Scanning Electron Microscopy of Apothecia of Sclerotinia trifoliorum Erikss. and Related Species

The technique of scanning electron microscopy (SEM) was used to study the microanatomy of apothecia of Sclerotinia trifoliorum Erikss., S. sclerotiorum (Lib.) de Bary, and S. minor Jag., In the sterile tissue zones of apothecia, the only interspecific difference detected was that one in the structure of the apothecial margin of small- and large-sclerotium Sclerotinia spp., The ectal excipulum, at the apothecial margin in S. minor appeared to be comprised of textura globulosa with globose cells. Whereas, in S. trifoliorum and S. sclerotiorum it appeared to be comprised of textura porrecta with prosenchymatous cells.
The other interspecific difference reported in this paper is a dimorphism in spore size (ratio 4:4) in asci of S. trifoliorum. Otherwise, the microanatomy of ascocarps of S. trifoliorum and S. sclerotiorum was identical to that one of S. minor. Differences in the density of hyphae in the medullary excipulum in the apothecial disks of the three species studied were not detected. The presence of a membranous film over the hymenium of the ascocarp of S. trifoliorum or, any other species, as well as the bundlelike structure of the apothecial stipes in the studied species, as described in earlier reports, was also not found.     

The structure and histochemistry of sclerotia ofSclerotinia minor Jagger

Summary A detailed histochemical investigation was carried out on rind, cortical and medullary hyphae of sclerotia ofSclerotinia minor Jagger. Four developmental stages, including mature sclerotia, were studied. The walls and septa of all hyphae contained chitin and -1,3 glucans, while those of the rind contained in addition, a melanin-like pigment. An extracellular matrix, which accumulated around cortical and medullary hyphae, consisted primarily of -1,3 glucans, although another polysaccharide, which could not be identified by histochemical methods, was also present. Phenolic material was deposited around the extracellular matrix and in the few interhyphal spaces that remained at maturity. Glycogen was present throughout the cytoplasm of hyphae of the cortex and medulla, at all stages of their differentiation. Polyphosphate granules were laid down within small vacuoles and as sclerotia matured, became most common in the cortical region. Protein bodies developed rapidly in cortical and medullary hyphae until at maturity, they were the most obvious interhyphal feature. These bodies were either round or elongated in shape, the elongated ones often lying parallel to the long axis of the hyphae, and in close association with strands of endoplasmic reticulum. No lipid reserves were detected.Mrs. S.Lowry.     

Morphological characteristics of sclerotia formed from hyphae of <Emphasis Type="Italic">Grifola umbellata</Emphasis> under artificial conditions

The morphological characteristics of sclerotia were induced in cultures of the fungus Grifola umbellata by introducing an unidentified companion fungus were studied by light microscope, scanning and transmission electron microscope (SEM and TEM). Light microscope and SEM investigations of developing sclerotia revealed that aerial mycelial hyphae diminished with age, and mature sclerotia had two tissue layers, the rind and medulla. The medulla was comprised of thin and thick-walled hyphae of varying diameter. The thick-walled cells always formed below the hyphal tips. Retraction of the cytoplasm was accompanied by the thickening of cell wall. There were crystalline initials in the newly formed sclerotium. Crystalline initials were always formed in the tip of medullary hyphae and were not of regular shape. A series of changes occurred in the cells in which the crystalline initials would be formed, such as enlargement of size, formation of one or several large vacuoles. Crystalline initials developed via amorphous materials in the cytoplasm deposited in the vacuoles. At last crystalline initials was released by degradation of the cell wall.     

Antifungal effects of essential oils from oregano and fennel on Sclerotinia sclerotiorum

AIMS: The antifungal effects of essential oils of oregano (Origanum syriacum var. bevanii) and fennel (Foeniculum vulgare) were evaluated against Sclerotinia sclerotiorum. Effects of the essential oils on morphological structures of hyphae and sclerotia were studied under light and scanning electron microscopes (SEM). METHODS AND RESULTS: Inhibitory effects of volatile and contact phases of the essential oils used were determined on hyphae and sclerotia. Both essential oils have a marked antifungal effect against S. sclerotiorum. Soil amendment with essential oils has significant effect on reducing sclerotial viability. Both essential oils significantly inhibited the fungal growth in soil, thereby increasing the number of surviving tomato seedling by 69.8% and 53.3%, respectively. Light and SEM observations on pathogen hyphae and sclerotia revealed considerable morphological alterations in hyphae and sclerotia. CONCLUSIONS: The significant reduction in the mycelial growth and germination of sclerotia would greatly reduce the pathogen inoculum source. This may influence the rate of disease development in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the reduction in the number of diseased plants in infested soil amended with essential oils, we concluded that oregano and fennel essential oils could be used as possible bio fungicides alternative to synthetic fungicides against phytopathogenic fungi.     

Calcineurin is required for sclerotial development and pathogenicity of Sclerotinia sclerotiorum in an oxalic acid-independent manner

Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Calcineurin, a Ser/Thr phosphatase linked to several signal-transduction pathways, plays a key role in the regulation of cation homeostasis, morphogenesis, cell-wall integrity, and pathogenesis in fungi. We demonstrate that calcineurin expression in S. sclerotiorum is altered in a phase-specific manner during sclerotial development. Inhibition of calcineurin by FK506, cysclosporin A, or inducible antisense calcineurin expression impaired sclerotial development at the prematuration phase and increased germination of preformed sclerotia. Induction of antisense calcineurin expression in S. sclerotiorum resulted in reduced pathogenesis on tomato and Arabidopsis. However, secretion of oxalic acid, a key virulence factor of S. sclerotiorum, was not altered. Inhibition of calcineurin conferred a reduction in cell wall beta-1,3-glucan content and increased sensitivity to cell-wall-degrading enzymes and to the glucan synthase inhibitor caspofungin. Thus, calcineurin plays a major role in both sclerotial development and pathogenesis of S. sclerotiorum and, most likely, other phytopathogens.     

Changes during development in the permeability of sclerotia ofSclerotinia minor to an apoplastic tracer

Summary Sclerotia ofSclerotinia minor produced in culture are permeable to the apoplastic tracer sulphorhodamine G (SR) in early stages of their development, but become impermeable as the rind differentiates at the onset of maturation. Reduction in permeability corresponds with deposition of the dark brown pigment in the rind cell walls rather than initiation of the rind as a distinct surface layer. Fluorochrome permeation into cut sclerotia indicates that, while the rind is the primary barrier, the walls and extracellular matrix of the cortex and medulla of mature sclerotia also impede SR movement. Some cells take up fluorochrome into the protoplast. This indicates enhanced proton pumping activity at the cell surface, which suppresses ionisation of the fluorochrome, allowing it to cross the plasma membrane and accumulate in the hyphae. In intact sclerotia such hyphae are very rare and were detected only at one stage of development. However, in cut sclerotia at the two earliest stages of development most of the hyphae near the cut surface accumulated SR and it is possible that this is due to proton pumping activity induced by wounding.Abbreviations ECM extracellular matrix - SR sulphorhodamine G     

THE MORPHOGENESIS AND POSSIBLE EVOLUTIONARY ORIGINS OF FUNGAL SCLEROTIA

1. Fungal sclerotia are able to survive adverse conditions for long periods and they are formed by many important plant pathogens. An understanding of the factors involved in their initiation and development may lead to a method of repressing their formation in nature, thereby reducing the chances of survival of fungi that depend on them as persistent resting stages in their life-cycles. Also, data on sclerotial morphogenesis may be applicable to other multihyphal fungal structures. 2. There are three types of sclerotial development. The most primitive and least common is the loose type, which is illustrated by Rhizoctonia solani. The sclerotium forms by irregular branching of the mycelium followed by intercalary septation and hyphal swelling. When mature, it consists of loosely interwoven hyphae that are rich in food reserves and darkly pigmented. The main types of development are terminal and lateral. The former develops from the coalescence of initials that are produced by a well-defined pattern of branching at the tip of a hypha or tips of closely associated hyphae, e.g. Botrytis cinerea. Lateral sclerotia are formed by the interweaving of side branches of one or several main hyphae. When only one main hypha is involved the sclerotium is of the lateral, simple type, e.g. Sclerotinia gladioli. If several main hyphae give rise to a sclerotium, the term strand type has been used. Sclerotium rolfsii is the classical example. 3. There is a considerable literature on the effects of environmental conditions on the initiation, development and maturation of sclerotia but few attempts have been made to interpret the data. Phenolics and/or polyphenol oxidases have been found to be connected with morphogenesis of the protoperithecium of Neurospora crassa, the perithecium of Podospora anserina and of Hypomyces sp. and the basidiocarp of Schixophyllum commune. A close correlation has been shown between melanin synthesis and microsclerotial development by Verticillium but there appears to be no literature on the role of phenolics and polyphenol oxidases in the morphogenesis of sclerotia. Possibly these substances may inhibit growth of the apices of main hyphae by changing the permeability of the membrane, by inducing a thickening of the cell wall at the tip or by reducing the plasticity of the wall. Such a check in growth could trigger-off the formation of initials close to the margin of the colony or elsewhere in the culture. Sulphydryl groups and disulphide bonds are of great significance in morphogenesis of organisms and are probably involved in sclerotial initiation. The formation of a large number of hyphal branches is a prerequisite for sclerotial initiation and mycelial branching is possible only if there is plasticity of hyphal walls. The ability of the wall to be moulded is possibly related to changes in the sulphur linkages of the protein of the protein-carbohydrate complexes of the cell wall and could be influenced by sulphur availability or the activity of specific enzymes. 4. After a sclerotial primordium has been initiated, further increase in size will depend on the continued, active translocation of nutrients to the site of development. Movement of nutrients to sclerotia is through a few translocatory hyphae. Presumably, nutrients will continue to move into the young sclerotium as long as a concentration or pressure gradient is maintained. Energy and substances for the formation of new branches are supplied in this way and as the requirements for hyphal branches are reduced, excess nutrients become available for conversion to inactive or insoluble reserves and for exudation. The exudates are often complex, consisting of proteins, including enzymes, lipids and carbohydrates. Many sclerotia have a mucilaginous matrix in which the medullary hyphae are embedded. Sclerotium-forming, fungal species that are not regarded as having such a matrix appear to secrete a layer of mucilage over the surface of sclerotial hyphae. This mucilage could have a morphogenetic function and serve as an adhesive which loosely binds hyphae together. More permanent unions are by hyphal fusions or anastomoses. 5. The sclerotium matures within a few days of attaining its maximum size. The rind effectively seals off the medullary hyphae from the surroundings and the translocatory hyphae cease to function. Thus the sclerotium is isolated both physiologically and nutritionally. The endogenous reserves enable the structure to exist in the absence of exogenous nutrients and then, when conditions become suitable, to germinate. 6. The sclerotium appears to provide an example of convergent evolution whereby analogous structures, which have become adapted to resist adverse conditions, have evolved. Data are available mainly for Typhula spp. and ScZerotinia spp. Sclerotia may be degenerate sexual reproductive structures, hyphal aggregates that have developed from closely interwoven conidiophores and undifferentiated conidia or they may be modified vegetative structures.     

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.     

Effect of Sclerotial Damage of Sclerotinia sclerotiorum on the Mycoparasitic Activity of Trichoderma hamatum

Damaged sclerotia of Sclerotinia sclerotiorum buried in soil infested with Trichoderma hamatum isolate TMCS - 3 were degraded rapidly when the medulla of sclerotia was com pletely exposed by the feeding activity of larvae of the fungus gnat Bradysia coprophila. These heavily damaged sclerotia also enhanced , in vitro, the growth of TMCS - 3 . Growth of TMCS - 3 in liquid culture was studied using different carbon sources as substrates , including sclerotia of S. sclerotiorum. Significantly more biomass of TMCS - 3 was recovered using sclerotia as a substrate compared to other carbon sources tested . Exudates from sclerotia whose melanized rinds had been completely removed by feeding larvae accelerated the germination of conidia of TMCS - 3 . Concentrations of amino acids , carbohydrates and proteins in the sclerotial exudates were not increased as damage to sclerotia was increased . Exudation of electrolytes was higher in undamaged than damaged sclerotia . Glucanase activity of TMCS - 3 was slightly increased when the fungus was exposed to damaged sclerotia . However , chitinase activity was not increased by damaging the sclerotia . Larval damage altered the sclerotia not only physically but also chemically , thereby enhancing the activity of the fungus T. hamatum.     

A fungal cell wall integrity-associated MAP kinase cascade in Coniothyrium minitans is required for conidiation and mycoparasitism

Coniothyrium minitans is an important biocontrol agent against Sclerotinia diseases. Previously, a conidiation-deficient mutant ZS-1T1000 was screened out from a T-DNA insertional library of C. minitans. CmBCK1, encoding MAP kinase kinase kinase and homologous to BCK1 of Saccharomyces cerevisiae, was disrupted by T-DNA insertion in this mutant. Targeted disruption of CmBCK1 led to the mutants undergoing autolysis and displaying hypersensitivity to the cell wall-degrading enzymes. The △CmBCK1 mutants lost the ability to produce pycnidia and conidia compared to the wild-type strain ZS-1. △CmBCK1 mutants could grow on the surface of sclerotia of Sclerotinia sclerotiorum but not form conidia, which resulted in much lower ability to reduce the viability of sclerotia of S. sclerotiorum. Furthermore, CmSlt2, a homolog of Slt2 encoding cell wall integrity-related MAP kinase and up-regulated by BCK1 in S. cerevisiae, was identified and targeted disrupted. The △CmSlt2 mutants had a similar phenotype to the △CmBCK1 mutants. The △CmSlt2 mutants also had autolytic aerial hyphae, hypersensitivity to cell wall-degrading enzymes, lack of conidiation and reduction of sclerotial mycoparasitism. Taken together, our results suggest that CmBCK1 and CmSlt2 are involved in conidiation and the hyperparasitic activities of C. minitans.     

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.     

Mycoparasitism by Coniothyrium minitans on Sclerotinia sclerotiorum and its Effect on Sclerotial Germination

Scanning electron microscopy showed that hyphae of Coniothyrium minitans produced appressorium-like swellings when they came in contact with Sclerotinia sclerotiorum in dual culture on PDA. The parasitized hyphae gradually skrank and collapsed, and hyphae of the mycoparasite were found inside the host hyphae. The mycoparasite hyphae grew inter- and intracellularly within the sclerotia of S. sclerotiorum. In the later stages of parasitism, hyphae of the mycoparasite proliferated extensively within the sclerotia and formed pycnidia near the sclerotial surface. At this stage, the sclerotia became flattened, soft and disintegrated. Sclerotia parasitized by C. minitans failed to germinate either myceliogenically or carpogenically.     

Factors Affecting Biological Control of Sclerotinia sclerotiorum by Fungal Antagonists

Studies were conducted to determine the effects of soil moisture (9, 16 or 24% w/w) and temperature (5, 15, 20 or 25°C) on the control of sclerotia of Sclerotinia sclerotiorum by five fungal agents in sterile and natural field soil. All five biocontrol agents were effective in reducing the survival of sclerotia of S. sclerotiorum in sterile soil under dry (9% moisture) or wet (24% moisture) conditions at 20°C, but only Coniothyrium minitans was effective in natural soil. Coniothyrium minitans was the most effective in reducing sclerotial viability at the temperature range of 15–25°C. Trichoderma virens was effective against sclerotia of S. sclerotiorum to a lesser extent than C. minitans , and in non-autoclaved soil, it performed best at 25°C. Although Epicoccum purpurascens , Talaromyces flavus and Trichothecium roseum were effective against sclerotia of S. sclerotiorum in some instances, they were less effective than C. minitans and T. virens . Sclerotia of S. sclerotiorum conditioned for myceliogenic germination were more vulnerable to attack by the biocontrol agents than dormant sclerotia. The implications are discussed with respect to enhancement of biological control of crop diseases caused by S. sclerotiorum in different geographic regions.     

Biological control of Sclerotinia disease by Aspergillus sp. on oilseed rape in the field

Sclerotinia sclerotiorum causes serious yield losses to many crops worldwide. Aspergillus sp. Asp-4, previously shown to inhibit germination of sclerotia of S. sclerotiorum in vitro and in the field, was evaluated in field trials for suppression of this pathogen on oilseed rape. Spray application of Asp-4 to the soil prior to sowing rice in a rice–oilseed rape rotation resulted in a significant reduction in incidence of Sclerotinia stem rot on oilseed rape compared with the non-treated control in two field trials. This application of Asp-4 also resulted in a significant reduction in germination of sclerotia relative to the non-treated control in these field trials, suggesting that this reduction in sclerotial germination led to disease control. Microscopic examination demonstrated that Asp-4 could effectively colonise external and internal portions of sclerotia of S. sclerotiorum in vitro. Incubation of Asp-4 with sterile sclerotial material induced production of β-glucanase and chitinase activities by this isolate; β-glucanase and chitinase being potentially capable of degrading the glucan and chitin polymeric components of sclerotia. Incubation of Asp-4 with sterile sclerotial material also resulted in a significant reduction in dry weight of this sclerotial material relative to the non-treated control in 96?h in vitro experiments. Experiments reported here indicate that Aspergillus sp. Asp-4 has promise as a biological control agent for S. sclerotiorum on oilseed rape. Experiments reported here suggest that disease control results from inhibition of germination of sclerotial resting structures due to mycoparasitic colonisation by Asp-4.     

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.     

Identification and characterization of Sclerotinia sclerotiorum NADPH oxidases

Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Sclerotinia sclerotiorum, an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. sclerotiorum NADPH oxidases (SsNox1 and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNox1 and SsNox2. Silencing of SsNox1 expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. ΔSsnox1 strains had reduced ROS levels, were unable to develop sclerotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. sclerotiorum.