Identification of genes up-regulated during conidiation of Fusarium oxysporum through expressed sequence tag analysis

Fusarium oxysporum produces three kinds of asexual spores, microconidia, macroconidia, and chlamydospores. F. oxysporum produces microconidia and macroconidia in carboxymethyl cellulose-added liquid medium (CMCLM) and exhibits vegetative growth without conidiation in complete liquid medium (CLM). The cDNA libraries were constructed using mRNAs from CLM and CMCLM cultures. A total of 1288 and 1353 clones from CLM (vegetative growth) and CMCLM (conidiation) libraries, respectively, were sequenced, and 641 and 626 unique genes were identified. Of these unique genes, only 130 ( approximately 20%) were common in the two libraries, indicating different patterns of gene expression during vegetative growth and conidiation. The expression levels of 496 CMCLM-specific genes were compared during vegetative growth and conidiation by cDNA dot-blot differential hybridization and real-time quantitative PCR analyses, and 42 genes were identified to display >5-fold increases in mRNA abundance during conidiation. These genes provide ideal candidates for further studies directed at understanding fungal conidiogenesis and its molecular regulation.     

Variability in Indian Isolates of Arthrobotrys dactyloides Drechsler: A Nematode-Trapping Fungus

Five isolates of Arthrobotrys dactyloides (A, B, C, D, and E) were isolated from different locations of India. Their variability in relation to morphology, radial growth, and nematode capturing or trap-forming ability was observed. All of the five isolates produced two-celled slender conidia, whereas wider two- and three-celled conidia were produced by isolates A, C, D, and E only. The wider two- and three-celled conidia were not observed in cultures of isolate B. The isolate B produced macroconidia as well as microconidia. The microconidia were produced on separate conidiophores of smaller size. Macroconidia and microconidia were never produced on the same conidiophore, but the two types of conidiophores were produced on same or different hyphae. Similar to macroconidia, the microconidia also produced constricting rings of smaller size in presence of Meloidogyne graminicola. The constricting rings formed on microconidia did not capture second-stage juveniles of M. graminicola because of their smaller size. Among all the isolates, isolate B showed slow growth and higher nematode-capturing ability or trap-forming ability.     

Ultrastructural Studies of Microconidium Formation in Neurospora crassa

Microconidiating cultures of “peach-fluffy” (pe, fl; Y8743m, L; FGSC #569) were fixed at various times after the initiation of growth and examined with an electron microscope. Hyphae from which microconidia form are markedly vacuolated and show a much more extensive system of rough endoplasmic reticulum than young vegetative hyphae. A bulge in the hypha presages the start of microconidium formation, followed by the rupture of the outermost wall layers. A thick collar forms around the protruding microconidium due to extensive thickening of the inner wall layer of the parent hypha. At this stage, the cytoplasm of the developing microconidium is still continuous with that of the microsporophore cell from which it arises and is contained by a wall which is derived from the thickened collar. The microconidium is finally isolated from the cytoplasm of the microsporophore by a centripetal extension of the collar. Microconidia differ from macroconidia in having a more extensive endoplasmic reticulum and fewer mitochondria, in addition to being smaller and having a single nucleus.     

New species ofAcremonium, Cylindrocarpon andVerticillium from soil in the Bonin (Ogasawara) Islands, Japan

Three new soil fungi from the Bonin (Ogasawara) Islands, Japan are described:Acremonium macroclavatum, characterized by large clavate guttulate conidia;Cylindrocarpon boninense, characterized by 3–7-septate clavate macroconidia, terminal or intercalary chlamydospores, rarely produced unicellular clavate microconidia; andVerticillium hahajimaense, characterized by conidial heads bearing cylindrical conidia, and catenulate chlamydospores.     

Ultrastructural Studies on the Microconidia of Botrytis cinerea Pers. and their Phialoconidial Development

The genesis and ultrastructure of microconidia of Botrytis cinerea Pers. were studied, using TEM and SEM. Microconidia are produced in flask-shaped, terminal or lateral phialides. The first microconidium develops holoblastically, subsequent microconidia are formed enteroblastically, in basipetal succession from a fixed conidiogenous locus. Mature microconidia are characterized by a two-layered spore wall covered by mucilage, a single, sickle-shaped nucleus, one or two large lipid bodies, a few mitochondria of the cristae-type, ribosomes, sparse endoplasmatic reticulum close to the plasmalemma and a small amount of cytoplasma. The basal septum which is surrounded by a collar-like rim is perforated by a simple central pore with a pore plug. Differences in genesis and ultrastructure of microconidia compared with macroconidia are discussed in relation to the function of these two spore types.     

Identification and pathogenic characterization of endophytic <Emphasis Type="Italic">Fusarium</Emphasis> species from cowpea seeds

Isolates of Fusarium were obtained and identified from seeds of cowpea, Vigna unguiculata (L.) Walp., by means of blotter tests and slide cultures. Species were differentiated according to the morphology of the macroconidia, microconidia and their arrangement in chains or false heads, the size and type of conidiophore, and the presence or absence of chlamydospores. The species were identified as F. semitectum, F. equiseti, F. oxysporum, F. solani, F. anthophilum, F. sporotrichioides, F. moniliforme, and Fusarium sp. Among the species, F. semitectum was the most frequently detected. None of these species were pathogenic when inoculated in susceptible cowpea cultivar (BR 17- Gurgueia). But, an isolate of F. oxysporum f. sp. tracheiphilum used as a standard of comparison for pathogenicity (control) induced symptoms of yellowing, vascular wilting, and death of a susceptible cowpea cultivar under the same environmental conditions.     

两种捕食线虫丝孢菌形态多样性研究

多头节丛孢[Arthrobotrys polycephala(Drechsler)Rifai]和附胞隔指孢[Dactylella haptospora(Drechsler)Zhang et al.)是两种极不常见的捕食线虫丝孢菌,A.polycephala为国内新纪录种。通过对这两种菌的形态观察发现:A.polycephala的大、小分生孢子均可萌发,后代同母体,小分生孢子一般为单端萌发,大分生孢子为两端萌发,并且小分生孢子可以继续发育形成大分生孢子。这两种菌均形成类似厚垣孢子的结构。     

Infectivity of Chlamydospores vs Microconidia of Fusarium oxysporum f.sp. lycopersici on Tomato

The effect of nature of inoculum on disease induced by Fusarium oxysporum f.sp. lycopersici on tomato was tested. Chlamydospores produced in soil 30 days after inoculation induced a more severe disease than microconidia indicating a higher inoculum potential of chlamydospores.
The method proposed produces easily an inoculum of F. oxysporum f.sp. lycopersici which infects the plants consistently and induces a relatively high disease severity.     

Autophagy during conidiation and conidial germination in filamentous fungi

Filamentous fungi form aerial hyphae on solid medium, and some of these differentiate into conidiophores for asexual sporulation (conidiation). In the filamentous deuteromycete, Aspergillus oryzae, aerial hyphae are formed from the foot cells and some differentiate into conidiophores, which are composed of vesicles, phialides and conidia. Recently, we isolated the yeast ATG8 gene homologue Aoatg8 from A. oryzae, and visualized autophagy by the expression of an EGFP (enhanced green fluorescent protein)-AoAtg8 fusion protein and DsRed2 protein in this fungus. Furthermore, by constructing the Aoatg8 deletion and conditional mutants, we demonstrated that autophagy functions during the process of differentiation of aerial hyphae, conidiation and conidial germination in A. oryzae. Here, we discuss the contribution of autophagy towards the differentiation and germination processes in filamentous fungi.     

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。     

Fusarium foetens, a new species pathogenic to begonia elatior hybrids (Begonia x hiemalis) and the sister taxon of the Fusarium oxysporum species complex

A new disease recently was discovered in begonia elatior hybrid (Begonia × hiemalis) nurseries in The Netherlands. Diseased plants showed a combination of basal rot, vein yellowing and wilting and the base of collapsing plants was covered by unusually large masses of Fusarium macroconidia. A species of Fusarium was isolated consistently from the discolored veins of leaves and stems. It differed morphologically from F. begoniae, a known agent of begonia flower, leaf and stem blight. The Fusarium species resembled members of the F. oxysporum species complex in producing short monophialides on the aerial mycelium and abundant chlamydospores. Other phenotypic characters such as polyphialides formed occasionally in at least some strains, relatively long monophialides intermingled with the short monophialides formed on the aerial mycelium, distinct sporodochial conidiomata, and distinct pungent colony odor distinguished it from the F. oxysporum species complex. Phylogenetic analyses of partial sequences of the mitochondrial small subunit of the ribosomal DNA (mtSSU rDNA), nuclear translation elongation factor 1α (EF-1α) and β-tubulin gene exons and introns indicate that the Fusarium species represents a sister group of the F. oxysporum species complex. Begonia × hiemalis cultivars Bazan, Bellona and Netja Dark proved to be highly susceptible to the new species. Inoculated plants developed tracheomycosis within 4 wk, and most died within 8 wk. The new taxon was not pathogenic to Euphorbia pulcherrima, Impatiens walleriana and Saintpaulia ionantha that commonly are grown in nurseries along with B. × hiemalis. Inoculated plants of Cyclamen persicum did not develop the disease but had discolored vessels from which the inoculated fungus was isolated. Given that the newly discovered begonia pathogen is distinct in pathogenicity, morphology and phylogeny from other fusaria, it is described here as a new species, Fusarium foetens.     

Successful induction and recognition of conidiation,conidial germination and chlamydospore formation in pure culture of Morchella

Morels, fungi from the genus Morchella, are popular edible mushrooms. However, little knowledge of their asexual reproduction and inaccessible pure mitospores hamper illumination of their life cycle. Herein, we successfully induced conidiation, conidial germination and chlamydospore formation in pure culture of Morchella sextelata. Conidiation proceeded via four morphologically distinct stages: development of the conidiophore stalk, stalk branching, phialide differentiation, and conidium production. Terminal and intercalary chlamydospores were formed on conidial hyphae. The development of conidiophores occurred earlier, with more conidia produced, in cross-mating cultures than in single-spore cultures. Mature conidia were spherical and 2.5–8 μm in diameter, with a vast majority (nearly 99%) 2.5–5 μm in diameter. Each conidium contained one to three nuclei (80.2% conidia contained one nucleus, 19.1% contained two nuclei, and 0.7% contained three nuclei). The conidial nucleus diameter was 1–2 μm. The nuclear mitosis in detached conidia that was observed may benefit their colony initiation. Additionally, morel conidia formed conidial anastomosis tubes. Conidia (mitospores) likely not only function as spermatia, but also as reproductive propagules in Morchella. Further research is imperative to elucidate the relationship between the conidia and chlamydospores, and their unique function in the morel life cycle.     

Cylindrocarpon pauciseptatum sp. nov., with notes on Cylindrocarpon species with wide, predominantly 3-septate macroconidia

A Cylindrocarpon species with up to 10 μm wide, straight and predominantly 3-septate macroconidia, subglobose to ovoidal microconidia and chlamydospores, is described as Cyl. pauciseptatum. It is most similar to Cyl. austrodestructans but no chlamydospores and microconidia are formed in the latter. Similar macroconidia also occur in Cyl. theobromicola, which forms oval to ellipsoidal microconidia at least sparsely and has slightly curved macroconidia, and Cyl. destructans var. crassum, which forms abundant 1-celled microconidia. DNA sequence data of the internal transcribed spacer regions 1 and 2 plus the 5.8S rDNA and the partial beta-tubulin gene were used for phylogenetic inferences. Cylindrocarpon pauciseptatum and Cyl. macrodidymum are monophyletic and are closely related to other species of Cylindrocarpon sensu stricto including members of the Cyl. destructans (teleomorph, Neonectria radicicola) species complex, which accommodates Cyl. liriodendri (teleomorph, Neon. liriodendri), Cyl. destructans var. crassum and Cyl. austrodestructans (teleomorph, Neonectria austroradicicola comb. nov.). Cylindrocarpon theobromicola is distantly related to species of Cylindrocarpon sensu stricto or Neonectria sensu stricto. It clustered among cylindrocarpon-like species with curved macroconidia, of which some belong to the Neon. mammoidea group. Relatively voluminous cells in sporodochial conidiophores of Cyl. theobromicola resembled those described for Campylocarpon, which is closely related to members of the Neon. mammoidea group including Cyl. theobromicola. Cylindrocarpon pauciseptatum has been isolated from roots of Vitis spp. in South-eastern Europe (Slovenia) as well as New Zealand, where it also occurs on roots of Erica melanthera.     

Strahleninduzierte Mutationen bei Dermatophyten III. Untersuchungen über den Zusammenhang der Virulenz mit der Mikromorphologie und der keratinolytischen Aktivität

Zusammenfassung 1. Aus morphologisch normaler Kultur vonMicrosporon gypseum wurden die UV-Mutanten folgender acht Kategorien selektiert: 1. Kulturen, die Makrokonidien, Mikrokonidien und Chlamydosporen bilden, 2. Kulturen mit Makrokonidien und Mikrokonidien, 3. Kulturen mit Makrokonidien und Chlamydosporen, 4. Kulturen mit Makrokonidien und Chlamydosporen, 5. Kulturen nur mit Makrokonidien, 6. Kulturen nur mit Mikrokonidien, 7. Kulturen nur mit Chlamydosporen, 8. Kulturen vollständig ohne Sporen.2. Alle Kulturen dieser Mutantenkategorien zeigten in vitro die keratinolytische Aktivität und die Griseofulvinempfindlichkeit. Bei den Mutanten wurden nur quantitative Unterschiede in diesen Eigenschaften beobachtet. Manche mutierten Kulturen waren mehr als zehnmal griseofulvinsensitiver als die Ausgangskulturen.3. Aus den virulenten Kulturen vonM. gypseum wurden avirulente Kulturen isoliert.4. Die Kulturen, welche nur Chlamydosporen bildeten und solche, die überhaupt keine Sporenbildung aufwiesen, waren in allen Fällen avirulent. Die konidienbildenden (Makrokonidien, Mikrokonidien) mutierten Kulturen waren in den meisten Fällen virulent.5. Es wurde kein Zusammenhang zwischen der keratinolytischen Fähigkeit in vitro und der Virulenz der Kultur in vivo gefunden. Die in vitro erhöhte keratinolytische Aktivität des DermatophytenM. gypseum determiniert selbst nicht seine Virulenz.

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Summary 1. From the morphologically normal strain ofMicrosporon gypseum eight groups of mutants were selected: 1. strains with production of macroconidia, microconidia and chlamydospores, 2. strains with macroconidia and microconidia, 3. strains with macroconidia and chlamydospores, 4. strains with microconidia and chlamydospores, 5. strains with macroconidia only, 6. strains with microconidia only, 7. strains with chlamydospores only, 8. mycelial strains without production of spores.2. All the strains of the eight group were keratinolytically active in vitro and sensitive to griseofulvin. Quantitative differences, in the features evaluated, were only found. Some mutants were more than 10 times more sensitive to griseofulvine in comparison with the wild strains.3. Avirulent strains were isolated from the virulent wild strainM. gypseum.4. The strains producing only chlamydospores and mycelial strains without sporulation were avirulent in all cases. The greater part of conidia producing strains remained virulent.5. No relationship between the degree of keratinolytic activity in vitro and virulence in vivo was found. The virulence itself cannot be determined only by an increase in keratinolytic activity in vitro.

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Diese Mitteilung wurde auf dem III. Internationalen Dermatologischen Symposium in Bratislava (4.–6.X.1966) vorgetragen.     

A NEW FUNGUS ON CYPERUS IRIA

Thind , K. S., and G. S. Rawla . (Panjab U., Chandigarh, India.) A new fungus on Cyperus iria. Amer. Jour. Bot. 48(10): 859–862. Illus. 1961—Duosporium cyperi gen. et sp. nov., collected on Cyperus iria Linn., is characterized by the production of 2 types of conidia, macroconidia and microconidia. The macroconidia are straight, cylindrical, triseptate, with their middle cells dark brown, long, thick-walled, and end cells much paler or subhyaline, short, thin-walled, the middle and the end cells being equally wide. The microconidia are nonseptate, spherical, dark brown, prominently verrucose, and produced apically on the conidiophores like the macroconidia; occasionally also formed as secondary conidia on germinating 3-septate macroconidia in culture. The formation of 2 distinct types of conidia in Duosporium and the features of its macroconidia characteristically differentiate it from its close relative Helminthosporium, although Duosporium. cyperi is not unique among fungi in producing 2 types of conidia.     

The Aspergillus nidulans putative kinase, KfsA (kinase for septation), plays a role in septation and is required for efficient asexual spore formation

In Aspergillus nidulans nuclear division and cytokinesis are coupled processes during asexual sporulation. Metulae, phialides and conidia contain a single nucleus. Here we describe the role of a putative Saccharomyces cerevisiae Kin4-related kinase, KfsA (kinase for septation) in the control of septum formation in A. nidulans. The kfsA deletion caused an increase in the number of conidiophores with septa in their stalks from 20% in wild type to 60% in the mutant strain. Interestingly, 7% of metulae contained two nuclei and the corresponding phialides remained anucleate, suggesting septum formation before proper segregation of nuclei. This points to a checkpoint control of KfsA, which prevents septum formation before nuclear separation. KfsA localized to the cortex and septa in hyphae and in conidiophores but not to the spindle-pole bodies, as it was shown for Kin4 in yeast. KfsA appeared at septa after actin disappeared, suggesting an additional role of KfsA late during septum formation.