Morphology,development and cytology of Taphrina maculans Butler

Morphology, development and nuclear behavior of the ascogenous stroma and asci in the infection spots have been described inTaphrina maculans Butler. The fungus forms subcuticular and intercellular mycelium in the leaf tissues and the ascogenous layers originate through division of the subcuticular hyphal cells in the infection sites. Germination of ascogenous cells starts with their elongation in the uppermost layer forming asci and ascospores without formation of stalk cells. Meiosis of the fusion (diploid) nucleus occurs in the young ascus as in otherTaphrina species devoid of stalk cells. The haploid chromosome complement in this species consists of 3 chromosomes (n=3). All the cells in the stromatic layer are potential ascogenous cells and ascus formation continues, until all of them are exhausted in the infection spot. Eight ascospores are normally formed in each ascus, but multi-plication of ascospores may occurin situ later. Three morphologically distinct types of ascus opening are encountered, which are apparently not correlated with prevalent environment. Multiplication of ascospores after their discharge from mature asci occurs by budding proceded by a mitotic division of the spore nucleus. Blastospores (budded cells) germinate into short hyphae and binucleate condition of cells originates by mitotic division of the nucleus. Occurrence of giant cells containing 2 nuclei is often observed. Possible origin of Uredinales fromTaphrina-like ancestors has been indicated due to their close resemblance.     

Studies on taphrina maculans butler,inciting leaf spot of turmeric (curcuma longa L.). I. Isolation of the pathogen

Summary T. maculans inciting leaf spot of turmeric is difficult to be isolated into a pure culture. The best stage of the spot development on turmeric which yielded successful isolations was either the initial or the middle stage expressing yellowish necrotic areas. The successful course of development of asci, ascospore and conidia has been worked out. By streaking ascospore and conidial suspensions, the spot bearing portions of leaves on the inner side of the lid thus placing them in hanging position above the plates poured with cleared and acidified potato dextrose agar or turmeric leaf decoction agar or by streaking sterile water washings from the chocolate brown spots on these media, it was possible to isolate successfully the pathogenic culture ofT. maculans at 20° C.Condensed from the thesis submitted by the senior author to the University of Poona for M. Sc. (Agri.) under the guidance of the junior author. Respectively Assistant Professor of Plant Pathology, College of Agri. Poona and Wheat Rust Mycologist, Mahabaleshwar, India.     

Studies on taphrina maculans butler inciting leaf spot of Turmeric (Curcuma Longa L.). III. Perpetuation and transmission of the disease

Summary The pathogenT. maculans inciting leaf spot of turmeric perpetuates in the form of ascospores and conidia and incites first infection on the lowermost leaves, in October and November when the atmospheric humidity prevails about 80 % with 21°–23° C temperatures. The secondary infection is due to availability of large potential of inoculum of ascospores and conidia frequently and periodically produced by the pathogen under cool temperature conditions (20°–24° C) with about 80 % atmospheric humidity. Plant debris, rhizomes etc. of the previously affected crop or soil from the fields where turmeric crop was taken in the previous season do not serve the primary source of infection.Condensed from the Thesis submitted by the senior author to the University of Poona for M. Sc. (Agri.) under the guidance of the Junior author.Respectively Assistant Professor of Plant Pathology.College of Agriculture, Poona and Wheat Rust Mycologist, Mahableshwar, India.     

CHROMOSOME NUMBERS IN THE HYPOCREALES. II. ASCUS DEVELOPMENT IN NECTRIA CINNABARINA

The cytology of ascus development in Nectria cinnabarina was investigated with the orceinsmear technique, from crozier formation to ascospore maturation. At prophase I synapsis occurs while the chromosomes are still contracted, and the nucleus passes through dictyotene, a diffuse stage rarely seen in plants. A haploid complement of five chromosomes has been precisely determined. The first two divisions in the ascus constitute meiosis, and the third (mitotic) is followed by ascospore delimitation. A fourth division takes place in the ascospore, which is subsequently divided by a septum into two uninucleate cells. Of all species of Nectria thus far investigated N. cinnabarina is the only species in which additional nuclear divisions in the ascus do occur, accounting for the multinucleate condition in the ascospore cells. The bearing that this distinctive nuclear condition has on phylogeny and evolution in the Hypocreales is discussed.     

Anatomy and cytology of Taphrina entomospora during infection of Nothofagus

Taphrina entomospora is one of the few species of the genus described on native plants of the Southern Hemisphere and also one of the few leaf pathogens known on Nothofagus species. The anatomical changes it produces on N. pumilio leaves, and its morphology, cytology, and sporogenesis were studied. The fungus is a perennial species that overwinters as mycelium in the foliar buds and infects the developing leaves, so the whole blade develops the disease symptoms. Interveinal areas of the leaves become chlorotic, thickened and rounded. Palisade parenchyma fails to develop, with spongy parenchyma developing as packed, rounded, isodiametric cells with little intercellular space. The mycelium is subcuticular, dikaryotic, and produces ascogenous hyphae, asci, and ascospores as described for other species in the genus. Before ascus discharge, ascospores bud in a regular, unique way. The life-cycle of T. entomospora is compared with other representative taxa in the genus and the distribution of this pathogen is discussed.     

Taphrina maculans reduces the therapeutic value of turmeric (Curcuma longa)

Rhizomes of turmeric are used in several culinary preparations. They have been used as household remedies since time immemorial. Phenolics are found in plants only and they play a great role in human health. The high performance liquid chromatography of different parts of healthy and infected plants reveals that the phenolic acid content is reduced in diseased tissues including rhizomes which are commonly consumed by human beings in several countries of the world. Results also showed that the infected leaves had maximum phenolic acids as compared to healthy leaves which indicate that due to infection the amount of phenolic acids increased. We report for the first time that leaf spot (Taphrina maculans) of turmeric (Curcuma longa), which is an important disease, reduces the quantity and number of phenolics thereby damaging the therapeutic properties of turmeric.     

Ascospore aggregation and oxylipin distribution in the yeast Dipodascopsis tothii

Upon cultivation of the yeast Dipodascopsis tothii in its sexual stage, small ascospores are released individually from the ascus tip, which then assemble in sheathed cluster balls. In contrast to Dipodascopsis uninucleata, this yeast produced smooth bean shaped ascospores with sheath-like appendages that assemble in a disordered sheathed ball of ascospores outside the ascus. Strikingly, upon release, the ascus tip contained 3-hydroxy oxylipins, while the released ascospore clusters contained little or no 3-hydroxy oxylipins as indicated by immunofluorescence microscopy. In D. uninucleata, these oxylipins are concentrated on the spore surface and interspore matrix, but not on the ascus tip.     

The origin of ascospore-delimiting membranes in Taphrina deformans

Summary The origin of ascospore-delimitig membranes in Taphrina deformans has been studied in material fixed in KMnO₄ and stained either in lead citrate or selectively with a phosphotungstic acid-chromic acid mixture (PTA-CA). Structural continuities exist between the ascus plasmalemma and the delimiting membranes. Both of these membrane systems stain preferentially with PTA-CA while other cell membranes do not stain. The spore-delimiting membranes are formed by invagination of the ascus plasmalemma at specific sites adjacent to nuclei. An ascus vesicle is not formed.     

Chemical suppression of the sexual stage of Leptosphaeria maculans on oil-seed rape and turnip seed crop straw

A selection of fungicides, herbicides and surfactants and urea were tested for their effect on the production of pseudothecia and ascospore release of Leptosphaeria maculans present on oil-seed rape straw and turnip seed crop straw. The fungicides ethyl mercury phosphate, triarimol, fenarimol, carbendazim, tridemorph and benomyl, each at 1 g/litre, the herbicides dinoseb and diquat, each at 10 g/litre the surfactants Bradasol, Cetrimide, Deciquam 222, Hyamine 1622 and Maxonol N, each at 50 g product/litre, and urea at 150 g/litre, applied to straw before pseudothecia had formed were more than 90% effective in preventing further development. These chemicals were also effective in preventing further ascospore production when applied to straw bearing mature pseudothecia but only dinoseb and urea prevented the release of mature ascospores. The results suggest that it may be possible to break the life cycle of L. maculans by chemical treatment and thereby obviate the need for subsequent control measures.     

Influence of meteorological parameters on Leptosphaeria maculans and L. biglobosa spore release in central and eastern Poland

Leptosphaeria maculans and L. biglobosa are fungal pathogens able to cause allergic reactions in humans and infect plants of Brassica species. The rate of their development and subsequent spore release depend on weather conditions. The aim of this paper was to pinpoint the exact meteorological conditions triggering the release of L. maculans and L. biglobosa ascospores in central and eastern Poland. Multiple regressions indicated that the frequency and amount of rainfall over short periods were important in mediating spore release. The first ascospore event depended mainly on the number of rainy days during the first 10 days of July and the cumulative precipitation during July and September. The most important variables for maximum spore release were cumulative rainfall in the beginning of July and the end of September, as well as the number of days with precipitation events in the first 10 days of August. The results highlighted for the first time the importance of the days preceding the collection of oilseed rape plants from the field. Higher moisture content of senescing but still living stems play a crucial role in the early start of the ascospore season and the maximum release of ascospores. This was not yet considered to date.     

Ultrastructure of ascospore delimitation in freeze substituted samples ofAscodesmis nigricans (Pezizales)

Summary Freeze substitution proved to be a valuable technique for studying the early stages of ascosporogenesis inAscodesmis nigricans. Our observations indicate that the ascus vesicle originated from the ascus plasma membrane. Invaginations of the plasma membrane produced ascus vesicle initials consisting of two closely spaced unit membranes. The appearance of the outer leaflet of each of these membranes was identical to that of the inner leaflet of the ascus plasma membrane. Apparent points of continuity between ascus vesicle initials and the plasma membrane were observed. Ascus vesicle initials accumulated in the ascus cytoplasm near the plasma membrane and then coalesced to form the ascus vesicle, a peripheral, cylinder-like structure consisting of two closely spaced unit membranes that extended from the ascus apex to the ascus base. The ascus vesicle then became invaginated in a number of regions and subsequently gave rise to eight sheet-like segments, or ascosporedelimiting membranes, that encircled uninucleate segments of cytoplasm forming ascospore initials. Like the ascus vesicle, each ascospore-delimiting membrane consisted of two closely spaced unit membranes, the inner of which became the ascospore plasma membrane. The ascospore wall then developed between the spore plasma membrane and the outer membrane. Many details of ascospore maturation were clearly visible in freeze substituted samples.     

Studies on the development of perithecium of Ceratocystis stenoceras

The development of the perithecium of Ceratocystis stenoceras was observed by a light microscope and by a scanning electron microscope.The fungus has developed dark brown perithecia on wheat agar medium in three days of incubation. Perithecial primordia appeared as tightly knotted coils. At the center of it an oval ascogonium was observed. The ascogonium was developed from a lateral wall of a hypha, and the hyphae covering the ascogonium branched at the basal part where the ascogonium was attached. These hyphae branched repeatedly in the developmental growth to cover the ascogonium, and it was finally covered tightly. The plasmogamy of this fungus is much probably performed by the gametangial contact. As the stage proceeded, the ascogonium elongated, the terminal and the basal portions of it swelled and cleavage of the ascogonium resulted. Each of the cleaved ascogonia germinated continuously and stretched out the ascogenous hyphae. About that time the cells consisting of perithecia were vacuolated from the center and successively dissolved, so that a space was formed in the center of the body. Ascogenous hyphae continued to develop downwards, and their end were fixed to the inner wall of the body.The upper portion of the hyphae converged to the center of the body and the ascogenous hyphae became the supporting tissue for ascus formation.Hook formation was observed prior to the ascus formation. After completion of karyogamy by hook formation, the fissure appeared on the ascus and the end portion was released. The released portion included eight ascospores. The ascus had a smooth surface and no special structure was seen on the top. As the asci were matured, they evanesced by themselves and concurrently ascospores came out. Finally the body was massively filled with ascospores.     

Mapping 3-hydroxy oxylipins on ascospores of <Emphasis Type="Italic">Eremothecium sinecaudum</Emphasis>

3-Hydroxy oxylipins were uncovered on ascospores of Eremothecium sinecaudum using immunofluorescence microscopy. This was confirmed by gas chromatography mass spectrometry. These oxylipins were observed only on ascospore parts characterised by nano-scale surface ornamentations simulating a corkscrew as demonstrated by scanning electron microscopy. Conventional ascospore staining further confirms its hydrophobic nature. Using confocal laser scanning microscopy we found that the corkscrew part with spiky tip of needle-shaped ascospores may play a role in rupturing the ascus in order to affect its release. Through oxylipin inhibition studies we hypothesise a possible role for 3-hydroxy oxylipins in facilitating the rupturing process.     

Effects of temperature on germination and hyphal growth from ascospores of A-group and B-group Leptosphaeria maculans (phoma stem canker of oilseed rape)

Ascospores of both A‐group and B‐group Leptosphaeria maculans germinated at temperatures from 5–20°C on distilled water agar or detached oilseed rape leaves. After 2 h of incubation on water agar, some A‐group ascospores had germinated at 10–20°C and some B‐group ascospores had germinated at 5–20°C. The percentages of both A‐group and B‐group ascospores that had germinated after 24 h of incubation increased with increasing temperature from 5–20°C. The observed time (Vo₅₀) which elapsed from inoculation until 50% of the spores had germinated was shorter for B‐group than for A‐group ascospores. Germ tube length increased with increasing temperature from 5–20°C for both ascospore groups. Germ tubes from B‐group ascospores were longer than germ tubes from A‐group ascospores at all temperatures tested, but the mean diameter of germ tubes from A‐group ascospores (1.8 μm) was greater than that of those from B‐group ascospores (1.2μm) at 15°C and 20°C. The average number of germ tubes produced from A‐group ascospores (3.8) was greater than that from B‐group ascospores (3.1) after 24 h of incubation at 20°C, on both water agar and leaf surfaces. Germ tubes originated predominantly from interstitial cells or terminal cells of A‐group or B‐group ascospores, respectively, on both water agar and leaf surfaces. Hyphae from A‐group ascospores grew tortuously with extensive branching, whilst those from B‐group ascospores were predominantly long and straight with little branching, whether the ascospores were produced from oilseed rape debris or from crosses between single ascospore isolates, and whether ascospores were germinating on water agar or leaf surfaces.     

Cytological and genetic studies of the life cycle of Saccharomycopsis lipolytica

Summary In the alkane yeast Saccharomycopsis lipolytica (formerly: Candida lipolytica) the variability in the ascospore number is caused by the absence of a correlation between the meiotic divisions and spore wall formation. In four spored yeasts, after meiosis II, a spore wall is formed around each of the four nuclei produced by meiosis II. However, in the most frequently occurring two spored asci of S. lipolytica, the two nuclei are already enveloped by the spore wall after meiosis I due to a delay of meiosis II. This division takes place within the spore during the maturation of the ascus. In this case germination of the binucleate ascospore is not preceded by a mitosis. It follows that the cells of the new haploid clones are mononucleate. In the three spored asci, which occur rarely, only one nucleus is surrounded by a spore wall after meiosis I; the other nucleus undergoes meosis II before the onset of spore wall formation. The result is one binucleate and two mononucleate spores. In the one spored asci the two meiotic divisions occur within the young ascospore, i.e. spore wall formation starts immediately after development of the ascus. These cytological observations were substantiated by genetic data, which in addition confirmed the prediction that binucleate spores may be heterokaryotic. This occurs when there is a postreduction of at least one of the genes by which the parents of the cross differ. This also explains the high frequency of prototrophs in the progeny on non-allelic auxotrophs since random spore isolates are made without distinguishing between mono-and binucleate spores. The possibility of analysing offspring of binucleate spores by tetrad analysis is discussed. These findings enable us to understand the life cycle of S. lipolytica in detail and we are now in a position to start concerted breeding for strain improvement especially with respect to single cell protein production.     

New fungal genera, Tectonidula gen. nov. for Calosphaeria-like fungi with holoblastic-denticulate conidiogenesis and Natantiella gen. nov. for three species segregated from Ceratostomella

Two morphologically similar groups of ascomycetes with globose to subglobose perithecia, elongate necks, unitunicate asci floating freely at maturity, and hyaline ascospores currently placed in Calosphaeria s. lat. and Ceratostomella s. lat., respectively, are studied. The Calosphaeria-like fungi have groups of perithecia growing between cortex and wood, arranged in circular groups with converging necks and piercing the cortex in a common point; the asci with a shallow apical ring and U- to horseshoe-shaped hyaline ascospores are compared with Calosphaeria pulchella, the type species of the genus. Conidiogenesis of the investigated Calosphaeria-like fungi is holoblastic-denticulate; ramichloridium-like and sporothrix-like conidiophores and conidia were formed in vitro. Ascospore and ascus morphology, structure of the ascal apex, ascogenous system, mode of conidiogenesis and the large subunit rRNA sequences of this group differ considerably from C. pulchella and both groups are unrelated. Thus a new genus, Tectonidula, is described with two accepted species, T. hippocrepida and T. fagi; they are separated by ascospore and ascus morphology and holoblastic-denticulate conidiogenesis from the core species of Calosphaeria. The placement of Tectonidula among perithecial ascomycetes is discussed. The relationship of Tectonidula with Barbatosphaeria and two ramichloridium-like hyphomycetous genera Rhodoveronaea and Myrmecridium is investigated. Three species formerly attributed to Ceratostomella are studied. The revision of the herbarium type specimen and fresh material of Ceratostomella ligneola revealed that it is conspecific with Ceratostomella ampullasca and Ceratostomella similis. The LSU phylogeny clearly separated C. ligneola from Ceratostomella s. str. and morphologically similar Lentomitella. On the basis of molecular sequence data and detailed comparison of morphology of asci, ascospores and ascogenous system the genus Natantiella is described for C. ligneola with C. ampullasca and C. similis as its synonyms. Natantiella produced sterile mycelium in vitro.     

Epidemiologische Untersuchungen bei der Wurzelhals- und Stengelfäule des Rapses, verursacht durch Phoma lingam

Epidemiological investigations in the root and collar rot of oil seed rape, caused by Phoma lingam In this part of investigations on Phoma lingam (Tode ex. Fr.) Desm., epidemiological experiments were carried out on infection rates of oil seed rape and the fungus spectrum present during the vegetation period as well as on spore dispersal of Leptosphaeria maculans (Desm.) Ces. Et de Not.), the sexual stager of p. lingam. The infection at the stalk base can take place in autumn and about 10 to 40% were found to have P. lingam inside the tissue with higher inoculum potential P. lingam was more frequently isolated at the connection points of the lower leave than at the height of the soil level. The fungus spectrum and the frequency of the fungi isolated changed in course of the vegetation period. Whilst P. lingam and F. tabacinum increased and were isolated at high numbers, Alternaria spp. And P. eupyrena were found less at the beginning and at the end of the season. Two to three other species and further three to four were isolated at low level respectively occurred less common. The pathogenic importance of some of these species is not yet determined. The low level of leaf infection occurring in the federal Republic of Germany may have several reasons as e.g. the short phase of ascospore ejection and the low temperature in November and December. Furthermore, deep ploughing early in autumn for wheat sowing decreased inoculum potential as could be observed in sore catches obtained in several years. The development of the perithecia and the beginning of sport discharge was dependent on the rain fall. Generally, spore flight started a few hours after the rain fall began, but sometimes a high relative humidity seemed to be sufficient to initiate spore discharge. There was the tendency of spores been more discharged at slight winds (2–3) and consequently, because of winds being slightly stronger in the afternoon and evening, spores were more found in these daytimes.     

Assessing Quantitative Resistance against Leptosphaeria maculans (Phoma Stem Canker) in Brassica napus (Oilseed Rape) in Young Plants

Quantitative resistance against Leptosphaeria maculans in Brassica napus is difficult to assess in young plants due to the long period of symptomless growth of the pathogen from the appearance of leaf lesions to the appearance of canker symptoms on the stem. By using doubled haploid (DH) lines A30 (susceptible) and C119 (with quantitative resistance), quantitative resistance against L. maculans was assessed in young plants in controlled environments at two stages: stage 1, growth of the pathogen along leaf veins/petioles towards the stem by leaf lamina inoculation; stage 2, growth in stem tissues to produce stem canker symptoms by leaf petiole inoculation. Two types of inoculum (ascospores; conidia) and three assessment methods (extent of visible necrosis; symptomless pathogen growth visualised using the GFP reporter gene; amount of pathogen DNA quantified by PCR) were used. In stage 1 assessments, significant differences were observed between lines A30 and C119 in area of leaf lesions, distance grown along veins/petioles assessed by visible necrosis or by viewing GFP and amount of L. maculans DNA in leaf petioles. In stage 2 assessments, significant differences were observed between lines A30 and C119 in severity of stem canker and amount of L. maculans DNA in stem tissues. GFP-labelled L. maculans spread more quickly from the stem cortex to the stem pith in A30 than in C119. Stem canker symptoms were produced more rapidly by using ascospore inoculum than by using conidial inoculum. These results suggest that quantitative resistance against L. maculans in B. napus can be assessed in young plants in controlled conditions. Development of methods to phenotype quantitative resistance against plant pathogens in young plants in controlled environments will help identification of stable quantitative resistance for control of crop diseases.     

Programmed ascospore death in the homothallic ascomycete Coniochaeta tetraspora

Immature asci of Coniochaeta tetraspora originally contain eight uninucleate ascospores. Two ascospore pairs in each ascus survive and mature, and two die and degenerate. Arrangement of the two ascospore types in individual linear asci is what would be expected if death is controlled by a chromosomal gene segregating at the second meiotic division in about 50% of asci. Cultures originating from single homokaryotic ascospores or from single uninucleate conidia are self-fertile, again producing eight-spored asci in which four spores disintegrate, generation after generation. These observations indicate that differentiation of two nuclear types occurs de novo in each sexual generation, that it involves alteration of a specific chromosome locus, and that the change occurs early in the sexual phase. One, and only one, of the two haploid nuclei entering each functional zygote must carry the altered element, which is segregated into two of the four meiotic products and is eliminated when ascospores that contain it disintegrate. Fusion of nuclei cannot be random-a recognition mechanism must exist. More study will be needed to determine whether the change that is responsible for ascospore death is genetic or epigenetic, whether it occurs just before the formation of each ascus or originates only once in the ascogonium prior to proliferation of ascogenous hyphae, and whether it reflects developmentally triggered alteration at a locus other than mating type or the activation of a silent mating-type gene that has pleiotropic effects. Similar considerations apply to species such as Sclerotinia trifoliorum and Chromocrea spinulosa, in which all ascospores survive but half the spores in each ascus are small and self-sterile. Unlike C. tetraspora, another four-spored species, Coniochaetidium savoryi, is pseudohomothallic, with ascus development resembling that of Podospora anserina.     

Orbilia ultrastructure, character evolution and phylogeny of Pezizomycotina

Molecular phylogenetic analyses indicate that the monophyletic classes Orbiliomycetes and Pezizomycetes are among the earliest diverging branches of Pezizomycotina, the largest subphylum of the Ascomycota. Although Orbiliomycetes is resolved as the most basal lineage in some analyses, molecular support for the node resolving the relationships between the two classes is low and topologies are unstable. We provide ultrastructural evidence to inform the placement of Orbiliomycetes by studying an Orbilia, a member of the only order (Orbiliales) of the class. The truncate ascus apex in the Orbilia is thin-walled except at the margin, and an irregular wall rupture of the apex permits ascospore discharge. Ascus, ascogenous and non-ascogenous hyphae were simple septate, with septal pores plugged by unelaborated electron-dense, non-membranous occlusions. Globose Woronin bodies were located on both sides of the septum. Nuclear division was characterized by the retention of an intact nuclear envelope, and a two-layered disk-shaped spindle pole body. The less differentiated nature of the spore discharge apparatus and septal pore organization supports an earliest diverging position of Orbiliomycetes within the subphylum, while the closed nuclear division and disk-shaped spindle pole body are interpreted as ancestral state characters for Ascomycota.