Ultrastructure and Development of Sclerotia of Botrytis cinerea Pers. in vitro

The development of sclerotia of Botrytis cinerea was examined at four stages during their maturation. The surface structure developed a network of profusely branched hyphae through their coalescence to a compact sclerotial body which was maturated by the deposition of melanin pigment. A characteristic feature of the hyphal cells of B. cinerea during the later stages of development was the presence of paramural bodies (plasmalemmasomes and lomasomes). Electrondense bodies with a limiting double-membrane congregated against the transverse septa of hyphal cells as sclerotia matured and may migrate from cell to cell through septal pores. We suggest that these and the lipid bodies found in hyphal cells may have a storage function in the resting sclerotia.     

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.     

The structure and histochemistry of sclerotia of Ophiocordyceps sinensis

The structure and histochemistry of sclerotia of Ophiocordyceps sinensis (synonym: Cordyceps sinensis) are described. The remains of the caterpillar epidermis and sometimes setae of the caterpillar were attached to the pigmented layer that is external to the rind of the sclerotium. The outer aerial hyphae and hyphae of the inner medulla were densely interwoven around the epidermis of the caterpillar; these eventually differentiated into the rind of the sclerotium. The medulla of the sclerotium consisted of three intergrading regions of hyphal density: high, low and a region of intermediate hyphal density. All hyphae of the medulla contained large quantities of protein, polysaccharide and polyphosphate; only the region of high hyphal density was rich in beta-1,3 glucans; the center of the sclerotium was almost devoid of hyphae and contained what are most likely the remains of caterpillar tissue. These features are compared with those of sclerotia of other fungi, and their possible significance is discussed.     

Structure of the outer surfaces of sclerotia of certain fungi

Summary The surfaces of sclerotia of Rhizoctonia solani, Botrytis cinerea and Sclerotinia rolfsii were examined with the Stereoscan electron microscope.The periphery of the sclerotium of R. solani consists of a loose net-work of hyphae which are not sufficiently thickened to withstand the extreme desiccation that takes place when the material is coated with gold-palladium alloy.The surface of the sclerotium of B. cinerea has many closely packed hyphal tips which project outwards from the centre of the structure. The thickening of the walls of the hyphae enable them to retain their shapes. A film was observed on the most exposed areas and this may have been dried-up melanin pigment.The outer skin of the sclerotium of S. rolfsii is an almost continuous layer which is thrown into ridges and troughs to give a wavy appearance.     

Characterization of the Sclerotinia sclerotiorum cell wall proteome

We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)‐anchored cell wall proteins and 30 non‐GPI‐anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes.     

桑椹肥大性菌核病病原菌生物学特性及流行性

【目的】对桑椹灾害性真菌病害——桑椹肥大性菌核病病原菌,即桑实杯盘菌(Ciboria shiraiana)的生物学特性进行研究,分析其流行性。【方法】采用人工接种、调查等研究方法,对C.shiraiana在无性生长阶段中菌丝侵染能力,菌核的休眠期,有性生长阶段中子囊孢子的结构、释放、数目以及萌发等进行研究,并对菌核萌发的物候期进行调查。【结果】C.shiraiana菌丝对桑雌花没有侵染能力;C.shiraiana菌核具有休眠期,低温处理6周以上的菌核才能萌发形成子囊盘;1个菌核可萌发1–15个子囊盘,直径为1.5 cm的子囊盘能产生高达(5.6–6.3)×10~7个子囊孢子;C.shiraiana子囊孢子在酸性环境中的萌发率明显高于在中性和碱性环境中的萌发率;C.shiraiana菌核萌发形成子囊盘产生子囊孢子的物候期,从1月下旬开始到4月中旬结束,其中在3月中旬萌发子囊盘的数目达到最高值。【结论】桑椹肥大性菌核病属于典型的流行性侵染病,在果桑栽培上容易造成毁灭性危害,生产上必须高度重视该病的防控。     

猪苓菌丝形成菌核伴生菌的发现及应用

在野生猪苓(Grifola umbellata (Pers.) Pilát)菌核生长穴中首次分离到猪苓菌丝形成菌核所必需的伴生菌(Grifola sp.).实验证明:纯培养的猪苓菌丝不能形成菌核,但其与伴生菌共培养,无论在实验室培养基上或用树棒栽培,猪苓菌核形成很快且发育正常;伴生菌是猪苓菌丝形成菌核的关键生物因子.另外,伴生菌菌丝和猪苓菌菌丝二者形态差别较大,前者多为细长的薄壁菌丝,后者多为细胞直径较大的薄壁和厚壁菌丝.     

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.     

The sclerotia of Coprinus lagopus

Summary Sclerotia of Coprinus lagopus were produced in the laboratory and observed microscopically from initiation to maturity. Isolated bulbous hyphal cells appeared below or on the agar surface as the primary sclerotial initials. Short interhyphal cells had expanded lateral walls and increased in number at their isolated locations, and were surrounded by a thick network of normal mycelium. Increasing in numbers, the bulbous cells developed a tightly compact colony devoid of agar if submerged below the agar surface and lacked normal hyphal filaments. The outer cells thickened formed cell lumina and developed as a protective rind to the mature sclerotium.     

An atypical forkhead‐containing transcription factor SsFKH1 is involved in sclerotial formation and is essential for pathogenicity in Sclerotinia sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen with a worldwide distribution. The sclerotia of S. sclerotiorum are pigmented multicellular structures formed from the aggregation of vegetative hyphae. These survival structures play a central role in the life and infection cycles of this pathogen. Here, we characterized an atypical forkhead (FKH)‐box‐containing protein, SsFKH1, involved in sclerotial development and virulence. To investigate the role of SsFkh1 in S. sclerotiorum, the partial sequence of SsFkh1 was cloned and RNA interference (RNAi)‐based gene silencing was employed to alter the expression of SsFkh1. RNA‐silenced mutants with significantly reduced SsFkh1 RNA levels exhibited slow hyphal growth and sclerotial developmental defects. In addition, the expression levels of a set of putative melanin biosynthesis‐related laccase genes and a polyketide synthase‐encoding gene were significantly down‐regulated in silenced strains. Disease assays demonstrated that pathogenicity in RNAi‐silenced strains was significantly compromised with the development of a smaller infection lesion on tomato leaves. Collectively, the results suggest that SsFkh1 is involved in hyphal growth, virulence and sclerotial formation in S. sclerotiorum.     

THE EFFECT OF SOME PHYSIOLOGICAL AND ENVIRONMENTAL FACTORS ON SCLEROTIAL ASPERGILLI

Rudolph , Emanuel D. (Ohio State U., Columbus.) The effect of some physiological and environmental factors on sclerotial Aspergilli. Amer. Jour. Bot. 49(1): 71–78. Illus. 1962.—The effect of varying conditions of carbon-nitrogen balance, temperature, pH, and light upon the formation of sclerotia by 6 species of Aspergillus (A. alliaceus, A. avenaceus, A. flavus, A. quercinus, A. sclerotiorum and A. wentii) was studied. On Czapek's agar, optimal growth as well as maximum production of sclerotia and conidia took place at high sucrose and nitrate concentrations. In general, fewer sclerotia were formed with glucose than with sucrose, and very poor growth took place with lactose. Sclerotia were formed best at temperatures that were optimal or below optimal for mycelial growth. The ranges of pH through which sclerotia were formed were narrower than those through which conidia and mycelia were formed. Light had no effect upon sclerotium formation. The formation of sclerotia in A. alliaceus was found to represent the strand-type development. A number of UV-induced strains and a spontaneous mutant strain of A. alliaceus showing varying amounts of sclerotium and conidium production are characterized. It is suggested that the sclerotia in Aspergillus are sterile stromata.     

Factors influencing the production of sclerotia in the wild rice (Zizania aquatica) pathogenSclerotium hydrophilum

Sclerotium hydrophilum was shown to be auxoheterotrophic for thiamine, with the addition of this vitamin being required for the induction of sclerotia on defined media, but riboflavin and pyridoxine also have a positive effect. In the absence of thiamine, an increase in glucose concentration lead to a decrease in the yield of sclerotia; however, the addition of thiamine negate this inhibition and, instead, as the glucose concentration increased a higher proportion of sclerotial initials matured. Overall it was found that thiamine, specifically the pyrimidine component of thiamine, is crucial for initiating sclerotium production, while glucose stimulates maturation. The effect of light on sclerotium production was found to be complex and dependent on the growth medium. Light is not required for either the induction or maturation of sclerotia, but continuous irradiation of developing cultures with either white light or black light induces an endogenous rhythm whereby sclerotia are formed every 48h. When exposed to alternating light/dark regimes mycelium that formed in the light does not mature sclerotia, but dark-formed mycelium does, even if it is subsequently exposed to light.     

THE SURVIVAL OF FUNGAL SCLEROTIA UNDER ADVERSE ENVIRONMENTAL CONDITIONS

1. Sclerotia have been found to survive under adverse environmental conditions for long periods. Their viability is retained at low and subzero temperatures, while it is progressively reduced at temperatures above about 15° C. Dry conditions and lack of competition from other organisms favour survival. Depletion of food reserves results in loss of resistance to environmental factors. 2. The majority of sclerotia consist of an outer layer of thickened, close-fitting, often pigmented cells that form a rind over a tissue of closely interwoven hyphae. A cortex and medulla is often discernible. 3. Features of sclerotia that are important in overcoming the harmful effects of desiccation and extremes of temperature include: the presence of a rind and sometimes an additional covering of either fungal or host tissue; the compact nature of the sclerotium; miscellaneous protectants on the surface of and in the hyphae; melanized hyphal walls; high intracellular osmotic concentrations; slow air-drying to lower moisture content. Also the buffering action of the soil and the protection afforded by vegetation and plant debris are important. 4. The active exudation of water, the accumulation and secretion of soluble carbohydrates during sclerotium development and maturation, and the formation of large amounts of mucilage are of significance in resistance to desiccation, extremes of temperature and radiations. 5. The nature and the pigmentation of the rind together with the site of sclerotium development may give protection against harmful radiations. 6. The loss of sclerotium viability caused by toxic chemicals in the soil and atmosphere or by the enzymic activities of other organisms is reduced by the rind and the melanization of hyphal walls. 7. Survival under starvation conditions is achieved by the accumulation of abundant food reserves (lipids and/or carbohydrates) and a low level of metabolic activity. In the presence of a compatible host exogenous sources of energy are sometimes used. 8. Although the sclerotium is highly adapted to survive adverse conditions for long periods, sometimes micro-environmental conditions may be so severe that only a few sclerotia can retain their viability. However, even a few such comparatively large bodies, rich in food reserves, have a considerable inoculum potential.     

Deficiency of the melanin biosynthesis genes SCD1 and THR1 affects sclerotial development and vegetative growth,but not pathogenicity,in Sclerotinia sclerotiorum

The fungus Sclerotinia sclerotiorum is a necrotrophic plant pathogen causing significant damage on a broad range of crops. This fungus produces sclerotia that serve as the long‐term survival structures in the life cycle and the primary inoculum in the disease cycle. Melanin plays an important role in protecting mycelia and sclerotia from ultraviolet radiation and other adverse environmental conditions. In this study, two genes, SCD1 encoding a scytalone dehydratase and THR1 encoding a trihydroxynaphthalene reductase, were disrupted by target gene replacement, and their roles in mycelial growth, sclerotial development and fungal pathogenicity were investigated. Phylogenetic analyses indicated that the deduced amino acid sequences of SCD1 and THR1 were similar to the orthologues of Botrytis cinerea. Expression of SCD1 was at higher levels in sclerotia relative to mycelia. THR1 was expressed at similar levels in mycelia and sclerotia at early stages, but was up‐regulated in sclerotia at the maturation stage. Disruption of SCD1 or THR1 did not change the pathogenicity of the fungus, but resulted in slower radial growth, less biomass, wider angled hyphal branches, impaired sclerotial development and decreased resistance to ultraviolet light.     

Morphological Transitions Governed by Density Dependence and Lipoxygenase Activity in Aspergillus flavus

Aspergillus flavus differentiates to produce asexual dispersing spores (conidia) or overwintering survival structures called sclerotia. Results described here show that these two processes are oppositely regulated by density-dependent mechanisms and that increasing the cell density (from 10¹ to 10⁷ cells/plate) results in the lowest numbers of sclerotial and the highest numbers of conidial. Extract from spent medium of low-cell-density cultures induced a high-sclerotium-number phenotype, whereas high-cell-density extract increased conidiation. Density-dependent development is also modified by changes in lipid availability. Exogenous linoleic acid increased sclerotial production at intermediate cell densities (10⁴ and 10⁵ cells/plate), whereas oleic and linolenic acids inhibited sclerotium formation. Deletion of Aflox encoding a lipoxygenase (LOX) greatly diminished density-dependent development of both sclerotia and conidia, resulting in an overall increase in the number of sclerotia and a decrease in the number of conidia at high cell densities (>10⁵ cells/plate). Aflox mutants showed decreased linoleic acid LOX activity. Taken together, these results suggest that there is a quorum-sensing mechanism in which a factor(s) produced in dense cultures, perhaps a LOX-derived metabolite, activates conidium formation, while a factor(s) produced in low-density cultures stimulates sclerotium formation.     

Sclerotial development, melanin production and lipid peroxidation bySclerotium rolfsii

Melanin pigments constituted 13.9% (W/W) of sclerotial walls ofSclerotium rolfsii. The lipid and ash contents in sclerotial walls were twice those in hyphal walls of the fungus. Progress in culture age and maturation of sclerotia were always accompanied by increased levels of lipid peroxidation products and melanin. Lipid peroxidation and melanin formation may thus proceed in parallel during sclerotial biogenesis and maturation. Both these processes are strongly affected by Fe²⁺ and by antioxidant vitamins (ascorbic acid), microelements (selenium) and mercapto compounds (glutathione). Myceliogenic germination and lytic activityvia melanin production can thus be affected by (anti)oxidants that could potentially be used for controlling sclerotia-producing fungi without using traditional toxic fungicides.     

Effect of copper-induced oxidative stress on sclerotial differentiation and antioxidant properties of Penicillium thomii PT95 strain

Penicillium thomii PT95 strain was able to form abundant orange, sand-shaped sclerotia in which carotenoids were accumulated. The aim of this work was to determine the effects of copper-induced oxidative stress on the sclerotial differentiation and antioxidant properties of PT95 strain. The results showed that the time of exudates initiation, sclerotial initiation and sclerotial maturation of PT95 strain were advanced in 1–2 days under the copper-induced oxidative stress growth conditions. The analytical results of sclerotial biomass, carotenoids content in sclerotia showed that copper-induced oxidative stress favored the sclerotial differentiation and biosynthesis of carotenoids. Under the copper-induced oxidative stress growth conditions, the total phenolics content and DPPH free radical scavenging activity of sclerotia of this fungus were decreased as compared with the control. However, the oxidative stress induced by a lower amount of CuSO₄ in media could enhance significantly the reducing power of sclerotia.