Mature Sclerotia Research Articles

A possible role for Rhizoglyphus robustus Nesbitt (Astigmata: Acaridae) in transmission of Sclerotium cepivorum Berk. (Deuteromycetes: Mycelia-Sterilia).

We evaluated the viability of Sclerotium cepivorum Berk. (causal agent of of garlic and onion white rot) mycelial and sclerotial fragments transported through excreta of Rhizoglyphus robustus Nesbitt fed on S. cepivorum. Excreta were obtained from mites fed on 1) mature, hydrated and fragmented sclerotia (mhfS); 2) mature, dehydrated and fragmented sclerotia (mdhfS); 3) whole mature sclerotia (wmS); 4) mycelium (M) and 5) potato dextrose agar culture media (PDA). After feeding, mite excreta were examined under compound and scanning electron microscopes for the presence of mycelial and sclerotial fragments. S. cepivorum mycelium growth and sclerotia formation were observed in three out of 60 excreta pellets from mites fed on M and mhfS and only in one excreta pellet out of 60 from mites fed on mature, dehydrated and fragmented sclerotia (mdhfS). Numerous fragments of mycelia and sclerotia on the mites' bodies, mainly on the sense organs of the ambulacral appendages and the mouthparts were identified by compound and scanning electron microscope observations. R. robustus may also influence white rot epidemiology as a consumer of S. cepivorum and spread and/or transmit the pathogen by carrying fungal fragments on its body externally.

Differentiation and Hydrogen Peroxide Production in Sclerotium rolfsii Are Induced by the Oxidizing Growth Factors, Light and Iron

Hydrogen peroxide is produced by Sclero- tium rolfsii during sclerotial differentiation, in re- sponse to oxidative growth conditions of light and Fe. An additive effect of (? light ? Fe)-induced oxida- tive stress is observed on H202 production levels and on sclerotia numbers during fungal development. High H202 production rates are correlated with high oxidative stress and are observed in less lipid-perox- idized young mycelia and sclerotia (sclerotia initials and early developing sclerotia) and not in differen- tiated, highly lipid-peroxidized older mycelia and sclerotia (developed and mature sclerotia). H202 production by undifferentiated mycelia and their ex- posure to exogenous H202 show a rate/dose-depen- dent correlation with degree of sclerotial differenti- ation (sclerotia numbers). Hydrogen peroxide and lipid peroxide concentration gradients are observed in fungal colonies during their transition from un- differentiated (mycelial) to differentiated (sclerotial) stage. Growth-stage-dependent changes in H202 pro- duction rates during development are not due to changes in glucose concentration and pH of the growth medium. Light/Fe-induced H202 production and its effect on S. rolfsii differentiation, are dis- cussed in the context of our hypothesis of oxidative stress-induced sclerotial differentiation.

Differentiation and hydrogen peroxide production in Sclerotium rolfsii are induced by the oxidizing growth factors, light and iron

Hydrogen peroxide is produced by Sclerotium rolfsii during sclerotial differentiation, in response to oxidative growth conditions of light and Fe. An additive effect of (± light ± Fe)-induced oxidative stress is observed on H2O2 production levels and on sclerotia numbers during fungal development. High H2O2 production rates are correlated with high oxidative stress and are observed in less lipid-peroxidized young mycelia and sclerotia (sclerotia initials and early developing sclerotia) and not in differentiated, highly lipid-peroxidized older mycelia and sclerotia (developed and mature sclerotia). H2O2 production by undifferentiated mycelia and their exposure to exogenous H2O2 show a rate/dose-dependent correlation with degree of sclerotial differentiation (sclerotia numbers). Hydrogen peroxide and lipid peroxide concentration gradients are observed in fungal colonies during their transition from undifferentiated (mycelial) to differentiated (sclerotial) stage. Growth-stage-dependent changes in H2O2 production rates during development are not due to changes in glucose concentration and pH of the growth medium. Light/Fe-induced H2O2 production and its effect on S. rolfsii differentiation, are discussed in the context of our hypothesis of oxidative stress-induced sclerotial differentiation.

401 Effects of Allyl Isothiocyanate on Mycelial Growth from Germinating Sclerotia of Sclerotium rolfsii

Isothiocyanates are volatile chemicals produced by damaged tissues of Brassica species. Allyl isothiocyanate (AITC), the predominant isothiocyanate in Indian mustard (B. juncea), has been shown to control pest in laboratory and field experiments. We investigated the effectiveness of AITC against the germination of sclerotia of Sclerotium rolfsii Saccardo, a common soilborne pathogen of tomato. Sclerotium rolfsii was cultured on PDA from a field isolate. Mature sclerotia were collected and placed in polyester mesh bags. Culture tubes (16 × 150 mm) were packed with 18 g clay loam soil. A sclerotia-bag was placed in each tube and covered with an additional 5 g soil. Soil was maintained at 60% field capacity for the duration of the experiment. AITC was injected into each tube through a septum. Treatments consisted of 0, 5.6, 11.2, 22.4, and 44.8 μmol AITC/L of atmosphere and an ethanol control. AITC in each tube was sampled using SPME and analyzed on GC-MS. Tubes remained sealed for 42 h at 30 °C. Sclerotia were then removed from tubes and bags and plated on PDA to determine viability. Radial growth was measured to determine the effects of AITC. Mycelial growth was negatively correlated to AITC concentration (P < 0.01). The highest concentration of AITC resulted in a 40.3% reduction in mycelial growth. Although the AITC concentrations used in this study did not kill sclerotia of S. rolfsii, they did suppress mycelial growth from germinating sclerotia. At higher concentrations, or mixed with other chemicals, AITC may prove to be an affective control for this pathogen.

Factors influencing the production of sclerotia in the wild rice (Zizania aquatica) pathogen Sclerotium 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.

Characterization of Mycorrhizal Isolates of Rhizoctonia solani from an Orchid, Including AG-12, a New Anastomosis Group

ABSTRACT Isolates of Rhizoctonia solani collected from mycorrhizal orchid (Pterostylis acuminata) plants and adjacent leaf litter were characterized. Of 23 selected isolates, 20 were members of a new anastomosis group (AG-12) and the rest were members of AG-6. There were no bridging anastomosis reactions observed between AG-12 and other AGs of R. solani. Among the 20 isolates of AG-12 evaluated, 18 vegetatively compatible populations were detected, indicating diversity within the AG. Mature cultures were dark brown, as were mature sclerotia. Some cultures produced alternating dark- and light-colored concentric rings, with sclerotia forming in the darker rings. Most cultures were appressed to the agar surface. In tests run to characterize pathogenic potential, selected mycorrhizal isolates of AG-12 and AG-6 did little damage to potato and barley seedlings, moderate damage to head lettuce seedlings, and more extensive damage to seedlings of cauliflower and radish. Isolates of AG-12 have not been observed to fruit in nature, and all attempts to induce formation of the teleomorph (Thanatephorus cucumeris) in the laboratory by selected isolates of AG-12 failed.

Factors influencing the production of penicillin V acylase byChainia, a sclerotialStreptomyces

Aerobic culture of a Streptomyces was found to produce penicillin V acylase (PVA) (PA, EC-3.5.1.11) extracellularly. Strain characteristics recorded on various media and presence of mature sclerotia led to its identification as Chainia, a sclerotial Streptomyces. The Chainia acylase hydrolyzed penicillin V (Pen V) preferentially. Maximum conversion of Pen V to 6-aminopenicillanic acid (6-APA) was observed when the 48 hour old fermented broth was used as an enzyme source. Fermentation parameters and the culture condition of the strain was optimized for high acylase activity.

Sclerotial biogenesis in the basidiomycete Sclerotium rolfsii. A scanning electron microscope study

Sclerotial biogenesis in Sclerotium rolfsii was studied using scanning electron microscopy. Three stages in sclerotial development were documented: sclerotial initials, development and mature stages. Initials developed from primordia, small tufts of mycelial hyphae held together very loosely. Each tuft grew in size and increased in hyphal density. Distinct structural details justifying further subdivision of the sclerotial initials stage were not observed. Differences among initials were determined temporally only with respect to size and degree of hyphal density. Sclerotia of the development stage already had assumed their spherical shape. An early and late developmental stage could be distinguished. Sclerotia of the former were covered with an extensive layer of loose aerial hyphae which obscured the sclerotial surface. Those of the latter stage had shed most of the aerial hyphae and exuded profusely. Mature sclerotia were compact, spherical and had shed completely the outer layer of aerial hyphae. The skin surface was rough and scaly.

Sclerotial Biogenesis in the Basidiomycete Sclerotium rolfsii. A Scanning Electron Microscope Study

Sclerotial biogenesis in Sclerotium rolfsii was studied using scanning electron microscopy. Three stages in sclerotial development were documented: sclerotial initials, development and mature stages. Initials developed from primordia, small tufts of mycelial hyphae held together very loosely. Each tuft grew in size and increased in hyphal density. Distinct structural details justifying further subdivision of the sclerotial initials stage were not observed. Differences among initials were determined temporally only with respect to size and degree of hyphal density. Sclerotia of the development stage already had assumed their spherical shape. An early and late developmental stage could be distinguished. Sclerotia of the former were covered with an extensive layer of loose aerial hyphae which obscured the sclerotial surface. Those of the latter stage had shed most of the aerial hyphae and exuded profusely. Mature sclerotia were compact, spherical and had shed completely the outer layer of aerial hyphae. The skin surface was rough and scaly.

Characterization of Anastomosis Group 11 (AG-11) ofRhizoctonia solani

Isolates of Rhizoctonia solani anastomosis group 11 (AG-11) were collected in Western Australia and Arkansas. Some isolates from each location fruited on 1.5% water agar, 2% V8-juice agar, and soil overlay plates and produced sexual structures typical of Thanatephorus cucumeris. Mycelium of isolates of AG-11 growing on potato-dextrose agar was white to light tan when young, but became brown to dark brown with age. Concentric rings of dark and light mycelium were visible in most cultures. Mature sclerotia were tan to light brown and were scattered over the agar surface. No anastomosis reactions (C0) were observed between paired isolates of AG-11 and anastomosis groups 1, 3-7, 9, and 10. C1 anastomosis reactions were observed between some isolates of AG-11 and some isolates of AG-2, -8, and -BI, indicating a distant or «bridging» anastomosis relationship with these three AG. Isolates of AG-11 were auxotrophic for thiamine. Isolates of AG-11 are known to damage lupine, soybean, and other plants in the field, and in greenhouse and growth chamber studies were shown to damage other crops, including cotton, radish, wheat, and potato

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

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.

Lectins in different members of the Sclerotiniaceae

Lectins were isolated from Sclerotinia minor, Sclerotinia trifoliorum, Sclerotinia miyabeana, Botrytis cinerea and three isolates of Sclerotinia sclerotiorum by affinity chromatography on immobilized d -galactose and characterized in some detail. The native lectins from all species are dimeric proteins composed of subunits of about 17 kDa which exhibit specificity towards methyl-α- d -galactopyranoside/ N -acetyl-α- d -galactosamine. Lectins from the different species can be distinguished by their serological behaviour in double immunodiffusion and ELISA assays. Whereas the lectins from the three S. sclerotiorum isolates are identical, those from S. trifoliorum, S. miyabeana and S. minor are closely related to but not identical to those from S. sclerotiorum . The lectin of B. cinerea is also related to the Sclerotinia lectins but to a lesser extent. In all the isolates the lectin content was very low in the undifferentiated mycelium but increased dramatically upon sclerotium development to reach maximal values in mature sclerotia where the lectin comprised up to 20% (w/w) of the total soluble sclerotial protein.

Studies on Cenococcum geophilum. II. Sclerotium morphology, germination, and formation in pure culture and growth pouches

Cenococcum geophilum sclerotium morphology, germination, and formation in pure culture and growth pouches is described. On agar, germination of most field-collected sclerotia occurred within 30 days. In vitro attempts to initiate sclerotia succeeeded to varying degrees for 20 of the 22 isolates (collected in three main habitats in Oregon) in agar or peat – vermiculite but not in liquid culture. Sclerotium formation was initiated in 1-month-old cultures by branching, lateral growth, and subsequent septation of hyphae. Surface cells of compact hyphae of young sclerotia elongated, became irregularly arranged, and melanized. In growth pouches, sclerotium initials developed on the paper wick from a hyphal mat formed from convoluted hyphae. Mature sclerotia appeared as small spherical structures with linear hyphae radiating out from the surface. Sections revealed a well-differentiated rind and a medulla with pseudoparenchymatous organization. Numerous nuclei and vacuoles were distributed in both rind and medullary hyphae. Key words: Cenococcum, sclerotium, initiation, in vitro.

Polysaccharide cytochemistry in maturing Aspergillus flavus sclerotia

Polysaccharide content and the dynamics of its accumulation was studied during sclerotial growth and differentiation in Aspergillus flavus. Using cytochemical methods and enzymic digestion with α-amylase on ultrathin sections, it was confirmed that glycogen represented the main polysaccharide storage material. During sclerotial development glycogen progressively accumulated in the cytoplasm in the form of α- and β-particles, while in the vacuolar system it appeared only as β-particles. In mature sclerotia most of the glycogen was found in the cytoplasmic rather than the vacuolar form and it is therefore suggested that the cytoplasmic form of glycogen is the main storage polysaccharide accumulated for survival and germination.

Isolation of a non-sclerotial mutant ofSclerotium rolfsii and its cellulolytic activity

A mutant strain EMS-1 ofSclerotium rolfsii lacking the ability to develop mature sclerotia was isolated following chemical mutagenesis of macerated sclerotia with ethyl methane-sulfonate. The mutant failed to form sclerotia even in the presence of lactose, threonine or iodo-acetic acid which promoted sclerotial development in the wild strain and the UV-8 mutant. EMS-1 exhibited higher (1.5 – 3.0 times) cellulase and hemicellulase activity compared to the wild strain. Possible correlation between sclerotial morphogenesis and cellulase and/or oxalic acid production is discussed.

Sclerotia of Aspergillus aculeatus

Sclerotial development in Aspergillus aculeatus is described for the first time. The sclerotium originates as a knot of interwoven hyphae. The mature sclerotium has a rind of flattened hyphae, below which are two cortical layers and a medulla. The outer cortical layer has isodiametric cells and the inner layer has somewhat radially elongated polyhedral cells. The thick walls of the cortical cells are perforated by simple septal pores. The medulla comprises a loosely interwoven mass of hyphae. Sclerotia remain pale luteous.

The Ultrastructure of Resting and Germinating Sclerotia of Botrytis allii

The structure of mature and germinating sclerotia of Bortrytis allii was studied by light and scanning electron microscopy. The mature sclerotium has (1) a narrow rind of rounded, rather thick-walled empty cells, and (2) a large central medulla of filamentous hyphae loosely arranged in a gelatinous matrix. Germinating sclerotia produced an abundance of conidiophores and conidia. The sequence of morphological events during sclerotial germination showed that the conidiophores emerging from the ruptured rind originated in the medualla.

The development, structure, and histochemistry of sclerotia of ectomycorrhizal fungi. I. Pisolithus tinctorius

Sclerotia were produced by growing Pisolithus tinctorius (Pers.) Coker and Couch in association with Pinus strobus L. and Pinus resinosa Ait. in plastic growth pouches. Developing and mature sclerotia were collected, fixed, and embedded for light microscopy and scanning and transmission electron microscopy. They were found to consist of an outer pigmented rind, an inner and outer cortex, and a large central medulla. Cortical and medullary areas were comprised of pseudoparenchyma which contained large deposits of glycogen, as well as protein and lipids. The structure of these sclerotia indicates that they are persistent propagules. Sclerotia may be important in nature for the recolonization of root tips after environmental stresses. They may also be useful for storing valuable strains of P. tinctorius and as a source of inoculum for experimental studies.

Studies on the ontogeny and ultrastructure of the sclerotium of Botrytis cinerea Pers. ex Nocca & Balbis

The ontogeny and ultrastructure of sclerotia of the fungus Botrytis cinerea Pers. ex Nocca & Balbis were studied by light, scanning, and transmission electron microscopy. Exudation droplets of various sizes and colour accumulated on sclerotial surfaces during development and eventually disappeared. A surface hyphal weft was present over sclerotia at maturity, forming a dense covering which often obscured the underlying rind. The rind consisted of highly vacuolated cells with thick, pigmented walls that remained intact even in old sclerotia. The cortex was poorly defined and usually consisted of only one layer of cells. The prosenchymatous medulla constituted the main volume of mature sclerotia. The ultrastructure of young sclerotial hyphae was similar to that of actively growing vegetative hyphae. Hyphae of mature sclerotia contained fewer nuclei and profiles of mitochondria and endoplasmic reticulum than young sclerotial hyphae. Electron-dense structures, tentatively identified as protein and polyphosphate bodies, were observed in hyphae of the cortex and medulla. Sclerotia of B. cinerea were structurally similar to those of Sclerotinia spp.

Changes of the cytoplasmic ultrastructure during development of sclerotia in Claviceps purpurea (Fr.) Tul.

The development of sclerotia of Claviceps purpurea was investigated by light and electron microscopy. During the first days after infection sterigma and conidiospores are formed. The spores show a moderately developed vacuolar system, they are thick walled and contain about 20% lipid (related to the cell volume) embedded in glycogen. The sterigma are cylindrical unicellular hyphae with electron dense cytoplasm and isolated strongly contrasted lipid droplets. In maturing sclerotia the hyphae become septated with increasingly thick cell walls and a large lipid content. The lipid forms small droplets in young cells, while in the mature sclerotium it occurs in the form of very large drops, occupying the major part of the cell. Simultaneously the composition of the lipid is changed. The mature cells have several nuclei. They are partially connected by osmiophilic substances, forming a network of intercellular spaces.