Sclerotial Cells Research Articles

Comparative transcriptome analysis of cells from different areas reveals ROS responsive mechanism at sclerotial initiation stage in Morchella importuna

Morels are some of the most highly prized edible and medicinal mushrooms, with great economic and scientific value. Outdoor cultivation has been achieved and expanded on a large scale in China in recent years. Sclerotial formation is one of the most important phases during the morel life cycle, and previous reports indicated that reactive oxygen species (ROS) play an important role. However, ROS response mechanisms at sclerotial initiation (SI) stage are poorly understood. In this study, comparative transcriptome analyses were performed with sclerotial and hyphal cells at different areas in the same plate at SI stage. Gene expression was significantly different at SI stage between sclerotial formation and mycelia growth areas. GO and KEGG analyses indicated more vigorous metabolic characteristics in the hyphae area, while transcription process, DNA repair, and protein processing were enriched in sclerotial cells. Gene expression related to H2O2 production was high in the hyphae area, while expression of H2O2-scavenging genes was high in sclerotial cells, leading to a higher H2O2 concentration in the hyphal region than in the sclerotium. Minor differences were observed in gene expression of H2O2-induced signaling pathway in sclerotial and hyphal cells; however, expression levels of the target genes of transcription factor MSN2, important in the H2O2-induced signaling pathways, were significantly different. MSN2 enhanced stress response regulation in sclerotia by regulating these target genes. Small molecular HSPs were also found upregulated in sclerotial cells. This study indicated that sclerotial cells are more resistant to ROS stress than hyphal cells through transcriptional regulation of related genes.

Involvement of autophagy and apoptosis and lipid accumulation in sclerotial morphogenesis of Morchella importuna

Sclerotial formation is a key phase of the morel life cycle and lipids have been recorded as the main cytoplasmic reserves in sclerotia of Morchella fungi without any experimental verification. In this study, the ultrastructural features of the undifferentiated mycelia stage (MS) and three main sclerotial differentiation states (sclerotial initial [SI], sclerotial development [SD] and sclerotial maturation [SM]) were compared by transmission electron microscopy. The nature of the energy-rich substance in hypha and sclerotium of Morchella importuna was qualitatively investigated by confocal laser scanning microscopy and quantitatively studied by extraction of lipids. Sclerotia were observed to form from the repeated branching and enlargement of either terminal hyphae or subordinate hyphal branches, indicating a complex type of sclerotial development. Autophagy and apoptosis were involved in the sclerotial metamorphosis of the cultivated strain of M. importuna. During the SI phase, the characteristic features of autophagy (vacuolation, coalescence of small vacuoles, existence of autophagosomes and engulfment of autophagosomes by vacuoles) were observed. At the SD phase, apoptotic characteristics (condensation of the cytoplasm and nucleus, shrinkage of plasma membrane, extensive plasma membrane blebbing and existence of phagosomes) could be seen in some developing sclerotial cells. In the final stage of sclerotial morphogensis, the sclerotial cells showed a necrotic mode of cell death. In addition, confocal laser imaging studies of live cells indicated that the energy-rich substance in morel hyphae and sclerotia was lipid. The lipid content in sclerotia was significantly more than that in hyphal cells. To the best of our knowledge, this is the first detailed ultrastructural description highlighting the involvement of autophagy and apoptosis in sclerotial metamorphosis of Morchella species and lipid accumulation during morel sclerotial development was also first experimentally verified. This work will promote a better understanding of the mechanism of morel sclerotial metamorphosis.

Rapid marker-assisted selection of antifungal Bacillus species from the canola rhizosphere

A marker-assisted approach was adopted to search for Bacillus spp. with potential as biocontrol agents against stem rot disease of canola caused by Sclerotinia sclerotiorum. Bacterial strains were isolated from the rhizosphere of canola and screened using multiplex PCR for the presence of surfactin, iturin A and bacillomycin D peptide synthetase biosynthetic genes. Among the 96 isolates screened, only CS-42 harbored all three genes and was subsequently identified as Bacillus cereus using 16S rRNA gene sequencing. This strain was found to be effective in significantly inhibiting the growth of S. sclerotiorum in vitro and in planta. Scanning electron microscopy studies at the dual culture interaction region revealed that mycelial growth was curtailed in the vicinity of bacterial metabolites. Complete destruction of the outmost melanised rind layer of sclerotia was observed when treated with the bacterium. Transmission electron microscopy of ultrathin sections challenged with CS-42 showed partially vacuolated hyphae as well as degradation of organelles in the sclerotial cells. These findings suggested that genetic marker-assisted selection may provide opportunities for rapid and efficient selection of pathogen-suppressing Bacillus strains for the development of microbial biopesticides.

Characterization of the Sclerotinia sclerotiorum cell wall proteome.

SummaryWe 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.

Cell wall structure of mushroom sclerotium (Pleurotus tuber-regium): Part 2. Fine structure of a novel alkali-soluble hyper-branched cell wall polysaccharide

The fine molecular structure of a novel alkali-soluble polysaccharide (SCA-I) from the mushroom sclerotial cell wall of Pleurotus tuber-regium was elucidated by 1-D and 2-D NMR spectroscopy as a hyper-branched glucan having a 64.9% degree of branching at O-6 and consisting a main chain with →1)-Glcp-(4→ linkages and short side chains having either →1)-Glcp-(3→ or →1)-Glcp-(6→ linkages. This partial structure of SCA-I was consistent with that obtained from methylation analysis. The molecular conformation of SDS-treated SCA-I was visualized for the first time by TEM and AFM. Microscopic images confirmed the highly branched structure of SCA-I as well as its multiple chain conformation when its compact structure was opened up by SDS treatment. This unique hyper-branched polysaccharide has the potential application in the design of carbohydrate-based nano-particles and drug delivery systems.

Sclerotial structures ofMorchella conicain agar media with different carbohydrates

In this study, the effects of various carbon sources for Morchella conica sclerotium formations were investigated under in vitro conditions. The formations of sclerotial initials, which improved from hyphae of Morchella conica , were obtained by using different carbohydrates. Vegetative mycelia were developed very well in agar media, containing malt extract (MEA), wheat (WA), potato dextrose (PDA) and complete medium yeast extract (CYM), however, moderate development occurred in defined media, containing glucose, sucrose, maltose and starch. The carbohydrate concentrations were prepared as 0.0%, 0.25%, 0.50%, 0.75%, 1.00% and 1.25%. Whole wheat agar and potato dextrose agar media consisting of glucose, sucrose and 0.25%, 0.75% and 1.25% starch excluded, while other cultural environments supported sclerotial formations. Sclerotial structures were examined morphologically and anatomically. The pigmentation changed from yellow to orange and brown. Time sclerotial formation varied from 10 days up to 7 months. Pigmentation, structure and quantity of sclerotia were taken as morphological criteria. Anatomical properties of sclerotia were obtained on light microscopy and scanning electron microscopy. Generally, sclerotial cells were very thick and spherical.

Interactions between the mycoparasite Pythium oligandrum and two types of sclerotia of plant-pathogenic fungi

The interactions between Pythium oligandrum hyphae and two types of sclerotia, i.e. the plano-convexoid sclerotium of Botrytis cinerea and the tuberoid sclerotium of Sclerotinia minor, were investigated by ultrastructural and cytochemical experiments. In the mycoparasitism of P. oligandrum, some differences in relation to sclerotium anatomy and the role of the rind layer in preventing invasion are documented. Both types of sclerotia showed neither alterations of the heavily melanised rind walls, nor direct rind wall penetration by P. oligandrum. This oomycete successfully entered B. cinerea sclerotia only through breaches at the junction of rind cells and corresponding to gaps in melanin deposits. On the other hand, none of these breaches was observed in the small sclerotia of S. minor, and P. oligandrum ingress in the sclerotia stopped at the inner rind layer. After the penetration of B. cinerea sclerotia by the mycoparasite, it extensively colonised the cortical and medulla areas by intercellular growth. The invaded tissues displayed pronounced alterations as well as some disorganisation of tissue in places. Colonisation was associated with severe chitin degradations of all host walls, which occurred even at some distance from P. oligandrum hyphae. The observation of wall thickenings in some P. oligandrum-hyphae suggests that the sclerotial cells constitute a harsh environment unsuitable for survival of the mycoparasite. These wall thickenings could be interpreted as P. oligandrum defence-like reactions.

Scanning Electron Microscopy of Sclerotia of Sclerotinia trifoliorum and Related Species

AbstractThe 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.

Stachybotrys elegans: a destructive mycoparasite of Rhizoctonia solani

Stachybotrys elegans colonizes hyphae as well as sclerotia of Rhizoctonia solani. Germinability of sclerotia, which had been incubated with the mycoparasite for 49 d, decreased from 84% to 5% as compared with 86% germinability of untreated sclerotia. Light and electron microscopic observations revealed that S. elegans grew toward and coiled around the host cells. In pure culture, cells of S. elegans were surrounded by extracellular fibrillar material. The production of this material was enhanced during contact and colonization of host hyphae only. This fibrillar material was not observed during interaction of the mycoparasite with host sclerotial cells. Penetration of host cells was accomplished by either unspecialized hyphal branches or pseudoappressoria. Penetration of host cells and emergence from them by the mycoparasite is apparently accomplished by both mechanical and enzymatic activity. Trophic hyphae of the parasite developed within the host cytoplasm, resulting in granulation of the cytoplasm and ultimately death of host cells. Papillae of unknown origin and chemical composition were frequently detected in colonized sclerotial cells. The above results indicate that mycoparasitism is the mode of action by which S. elegans attacks its host.

Developmental proteins in Aspergillus

One strain each of Aspergillus alliaceus, A. auricomus, A. parasiticus, A. flavus, and A. nomius were investigated for the presence of developmentally regulated sclerotial and conidial proteins. Total protein was extracted from mycelia, sclerotial initials, sclerotia, and conidia. Two groups of sclerotial proteins, a narrow range (12–22 kDa) of low-molecular-weight proteins with some polypeptides shared by pairs of strains, and a wide range (28–78 kDa) of higher-molecular-weight proteins which were unique for each strain, were observed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Developmental proteins were also observed in conidial extracts, in a wide molecular weight range (11–59 kDa). Isoelectric focusing and two-dimensional analysis yielded unique sclerotial and conidial protein banding patterns for each strain examined. In immunoblots of proteins from mycelial, sclerotial, and conidial extracts of all five strains and Sclerotinia sclerotiorum, only the sclerotial proteins of A. parasiticus, A. flavus, and A. nomius cross-reacted with antibodies raised to the major, lowmolecular-weight (12.2 kDa) sclerotial protein of A. flavus, p136s. It was determined by histochemical staining that the majority of protein in sclerotia was sequestered in the cytoplasm of sclerotial cells. p136s was localized within the cytoplasm of sclerotial cells by immunolabeling with anti-p136s and alkaline phosphatase-conjugated secondary antibodies. Our immunological results support current species concepts and sectional assignments of these species in the genus Aspergillus.

Parasitism of sclerotia of Sclerotium rolfsii by trichoderma harzianum

The ability of Trichoderma harzianum isolate 203 to attack the soil-borne plant pathogen Sclerotium rolfsii is apparently connected with the production by the isolates of chitinase and β-(1,3)-glucanase inside the attacked sclerotia during parasitism. SEM and TEM micrographs show that the mycoparasite degraded walls of sclerotial cells and the attacked cells lost their cytoplasmic contents. It is assumed that T. harzianum utilizes sclerotial cell contents thus enabling it to sporulate intensively on the sclerotial surface and inside the digested cells.

Extracellular and cell wall polysaccharides of Aspergillus alliaceus

Aspergillus alliaceus produces an α-linked extracellular polysaccharide containing d -galactosamine (70%), d -galactose (10.5%), d -glucose (10.5%) and acetate ion (2.6%). From mycelial and sclerotial cell walls an alkali-soluble α-glucan and an insoluble β-glucan-chitin complex have been isolated. The a-glucan amounts to 15% of the mycelial and 64% of the sclerotial cell walls, and the insoluble β-glucan-chitin complex amounts to 50% and 9.5% respectively.

Interactions between Rhizoctonia tuliparum sclerotia and soil microorganisms

Air-dried sclerotia of Rhizoctonia tuliparum were readily invaded by a number of soil fungi, particularly Mucor, Fusarium and Trichoderma species. Invasion was associated with an apparent rapid loss in viability as measured by germination on agar. However sclerotia were able to infect tulip bulbs, suggesting that germination on agar may not be a good measure of viability in the presence of fungal invaders. Non-air-dried sclerotia from culture were still viable after 2 years when buried in field soil but on the soil surface sclerotia were invaded by fungi resulting in a loss in viability. Some sclerotia (usually < 20%) recovered from infected bulbs were also invaded by fungi. When immersed in water air-dried sclerotia lost more than 60% of their dry weight through leakage. Infectivity of sclerotia dropped after one leakage cycle and viability after two. Materials which leaked from sclerotia included a previously undescribed broad spectrum antibacterial pyrone antibiotic. Soil bacterial populations increased rapidly when air-dried sclerotia were buried in soil. Dead non-antibiotic sclerotia persisted for at least 2 years in soil suggesting that the composition of the sclerotial cell wall may be important in survival.

Interactions between Rhizoctonia tuliparum and soil microorganisms

ABSTRACTRhizoctonia tuliparum Whetzel &amp; Arthur is a winter active soil‐borne pathogen, which causes the grey bulb rot of tulips and Dutch irises. The fungus survives by means of sclerotia.It was found that the sclerotia only germinated in unsterile soil at low temperatures (0–10 °C). Germination in sterile soil occurred over a wider range of temperatures (0–25 °C) but was inhibited at 20 °C after the addition of a suitable bacterial inoculum.An unidentified pyrone antibiotic was leaked by sclerotia which had been air‐dried and re‐wetted. This could be detected in vitro using an agar medium seeded with a spore suspension of Bacillus subtilis Cohn emend Prazmowski. In soil, however, material leaked by sclerotia stimulated both bacteria and fungi.Undried, air‐dried and oven‐dried (non antibiotic) sclerotia persisted equally well in soil over 2 yr. This suggests that the resistance to lysis of sclerotial cell walls may be important in the long term survival of this organism.

Fine structure of imbibed sclerotial cells of Claviceps purpurea (Fr.) Tul revealed by freeze‐etching

AbstractThe fine structure of the cortex of the natural sclerotium of Claviceps purpurea was studied. The cell wall of sclerotial cells is thickened due to overproduction of the fibrillar component of the wall. The intercellular spaces of the cortex tissue form a continuous system which is apparently instrumental in mediating communication between the growing sclerotium and the external milieu. The cytoplasmic membrane of imbibed sclerotial cells carriers abundant signs of secretory activity. Secretion vesicles, the content of which is discharged into external space, apparently contain exo 1,3‐glucanase. Cytoplasmic vesicles migrating toward the plugged pores of the thickened septa apparently involve hydrolase secretion too. Spherosomes with lipid content are a predominant component of the cytoplasm of sclerotial cells. The activity of the membrane systems of imbibed cells indicates that the mobilization of lipids sets in only after activation of the hydrolases. Findings of phagocytosis of lipid granules by vacuoles are relatively frequent so that lipolysis might proceed in the vacuoles. Alkaloids could not be detected with the aid of freeze‐etching.

Fine structure of imbibed sclerotial cells of Claviceps purpurea (Fr.) Tul revealed by freeze-etching.

The fine structure of the cortex of the natural sclerotium of Claviceps purpurea was studied. The cell wall of sclerotial cells is thickened due to overproduction of the fibrillar component of the wall. The intracellular spaces of the cortex tissue form a continuous system which is apparently instrumental in mediating communication between the growing sclerotium and the external milieu. The cytoplasmic membrane of imbibed sclerotial cells carriers abundant signs of secretory activity. Secretion vesicles, the content of which is discharged into external space, apparently contain exo 1,3-glucanase. Cytoplasmic vesicles migrating toward the plugged pores of the thickened septa apparently involve hydrolase secretion too. Spherosomes with lipid content are a predominant component of the cytoplasm of sclerotial cells. The activity of the membrane systems of imbibed cells indicates that the mobilization of lipids sets in only after activation of the hydrolases. Findings of phagocytosis of lipid granules by vacuoles are relatively frequent so that lipolysis might proceed in the vacuoles. Alkaloids could not be detected with the aid of freeze-etching.

Morphology, development, and cytochemistry of the hyphae and sclerotia of species in the Rhizoctonia complex

A detailed comparison of the gross morphology of hyphae, monilioid cells, and sclerotial formation and structure of 17 isolates of species in seven genera of the Rhizoctonia complex were studied. Cytochemistry of both hyphal and sclerotial cells was also studied. Gross morphology of hyphae was similar in many of the isolates and was considered of limited taxonomic value. Sclerotial formation in species of Thanatephorus, Waitea, Aquathanatephorus, and possibly Ceratobasidium was of the monilioid cell type. That of Athelia was of the hyphal strand type. Although initially similar, mature sclerotia in each of the genera showed great diversity. Most sclerotia of Thanatephorus were constructed of loosely arranged monilioid cells. Those in Ceratobasidium developed a marginal rind; Aquathanatephorus, a pitted surface and hyphal mat; Waitea, a bilaminate condition with elongate marginal and globose medullary cells; and Athelia, a complex tetralaminate structure. Distribution and concentration of carbohydrates, proteins, and lipids differed in the various genera. The strong dextrinoid reaction of the cytoplasm of Thanatephorus and Aquathanatephorus, accompanied by higher total lipids in cell walls and septa, was of significance. Stronger reactions for cellulose were found in species of Ceratobasidium, while cellulose appeared more concentrated in sclerotial cells than in hyphal cells of Thanatephorus. Lipids were deposited more abundantly in sclerotial cells than in hyphal cells.

Ultrastructure of sclerotia of Rhizoctonia solani Kühn invaded and decayed by soil microorganisms

Ultrastructural changes of sclerotia of Rhizoctonia solani Kühn undergoing microbial breakdown in soil were studied with two isolates differing in survival ability. Sclerotial cells of a short-lived isolate (F-15) lost their cytoplasmic contents more rapidly than those of long-lived isolate (B-5) and only the cell walls remained 8 months after burial in soil. In isolate F-15, incomplete septa lacking the complex septal pore apparatus were frequently found in empty cells. Bacterial-like organisms were detected in abundance within the amorphous layer surrounding sclerotial cells. They eroded the amorphous layer and the cell wall matrix. Disappearance of the cell wall resulted at least partially from exolysis caused by these microorganisms which were also found within empty cells. Penetration of the cell wall and sporulation by soil fungi were also frequently observed within sclerotial cells of F-15. Eventually, the empty sclerotial cell invaded by soil microorganisms collapsed and became loosely attached to one another. In contrast, the occurrence of bacterial-like organisms and microbial invasion were not as frequent with the sclerotia of the long-lived isolate (B-5). Instead, its sclerotia were characterized by many intact cells with thickened cell walls. Resistance of sclerotia of B-5 to microbial breakdown may be a result of thick cell walls or the ability of the fungus to repair decomposing cell walls.

Fine structure of sclerotial germination in Colletotrichum coccodes

Aged, dried sclerotia of Colletotrichum coccodes germinated by developing secondary, mucilage-covered acervuli at one or more points on the sclerotial surface. Sclerotial cells underwent structural transformations when placed on nutrient agar resulting in the formation of several ‘areas of activity’ within individual sclerotial cells. These areas of activity were further modified into secondary hyphae which grew through the intervening sclerotial cells to the exterior of the Sclerotium. Emergent hyphae were transformed into conidio-phores which abstricted conidia in the usual manner.

Water relations in sclerotia of some Sclerotinia and Sclerotium species

Experiments on the desiccation sclerotia of Sclerotinia sclerotiorum, Sclerotium delphinii and S. rolfsii demonstrate that the sclerotium rind does not effectively restrict water loss. Observations on rate of rehydration show that drying of the outer sclerotial tissues does not make them impermeable. The data suggest that survival of sclerotia under desiccating conditions is a property of the individual sclerotial cells.