intracellular cAMP levels.     

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.     

Hyphal growth and fragmentation of Penicillium chrysogenum in submerged cultures

A previously derived population model describing the average properties of hyphal elements in submerged cultures of filamentous fungi was revised, and a term for the influence fo spore germination on the average total hyphal length was added. The model was derived from a general balance for the distribution function for the hyphal elements. Based on experimental data and the derived model, simple kinetic expressions for spore germination, tip extension, branching, and hyphal break-up were set up. It is concluded that spore germination can be quantified by three parameters: (1) the time at which spore germination is initiated, (2) the time at which spore germination terminates, and (3) the fraction of viable spores in a spore suspension. The frequency of spore germination can be described with the B-distribution. For growth kinetics it is concluded that the branching frequency is closely correlated with the total hyphal length and that the average tip-extension rate can be described with saturation kinetics with respect to the hyphal length. Finally, the rate of fragmentation is linearly related to the energy input to the bioreactor, and related to the effective hyphal length. (c) 1995 John Wiley & Sons, Inc.