Chemical studies on the exudate and developing sclerotia of Sclerotinia sclerotiorum (Lib.) DeBary

Abstract

COLOTELO, N., J. L. SUMNER, and W. S. VOEGELIN. 1971. Chemical studies on the exudate and developing sclerotia of Sclerotirzia sckrotiorlrnl (Lib.) DeBary. Can. J. Microbiol. 17: 1189-1194. The exudate from sclerotia of 6-day-old cultures of Scle~.otinin sclerotiorirnz (Lib.) DeBary was found to contain cations, lipid:, ammonia, amino acids, proteins, and various enzymes. Disc-gel electrophoretic patterns of proteins, peroxidase, and polyphenoloxidase isoenzymes of the exudate differed from those of zclerotial extracts. Specific activities for catalase, peroxidase, polyphenoloxidase, and P-glucosidase enzymes of the exudate and sclerotial extracts were very similar. Cellulolytic and polygalacturonase activities for the exudate and sclerotial extracts were demonstrated. The nature and origin of the exudate are briefly discussed.Numerous small drops of clear liquid are frequently seen on the surface of developing sclerotia in culture, and within a short time these droplets increase greatly in size and may coalesce. Remsberg (1940) noted that the clear liquid from sclerotia of some Typhula species left a crystalline residue when dried on a microslide. This exudate has frequently been referred to by other workers but its chemical composition was not determined. Cooke (1969) fo~ind that the exudate from sclerotia of Sclerotiitia sclerotiorui~l and S. trifolioruin contained soluble carbohydrates, and referred to this exudate as being water that was actively excreted during the rapid development of sclerotia. The exudate from sclerotia of S. sclerotiorum has also been shown to have phenol oxidase activity and contain various salts, amino acids, and proteins (Jones 1970); however, details were not presented. A membrane enveloping the exudate on sclerotia of S. sclerotiorunt was reported by Colotelo et al. (1971) but the origin and nature of this membrane has not yet been determined.In conjunction with our studies on the enzyme changes associated with the development of sclerotia of S. sclerotiorum, the exudate was analyzed for those properties such as pH, dry matter content, inorganic ions, proteins, enzymes, free amino acids, lipids, and free ammonia, which are normally associated with sclerotia and mycelium. Where possible the properties of the exudate and sclerotial extracts were compared.The fungus, S . sclerotior~ml, originally isolated as sclerotia from diseased parsnips, was grown on a medium consisting of 300 g ground carrots, 16 g agar, and 700 ml deionized distilled water. A plug of inoculum, 7 mnl in diameter, from the periphery of a young colony grown o n the above medium was placed in the center of each Petri plate 138 mnl in diameter containing about 60ml of the carrot-agar medium. Cultures were incubated at 20°C.The liquid o n the surface of sclerotia of 6-day-old cultures was collected with a Pasteur pipette and analyzed immediately after being passed through a sterile 0.45-br Millipore filter. Sclerotia from these cultures were extratced using the following procedure and used for the electrophoretic stud...

Developmentally induced changes in the sclerotial proteome of Sclerotinia sclerotiorum

“…Another protein, peroxidase (spots 44 and 67), which was grouped in the cell defense category, exhibited a significant decrease between the initial and developmental stages. The abundance of catalase and polyphenoloxidase was not found to be significantly altered, although the occurrence of these enzymes has been reported in developing sclerotial extracts (Colotelo et al 1971). Spots 46, 59, and 63 corresponded to NADH: ubiquinone oxidoreductase (complex I) of the fungal respiration chain, an enzyme known to generate superoxide radical (O 2 À ) (Bai et al 2003).…”

“…These include the branching, interweaving and fusion of hyphae, translocation of nutrients and water, and the exudation of liquid droplets on the sclerotial surface (Chet & Henis 1975;Willetts & Bullock 1992). The biochemical composition of sclerotia has been shown to partially consist of carbohydrates, enzymes, free amino acids, and fatty acids (Cooke 1969;Jones 1970;Colotelo et al 1971). Additionally, melanin is the darkly pigmented polymer whose deposition accompanies sclerotial development and maturation, and serves a critical role in fungal persistence and longevity by affording protection against adverse environmental conditions (e.g., temperature extremes, UV and visible irradiation, desiccation, toxic metals, and antagonistic or competitive microbes) (Bell & Wheeler 1986).…”

Analysis of cDNA transcripts from Coniothyrium minitans reveals a diverse array of genes involved in key processes during sclerotial mycoparasitism

“…These were metallo (Families M14,M28 and M36;, serine (Families S09,S28 and S53; and cysteine (Family C15; CmEST-485) proteases (Table 1). S. sclerotiorum sclerotial exudate and the extract contain 1.0-2.8 mg/ml total protein and free amino acids particularly large quantities of aspartic acid and glutamic acid (Colotelo et al, 1971). Increased proteolytic activity suggests that C. minitans is actively engaged in the breakdown and uptake of these constituents of the sclerotia of S. sclerotiorum to derive the N required for its growth and sporulation during mycoparasitism.…”

“…These transporters are likely to be involved in the mobilisation of ions, amino acids, amines, proteins and fatty acids present in the exudates and sclerotia of S. sclerotiorum (Colotelo et al, 1971). Overall, expression of a wide range of genes associated with detoxification, stress response and nutrient utilisation by C. minitans during sclerotial colonisation implies that the mycoparasite has adapted various strategies to gain the ecological competence essential to thrive in this microenvironment.…”

“…Expression of several genes encoding enzymes involved in aromate catabolism [e.g. salicylate hydroxylase (CmEST-059, CmEST-221, CmEST-330, CmEST-410 and CmEST-449) and catechol 2,3 dioxygenase (CmEST-621 and CmEST-663)] suggests their involvement in the utilisation of amino acids, aromatic compounds, aminosugars, proteins and other substrates present in sclerotia and their exudates (Colotelo et al, 1971). Nearly 20% of the ESTs are of unknown function including some expressed by fungal plant pathogens or lack any matches in the databases and could play novel roles in sclerotial mycoparasitism by C. minitans.…”