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Cytochemical detection of a layer of mucilage around the hyphae of Cercosporella herpotrichoides
Notes and Brief Articles CYTOCHEMICAL DETECTION OF A LAYER OF MUCILAGE AROUND THE HYPHAE OF CERCOSPORELLA HERPO TRICHOIDES J.
REISS
Institute of Special Botany, University of Moine; Many studies have shown that the hyphae of fungi of different classes can possess an extracellular layer of mucilage: Sporotrichum sp. (Merzel, 1963), soft-rot fungi (Liese, 1964), blue-strain fungi (Schmid & Liese, 1965; Schmid & Baldermann, 1967) and some discomycetes (Moore, 1963; Moore, 1965). In most of these studies the slime has been detected in sections under the electron microscope and only Merzel (1963), Moore (1965) and Schmid & Baldermann (1967) have used cytochemical methods. In the case of the phytopathogenic Cercosporella herpotrichoides the occurrence of such a slime envelope has not yet been described but observations in the course of cytochemical localization of different enzymes in the cells of this organism (not yet published) led to the assumption that the hyphae of C. herpotrichoides might be covered by a layer of mucilage. Therefore a study was undertaken to detect this substance using cytochemical techniques. Moore (1965) pointed out that vegetable mucilage is a mixture of different polysaccharides having certain similarities with animal mucin. This term covers secretion products containing acid mucopolysaccharides. In cytochemical literature many methods for the localization of mucin have been described. Three techniques which are claimed to be specific with certain limitations were used in the present study. C. herpotrichoides was cultivated on malt agar (4 % malt extract, 0'5 % peptone, 2'5 % agar) in Petri dishes. After adequate growth, coverglasses were put into the agar in front of the hyphal tips. The hyphae were thus forced to grow on to the coverglasses, which were removed after 6 days. These preparations were subjected to staining procedures without prior fixation. As the preparations were examined under the microscope in water, additional stabilization procedures could be omitted. Three groups of stains were employed. Copper phthalocyanine dyes. The preparations were immersed for different periods in a solution of 1 g dye, 97 ml distilled water and 3 ml glacial acetic add (Steedman, 1950; Putt & Hukill, 1962; Moore, 1965). The dyes were alcian blue 8 GS (Chroma, Heidelberg, Germany; No. 10030), aldan yellow GXS (Chroma No. 10°31), aldan green 2 GX (Chroma No. 10032) and alcian green 3 BX (Chroma No. 15050). With the aldan yellow and greens the cells stained more or less uniformly yellowish or light green. An extracellular layer could not be observed. With the aldan blue solution, however, nearly all hyphae showed an extracellular blue envelope about o-5 {tm thick. The optimal staining period Trans. Br. mycol. Soc. 56 (3), (1971). Printed in Great Britain 30-2
482
Transactions British Mycological Society
was 15 min. After a further 45 min, globular intracellular structures were also stained blue. Toluidine blue 0 (Pearse, 1961; Moore, 1965). The coverglasses were treated with a 0'5 % aqueous solution of toluidine blue 0 (Chroma No. 11645), which stains mucin metachromatically. Only intracellular material was stained: round deep-blue granules in a light violet cytoplasm. Mucicarmine (Mayer, 1897; Moore, 1965). Mucicarmine solution (Chroma No. 20540) was diluted with distilled water (I :9, vjv). This working solution must be prepared daily. Even when staining the preparations for as long as 120 min the cells remained mostly unstained. Occasionally they were uniformly light pink. Treatment of the hyphae with undiluted staining solution produced the same result. Thus, only with the alcian blue 8 as technique could a distinct layer around the hyphae be stained. Quintarelli & Dellovo (1965) found this stain to be specific for acid polysaccharides, so it must be concluded that the layer around the hyphae of C. herpotrichoides contains those acid polysaccharides and can be defined as mucilage. The lack of staining after treatment with the alcian yellow and greens is probably due to an inadequate binding of these dyes with the fungal polysaccharides. The reason for the negative result with mucicarmine, which was shown to be specific for the gel of higher fungi (Moore, 1965), remains unknown. Probably the differences of composition between the mucin and the gel of higher fungi and the slime of C. herpotrichoides are so great that a binding of the latter substance with mucicarmine is impossible. I thank Mr Whittacker B.A. for his help in preparing the manuscript. REFERENCES
LIESE, W. (1964). Uber den Abbau verholzter Zellwande durch Moderfaulepilze. Hole als Roh- und Werkstoff 22, 289. MAYER, P. (1897). Uber Schleimfarbung, Mitteilungen ausder Zoologischen Station zu Neapel
12,
303.
MERZEL, J. (1963). Histochemical observations on polysaccharides in some species of Sporotrichum. Annales d'histochimie 8, 383. MOORE, E. J. (1965). Staining fungal gel with mucin techniques. Stain Technology 40, 23. MOORE, R. T. (1963). Fine structure of mycota. 1. Electron microscopy of the discomycete Ascodesmis. Nova Hedwigia 5, 263. PEARSE, A. G. E. (1961). Histochemistry, Theoretical and Applied. London: Churchill. PUTT, F. A. & HUKILL, P. B. (1962). Alcian green. A routine stain for mucins. Archives of Pathology 74, 169. QUINTARELLI, G. & DELLOVO, M. C. (1965). The chemical and histochemical properties of Alcian blue. IV. Further studies on the methods for the identification of acid glycoaminoglycans. Histochemie 5, I96. SCHMID, R. & BALDERMANN, E. (I967). Elektronenoptischer Nachweis von sauren Mucopolysacchariden bei Pilzhyphen. Naturunssenschqften 54, 52 I. SCHMID, R. & LIESE, W. (I965)' Zur Aubenstruktur der Hyphen von Blauepilzen, Phytopathologische Zeitschrift 54, 275. STEEDMAN, H. F. (1950). Alcian blue 8 as: A new stain for mucin. Quarterly Journal of microscopical Science 9 I , 477.
Trans. Br. mycol. Soc. 56 (3), (I97I). Printed in Great Britain
REISS
Institute of Special Botany, University of Moine; Many studies have shown that the hyphae of fungi of different classes can possess an extracellular layer of mucilage: Sporotrichum sp. (Merzel, 1963), soft-rot fungi (Liese, 1964), blue-strain fungi (Schmid & Liese, 1965; Schmid & Baldermann, 1967) and some discomycetes (Moore, 1963; Moore, 1965). In most of these studies the slime has been detected in sections under the electron microscope and only Merzel (1963), Moore (1965) and Schmid & Baldermann (1967) have used cytochemical methods. In the case of the phytopathogenic Cercosporella herpotrichoides the occurrence of such a slime envelope has not yet been described but observations in the course of cytochemical localization of different enzymes in the cells of this organism (not yet published) led to the assumption that the hyphae of C. herpotrichoides might be covered by a layer of mucilage. Therefore a study was undertaken to detect this substance using cytochemical techniques. Moore (1965) pointed out that vegetable mucilage is a mixture of different polysaccharides having certain similarities with animal mucin. This term covers secretion products containing acid mucopolysaccharides. In cytochemical literature many methods for the localization of mucin have been described. Three techniques which are claimed to be specific with certain limitations were used in the present study. C. herpotrichoides was cultivated on malt agar (4 % malt extract, 0'5 % peptone, 2'5 % agar) in Petri dishes. After adequate growth, coverglasses were put into the agar in front of the hyphal tips. The hyphae were thus forced to grow on to the coverglasses, which were removed after 6 days. These preparations were subjected to staining procedures without prior fixation. As the preparations were examined under the microscope in water, additional stabilization procedures could be omitted. Three groups of stains were employed. Copper phthalocyanine dyes. The preparations were immersed for different periods in a solution of 1 g dye, 97 ml distilled water and 3 ml glacial acetic add (Steedman, 1950; Putt & Hukill, 1962; Moore, 1965). The dyes were alcian blue 8 GS (Chroma, Heidelberg, Germany; No. 10030), aldan yellow GXS (Chroma No. 10°31), aldan green 2 GX (Chroma No. 10032) and alcian green 3 BX (Chroma No. 15050). With the aldan yellow and greens the cells stained more or less uniformly yellowish or light green. An extracellular layer could not be observed. With the aldan blue solution, however, nearly all hyphae showed an extracellular blue envelope about o-5 {tm thick. The optimal staining period Trans. Br. mycol. Soc. 56 (3), (1971). Printed in Great Britain 30-2
482
Transactions British Mycological Society
was 15 min. After a further 45 min, globular intracellular structures were also stained blue. Toluidine blue 0 (Pearse, 1961; Moore, 1965). The coverglasses were treated with a 0'5 % aqueous solution of toluidine blue 0 (Chroma No. 11645), which stains mucin metachromatically. Only intracellular material was stained: round deep-blue granules in a light violet cytoplasm. Mucicarmine (Mayer, 1897; Moore, 1965). Mucicarmine solution (Chroma No. 20540) was diluted with distilled water (I :9, vjv). This working solution must be prepared daily. Even when staining the preparations for as long as 120 min the cells remained mostly unstained. Occasionally they were uniformly light pink. Treatment of the hyphae with undiluted staining solution produced the same result. Thus, only with the alcian blue 8 as technique could a distinct layer around the hyphae be stained. Quintarelli & Dellovo (1965) found this stain to be specific for acid polysaccharides, so it must be concluded that the layer around the hyphae of C. herpotrichoides contains those acid polysaccharides and can be defined as mucilage. The lack of staining after treatment with the alcian yellow and greens is probably due to an inadequate binding of these dyes with the fungal polysaccharides. The reason for the negative result with mucicarmine, which was shown to be specific for the gel of higher fungi (Moore, 1965), remains unknown. Probably the differences of composition between the mucin and the gel of higher fungi and the slime of C. herpotrichoides are so great that a binding of the latter substance with mucicarmine is impossible. I thank Mr Whittacker B.A. for his help in preparing the manuscript. REFERENCES
LIESE, W. (1964). Uber den Abbau verholzter Zellwande durch Moderfaulepilze. Hole als Roh- und Werkstoff 22, 289. MAYER, P. (1897). Uber Schleimfarbung, Mitteilungen ausder Zoologischen Station zu Neapel
12,
303.
MERZEL, J. (1963). Histochemical observations on polysaccharides in some species of Sporotrichum. Annales d'histochimie 8, 383. MOORE, E. J. (1965). Staining fungal gel with mucin techniques. Stain Technology 40, 23. MOORE, R. T. (1963). Fine structure of mycota. 1. Electron microscopy of the discomycete Ascodesmis. Nova Hedwigia 5, 263. PEARSE, A. G. E. (1961). Histochemistry, Theoretical and Applied. London: Churchill. PUTT, F. A. & HUKILL, P. B. (1962). Alcian green. A routine stain for mucins. Archives of Pathology 74, 169. QUINTARELLI, G. & DELLOVO, M. C. (1965). The chemical and histochemical properties of Alcian blue. IV. Further studies on the methods for the identification of acid glycoaminoglycans. Histochemie 5, I96. SCHMID, R. & BALDERMANN, E. (I967). Elektronenoptischer Nachweis von sauren Mucopolysacchariden bei Pilzhyphen. Naturunssenschqften 54, 52 I. SCHMID, R. & LIESE, W. (I965)' Zur Aubenstruktur der Hyphen von Blauepilzen, Phytopathologische Zeitschrift 54, 275. STEEDMAN, H. F. (1950). Alcian blue 8 as: A new stain for mucin. Quarterly Journal of microscopical Science 9 I , 477.
Trans. Br. mycol. Soc. 56 (3), (I97I). Printed in Great Britain