Scanning Electron Microscopy of Sclerotia of Sclerotinia trifoliorum and Related Species

The microanatomy of immature 'white', 'slightly pigmented' and mature, 1-month-old 'black' sclerotia of Sclerotinia trifoliorum , S. sclerotiorum , and S. minor were studied by scanning electron microscopy (SEM). A surface mycelial network was present over sclerotia at maturity. Also dried exudate on the superficial, sclerotial cells at maturity was observed. At this stage of morphogenesis an outer layer of the wall of medullary hyphae was synthesized. Two zones (i.e., rind and medulla) of hyphal tissue in sections of mature sclerotia were distinguished. The wall of rind cells was thick and one-layered, whereas the wall of medullary hyphae was thick and bi-layered.
No lacunac (intercellular spaces) in sclerotial rind were found but the sclerotial medulla appeared to be lacunate in all three species. At the SEM level the structural organization of sclerotia of S. trifoliorum was identical to that one of sclerotia of S. sclerotiorum and S. minor. Thus, in the conducted investigation of the sclerotial stromata, a unique, structural characteristic of taxonomic importance to distinguish S. trifoliorum from the other Sclerotinia species was not found. Observations on the sclerotial morphogenesis in S. trifoliorum and the related species agree with and supplement the light and transmission electron microscope studies of other researchers.     

核盘菌属一新种——人参核盘菌

本文报道了采自人参(Panax ginseng C. A．Mey (保留名nom. Coiiscry))的核盘菌属一新种——人参核盘菌(Sclerotinla．Ginseng Wang C. R.,C. F. Chen et J. Chen)。该种在形态学以及可溶性蛋白、果胶(甲)酯酶和多聚半乳糖醛酸酶谱带等方面,均不同于已知种核盘菌(S. sclerotiorum (Lib．)de Bary),小核盘菌(S. minor Jagger)车轴草核盘菌(S．Trifoliorum Erikss．)和细辛核盘菌(S．Asari Wu et C．R. Wang)。模式标本(800719)保存于沈阳农业大学植物免疫研究室。     

Electrophoretic Karyotypes of Sclerotinia sclerotiorum

Electrophoretic karyotypes (EKs) of 83 isolates were variable within agricultural and natural populations of Sclerotinia sclerotiorum, as well as among S. sclerotiorum, Sclerotinia minor, and Sclerotinia trifoliorum. Variation in EKs was not observed within six mitotic or three meiotic lineages of isolates. EKs of 8 to 10 chromosome-sized DNAs were observed. Homologous and heterologous probes hybridized to four linkage groups.     

Ciborinia gentianae sp. nov., the causal organism of sclerotial flower blight of cut-flower gentians

A new Ciborinia causing sclerotial flower blight of cut-flower gentians (Gentiana triflora var. japonica and interspecific hybrids between related species or varieties) is described as Ciborinia gentianae on the morphological basis of sclerotia and apothecia. The characteristics of Ciborinia gentianae are (1) an abundant production of spermodochia in the hollow cavity of host stems; (2) flat and thin sclerotia produced beneath the epidermis and the inclusion of host vascular remnants within their medulla; (3) globose cells composed of ectal excipulum of apothecia; (4) elongated cells with a slight apical swelling in ectal excipulum at the apothecial margin; and (5) tetra nucleate ascospores. Asci and ascospores mounted in Melzer's reagent measured 156–208 × 8–12 μm and 11.8–15 × 5.5–7.1 μm, respectively.     

Electrophoretic studies of Australasian, North American and European isolates of Sclerotinia sclerotiorum and related species.

Forty-seven isolates of Sclerotinia species, collected from a variety of crops growing in Australia, New Zealand, North America and Europe, have been classified into three distinct groups on the electrophoretic patterns for soluble proteins, arylesterase, acid phosphatase, tetrazolium oxidase, glucose-6-phosphate dehydrogenase (NADP-linked) and reduced nicotinamide adenine dinucleotide phosphate dehydrogenase. There were only small intra-group differences. The electrophoretic patterns of an isolate of Whetzelinia (= Sclerotinia) tuberosa were characteristically different from those of the other isolates. These results support the findings from previous studies when ontogenetic, electrophoretic and mycelial-interaction criteria were used to group a smaller number of isolates from New South Wales, Australia. It is concluded that S. sclerotiorum, S. trifoliorum and S. minor are three distinct species.     

An approximation to the phylogeny of Sclerotinia and related genera

Phylogenies are constructed based on nuclear ribosomal internal transcribed spacer (ITS) DNA sequences from an ingroup consisting of 50 isolates representing 24 species of the discomycete family Sclerotiniaceae and an outgroup consisting of five related taxa of the same family. The ingroup taxa are: three Botrytis spp., two Botryotinia spp., one Ciborinia sp., one Dumontinia sp., one Grovesinia sp., six Myriosclerotinia spp., nine Sclerotinia spp. and one Sclerotium sp. The outgroup taxa are: one Ciboria sp., one Encoelia sp. and three Monilinia spp. The type species is included for all taxa except for Ciborinia and Encoelia. Several of the included taxa are important plant pathogens. The resulting phytogenies are discussed with regard to morphology, life history and taxonomy. A suspected relationship between Sclerotinia borealis and S. tetraspora , and Myriosclerotinia is rejected, while a suspected relationship between Ciborinia ciborium and Myriosclerotinia is strongly supported. Sclerotinia ulmariae , previously synonymized with Dumontinia tuberosa , is reinstated as an independent species of Dumontinia. Two new combinations, Dumontinia ulmariae and Myriosclerotinia ciborium , are proposed. The imperfectly known taxon Sclerotium cepivorum seems most closely related to Dumontinia. We conclude that Dumontinia , and Myriosclerotinia , as currently conceived, are monophyletic, and that Botryotinia along with Botrytis anamorphs probably also constitute a monophyletic lineage. The genus Sclerotinia is probably polyphyletic and characterized by symplesiomorphies rather than synapomorphies. Two putatively new taxa, Sclerotinia sp. 1, and Sclerotinia sp.2 are most closely related to S. minor, S. sclerotiorum and S. trifoliorum , and to S. borealis , respectively.     

Sclerotinia trillii, sp. nov., causing a new sclerotinia disease onTrillium in Japan

Sclerotinia trillii n. sp., which attacksTrillium tschonoskii andT. smallii in Hokkaido and northern Honshu, Japan, is described. The characters identifying this species with the genusSclerotinia are large tuberoid sclerotia, produced both on infected plants and in culture, which consisted of only mycelium (true sclerotia) and flesh apothecia produced on them. This species is distinguished fromS. sclerotiorum, S. minor, S. trifoliorum, andS. nivalis by relatively large sclerotia, cultural colony appearance, and red-brown to yellow-brown, relatively large apothecium, in addition to its parasitic nature onTrillium. Sclerotinia trillii is a psychrophilic having an optimum temperature for mycelial growth at 15–20°C.     

The biology ofSclerotinia sclerotiorum,S. trifoliorum,andS. minor with emphasis on specific nomenclature

The separation ofSclerotinia sclerotiorum (Lib.) de Bary, 5.trifoliorum Erikss., andS. minor Jagger into three distinct species has been based on traditional morphological and physiological criteria such as gross cultural characteristics, sclerotial size, ascus and ascospore dimensions, time of apothecial development in the field, and host association. However, these characteristics tend to be variable and some workers have concluded that the three fungi should be included in one species, 5.sclerotiorum. Recently, new data have been published on morphological, cultural, physiological, ontogenetic, enzyme pattern, mycelial interaction, and cytological characteristics of isolates ofSclerotinia spp. This information supplements, but does not replace, that available from more traditional taxonomic methods and helps to resolve the controversy on the taxonomy and nomenclature of these fungi. This article reviews the relevant literature on the biology of 5.sclerotiorum, S. trifoliorum, and 5.minor, with particular emphasis given to those differences between them that could be of significance regarding their specific nomenclature. After an introduction, mycelia, microconidia, sclerotia, apothecia, infection, control, and taxonomy and nomenclature are discussed. The authors conclude thatS. sclerotiorum, S. trifoliorum, andS. minor are distinct species. The characteristics used to distinguish between them are summarized in table form.     

THE CONFIRMATION OF THE VARIETY FABAE KEAY OF SCLEROTINIA TRIFOLIORUM ERIKSSON

There is confusion as to whether the Sclerotinia on Vicia faba is a normal strain of Sclerotinia trifoliorum as found on clover and related legumes or a distinct variety (var. fabae ). Pathologically the two fungi appear to be the same, and field observations indicate that beans in the rotation may aggravate the incidence of clover rot. A re-investigation of the morphology of the two fungi is made to assess the status of var. fabae.
The two fungi could not be distinguished by apothecial characters or cultural behaviour. Ascospore measurements confirm the existence of S. trifoliorum var. fabae , although 5. trifoliorum proper also occurs on beans. Var. fabae has not been found on any legume besides beans. Isolates from trefoil and sainfoin agree in spore size with S. trifoliorum on clover.
When isolates of S. trifoliorum are co-inoculated on the same Petri plate, they either do not react, or react to form a 'demarcation line'. Although a demarcation line is assumed to imply a genetic difference, non-reaction does not necessarily imply genetic identity. Non-reaction and the formation of a demarcation line also occur when var. fabae strains are co-inoculated together. When an isolate of S. trifoliorum is co-inoculated with an isolate of S. trifoliorum var. fabae an 'incompatibility line' is formed. Use can be made of this fact in identification.
On the basis of co-inoculations 5. trifoliorum and S. trifoliorum var. fabae are both shown to constitute a diverse range of biotypes. Segregation of biotypes in the ascus as a result of heterokaryosis in the ascus-initial is demonstrated. The view is expressed that heterokaryosis is the mechanism underlying the production of new biotypes.     

PHAEODISCUS,A NEW GENUS OF SCLEROTINIACEAE (INOPERCULATAE: DISCOMYCETES)

Phaeodiscus, a new genus of Sclerotiniaceae, is described with P. rhamnicola Batra as the single species. It is characterized by an ectal excipulum of subhyaline textura prismatica and textura porrecta; dichotomously branched, golden brown, aculeate paraphyses; and golden brown, narrowly ellipsoid, usually biguttulate ascospores. The two guttules fuse at maturity to form a single central guttule. The apothecia arise from pseudosclerotia consisting of numerous interconnected sclerotules. A number of other Sclerotiniaceae related to the new fungus are compared with it.     

Sclerotinia nivalis, sp. nov., the pathogen of snow mold of herbaceous dicots in Northern Japan

A newSclerotinia, previously reported asS. intermedia from Japan, is described asSclerotinia nivalis on the morphological basis of the sclerotial anamorph and teleomorph produced in culture. The characters assigning this species to the genusSclerotinia are the tuberoid sclerotia superficially produced on suscepts, the small sclerotia produced on aerial mycelium in culture, the interhyphal spaces in medullary tissue of sclerotia, and the globose cells constructing the ectal excipulum of apothecia. It is distinguishable fromS. sclerotiorum, S. minor, andS. trifoliorum by the intermediate sized sclerotia in culture, binucleate ascospores, the molecular mass of major proteins of sclerotia, and the patterns of esterase isozymes in sclerotial extracts. AlthoughS. nivalis causes snow mold of various dicots, it is a mesophile having an optimum temperature for mycelial growth of around 20°C. It attacks edible burdock(Arctium lappa), Chryhsanthemum morifolium, Ambrosia elatior, carrot(Daucus carota), Angelica acutiloba, Ajuga reptans, andPlantago lanceolata.     

The effect of benomyl sprays on Sclerotinia trifoliorum and yield of red clover

Benomyl sprays, applied monthly from September to January, increased the survival and dry-matter yield of red clover and decreased the numbers of apothecia of Sclerotinia trifoliorum. Two sprays increased dry-matter yield in the first harvest year, if applied between October and December, but decreased yield in the second harvest year.     

核盘菌弱毒株Ep-1PN培养性状的有性遗传特性

观察和测定了核盘菌(Sclerotinia sclerotiorum (Lib.) de Bary)弱毒株Ep-1PN单孢分离物的生长、菌落扩展和菌落形态等培养性状.结果发现来自7个子囊盘的1574个单孢分离物中有1560个分离物的培养性状与正常菌株的没有显著差异,与亲本相似的分离物只有6个,其它8个分离物介于弱毒株Ep-1PN与正常菌株之间.弱毒株Ep-IPN培养性状的有性遗传不遵循孟德尔核遗传规律.     

核盘菌弱毒株Ep-1PN培养性状的有性遗传特性*

观察和测定了核盘菌（Sclerotiniasclerotiorum（Lib．）deBary）弱毒株Ep-1PN单孢分离物的生长、菌落扩展和菌落形态等培养性状。结果发现：来自7个子囊盘的1574个单孢分离物中有1560个分离物的培养性状与正常菌株的没有显著差异,与亲本相似的分离物只有6个,其它8个分离物介于弱毒株Ep-1PN与正常菌株之间。弱毒株Ep-1PN培养性状的有性遗传不遵循孟德尔核遗传规律。     

Auriculora byssomorpha,a Tropical Lichen with a Remarkable Developmental Morphology*

The thallus, pycnidia, and the structure and ontogeny of the ascocarp in Auriculora byssomorpha are described and illustrated. The ontogeny of the apothecia is characterized by the repeated formation of new hymenia within the subhymenial layer. The development of the first hymenium is gymnocarpous but the de novo differentiation of new hymenia, each beneath the next older disintegrating one, gives rise to a peculiar type of secondary hemiangiocarpy. Portions of the decaying hymenia and adjacent subhymenial tissue frequently remain fastened to the apothecial margin and disc in the shape of ear-like appendages or as superimposed layers. In aged apothecia such decaying tissue may form a sheath around the apothecial margin and on the disc. The systematic position of the genus is briefly discussed.     

Incubation of excised apothecia enhances ascus maturation of Sclerotinia sclerotiorum

Synchronized maturation of ascospores of Sclerotinia sclerotiorum is desirable for establishing a transformation system, conducting genetic analyses of the pathogen, defining the precise epidemiological roles of ascospores and screening plant germplasm for resistance. In general, fresh apothecia collected from germinated sclerotia contained primarily immature or discharged asci. This study was undertaken to investigate whether maturation of asci and ascospores could be enhanced by incubation of excised apothecia and to determine the effects of factors such as temperature, excision time, light and ventilation on maturation of asci and ascospores in excised apothecia. Maturation of asci was compared between intact and excised apothecia that were incubated under similar conditions. Results demonstrated that temperature was an important factor affecting ascus maturation of S. sclerotiorum during incubation of excised apothecia, and the optimum temperature was around 21 C. After incubation at 21 C for 30 h, the percentage of undischarged mature asci in excised apothecia increased up to 70-80%. This increase was accompanied by a significant increase in ascospore production of up to 5 x 10(5) ascospores per apothecium. Detailed time course studies indicated that mature asci peaked at 30-36 h of postexcision incubation. Mature asci and the number of ascospores were higher in open incubation than in closed incubation, suggesting that accumulation of volatile substances was not required for ascus/ascospore maturation during postexcision incubation and ventilation could enhance the maturation process. Light also did not affect the maturation of asci during the incubation of excised apothecia. Germination rates for ascospores from excised apothecia under various treatments were similar to those from untreated apothecia but declined slightly with time postexcision. The incubation of excised apothecia promoted ascus maturation compared with intact apothecia.     

Evaluation of polyclonal-antibody-based immunoassays for detection of Sclerotinia sclerotiorum on canola petals, and prediction of stem rot

The potential for using polyclonal-antibody-based immunoassays for detection of Sclerotinia sclerotiorum on canola petals as part of a disease prediction model was investigated. A commercial ELISA kit designed for Sclerotinia homoeocarpa was evaluated for specificity to S. sclerotiorum in comparison to other Sclerotinia spp., and known phyllosphere fungi. This polyclonal-antibody-based kit cross-reacted with antigens from other Sclerotinia spp., and fungi, and absorbance values obtained from S. sclerotiorum-infested canola petals were poorly correlated with percentages of infested petals as determined by plating on semi-selective medium, except when petals were incubated at high humidity for 24 h at 20 degrees C-22 degrees C prior to ELISA evaluation. Additional polyclonal antibodies were prepared from mycelial and semi-purified cell wall antigens, and these antibodies were more specific to S. sclerotiorum than the ELISA kit. However, absorbance values obtained from S. sclerotiorum-infested canola petals were poorly correlated with percentages of infested petals as determined by plating on semi-selective medium. The results are discussed in relation to the use of polyclonal-antibody-based immunoassays for the prediction of epidemics or crop risk from sclerotinia stem rot of canola.     

Four new species of the genus Hymenoscyphus (fungi) based on morphology and molecular data

Four new species of Hymenoscyphus (H. brevicellulus, H. hyaloexcipulus, H. microcaudatus, and H. subsymmetricus) and a new Chinese record (H. subpallescens) are described. These five species share common characteristics: small apothecia (<3mm in diameter); hymenium whitish, pale yellow, to yellow in color; ectal excipulum of textura prismatica; asci arising from simple septa; ascospores scutuloid and guttulate; saprophytic nutrition; and leaf habitats, except for H. subsymmetricus, which grows on herbaceous stems. Phylogenetic analyses of internal transcribed spacer nuclear ribosomal DNA sequences, the universal DNA barcode for fungi, for 16 species in the genus indicated that these taxa were closely related to H. microserotinus, in accordance with their morphological features, but represented independent species. The distinguishing features of each new species from its relatives are discussed, and their phylogenetic relationships explored.     

Ophiostoma mitovirus 3a , ascorbic acid, glutathione, and photoperiod affect the development of stromata and apothecia by Sclerotinia homoeocarpa

Hypovirulence in Sclerotinia homoeocarpa is associated with infection by Ophiostoma mitovirus 3a (OMV3a). OMV3a is also present in asymptomatic isolates, with growth and virulence comparable to that of virus-free isolates. Hypovirulent isolates have impaired mitochondrial function resulting in increased activity of the alternative oxidase pathway, which is implicated in the reduction of reactive oxygen species in other fungi. In this study, hypovirulent, asymptomatic, and virus-free isolates were grown on potato dextrose agar amended with ascorbic acid or glutathione and were incubated under various photoperiods to determine the importance of reactive oxygen species, light, and OMV3a infection for differentiation of stromata and apothecia by S. homoeocarpa. Hypovirulent isolates did not form stromata or apothecia. Glutathione and darkness reduced stromata size and apothecia production by virulent and asymptomatic isolates. Apothecia formed under several different photoperiods, and ascorbic acid increased apothecia production. Ascospores were not detected in these apothecia. The results suggest that hypovirulence, light, and the superoxide radical are important factors in the formation of stromata and apothecia by S. homoeocarpa isolates. This is the first report of sterile apothecia production by North American isolates of S. homoeocarpa and provides a starting point for attempts to produce fertile apothecia.