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Distribution and properties of basic proteins in the phialide of Penicillium notatum during conidiation
Transactions British Mycological Society JACOBSEN, G. (1875)' Ueber phenylphorphorsauren und chloride derselben. Ber. dt. chem, Ges. 8, 1519-1523. HOFMANN, A, FREY, A., Orr, H., PETRZILKA, T. & TROXLER, F. (1958). Konstitutionsaufklarung und synthese von psilocybin. Experientia 14, 397-399. HOFMANN, A., HElM, R., BRACK, A. & KOBEL, H. (1958). Psilocybin, ein psychotroper Wirkstoff aus dem mexikanischen Rauschpilz Psilocybe mexicana Heim. Experientia 14, 10 7-10 9.
ofBiochemistry and Chemistry, Imperial College, London, S. W. 7
P. G. MANTLE and E. s. WAIGHT, Departments
DISTRIBUTION AND PROPERTIES OF BASIC PROTEINS IN THE PHIALIDE OF PENICILLIUM NOTATUM DURING CONIDIATION In a study of the cytochemical changes in the phialide of Penicillium notatum Westling during conidial development intense cytoplasmic staining in alkaline fast green was observed in the early stages of phialide differentiation which declined on maturation of the penicillus. Since it was also observed that cytoplasmic ribonucleic acid (RNA) distribution closely paralleled that of the basic proteins, the possible relationship between the two was followed throughout development. Penicillium notatum was grown on malt extract agar disks on coverslips for 3-5 days at 20 DC which gave a range of coverslip cultures consisting primarily ofyoung phialides after 3 days' growth to predominantly mature or senescent penicilli after 4 and 5 days' growth respectively. Coverslips were removed from the cultures and the adhering colonies were fixed in either 95 % alcohol, or acetic: alcohol 3: I, or 10 % neutral buffered formalin. Since the methyl green-pyronin and Feulgen techniques are not generally suitable for fungal material and gave inconsistent results in P. notatum, nucleic acids were stained with toluidine blue or trypan blue or by the acridine orange fluorescence method of Bertalanffy & Bertalanffy (1960). General protein staining was achieved by the mercury-bromophenol blue method at low pH and the coupled tetrazolium reaction (Danielli, 1947). Basic proteins were selectively stained by alkaline fast green FCF (pH 8· I) by the method of Alfert & Geschwind (1953), but since fading in permanent mounts was rapid, temporary preparations were made in glycerine jelly. Nucleic acids were removed, as applicable, with 5 % trichloroacetic acid (TCA) for 15 min at 900, with ribonuclease (RNase) or cold perchloric acid, or with deoxyribonuclease (DNase). Deamination was carried out by treatment with a mixture of equal volumes of 5 % TCA and 5 % sodium nitrite solutions at 20° for 30 min, a treatment known to destroy the e-amino groups of lysine but not the guanidino groups of arginine. Amino groups were blocked by fixation or post-fixation in 10 % neutral buffered formalin for 3 h at 20 0 • Arginine was detected by the Sakaguchi reaction. RNA and total protein methods revealed high concentrations of these substances in the young phialides with decreased amounts in the older structures. Removal of nucleic acids with TCA followed by alkaline fast Trans. Br. "tyeol. Soc. 53 (2), (1969). Printed in Great Britain
Notes and Brief Articles
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green staining gave a strong reaction in the cytoplasm of young phialides (PI. 23, fig. I). Mature phialides in which spore production was at a peak also showed relatively strong fast green staining (PI. 23, fig. 2), whereas the proportion of phialides showing strong staining decreased in older cultures (PI. 23, fig. 3). At all stages of phialide development the cytoplasm and nuclei of the conidia stained intensely. Selective removal of nucleic acids with RNase or perchloric acid or with DNase before fast green staining revealed that the nuclei of the mycelium and the phialides contained typical DNA-bound histone, but the cytoplasmic staining of the young phialides was due primarily to RNA-associated basic proteins. When tissues were deaminated before RNA removal, alkaline fast green staining was reduced but it was still appreciable. However, deamination after removal of RNA drastically reduced cytoplasmic staining, thus confirming the existence of RNAassociated basic proteins and indicating that they were rich in lysine. Arginine staining showed low activity in the mycelium, phialides and spores, strengthening the view that the alkaline fast green staining was due primarily to lysine-rich proteins. Formalin-fixed tissues subsequently subjected to RNA extraction with perchloric acid and then stained by the alkaline fast green procedure showed reduced cytoplasmic staining compared with the controls which were TCA-extracted and stained, indicating that at least a part of the staining by the latter method was due to basic proteins not associated with RNA. If formalin-fixed tissues were subjected to RNA extraction and then the exposed amino groups were subsequently blocked by a further period of formalin fixation, cytoplasmic fast green staining was abolished. Such procedures carried out on tissues originally fixed in acetic: alcohol gave similar results, but the general level of staining was appreciably reduced, indicating that the fixative had probably removed some of the basic protein. The use of 95 % alcohol as a fixative overcame this difficulty to some extent, but as this is a poor fixative for fungi it resulted in distorted preparations. It was difficult to reconcile the original observations of cytoplasmic basic proteins in Tetrahymena made by Alfert & Goldstein (1955) with the absence of appreciable Feulgen-stainable DNA in the cytoplasm. However, it is now well established cytochemically (Horn & Ward, 1957; Davenport & Davenport, 1965) and biochemically (Butler, Cohn & Simson, 1960; Lindsay, 1966; Shepherd & Noland, 1968) that such proteins can occur in association with cytoplasmic RNA. The present work clearly shows a decline in lysine-rich cytoplasmic ribonucleoprotein of the phialide during the phase of rapid mitotic division accompanying conidial production. At the end of conidial production only nuclear DNA-bound histone could be demonstrated by the fast green procedure at pH 8,1. It is apparent from the RNA and total protein observations that a rough parallel exists between the distribution of these and the cytoplasmic ribonucleoproteins during phialide development. Such observations are particularly interesting in the light of the Trans. Br, mycol. Soc. 53 (2), (1969). Printed in Great Britain 20
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findings of Righelato, Trinci, Pirt & Peat (1968) that there is no net synthesis of protein or RNA in chemostat cultures of P. chrysogenum after induction of conidiation in mycelial cultures. It seems that the induction mechanism alters the metabolism of the mycelium in such a manner that existing protoplasmic components are translocated to the phialides where materials appropriate to conidial formation are produced and accumulate. Transfer of proteins between the cytoplasm and the nucleus is now well established (Byers, Platt & Goldstein, 1963), and it is also known in Amoeba (Prescott & Bender, 1963) that migration to the cytoplasm of nuclear proteins bearing labelled basic amino acids occurs before mitosis and that these return to the nucleus at a late stage in cell division. The work of Hadley & Harrold (1958) indicates that spore production is exceedingly rapid once conidiation commences in P. notatum, and the accumulation of high concentrations of cytoplasmic ribonucleoproteins in the young phialide could be a means of providing an adequate pool of available basic proteins for subsequent transfer to daughter conidial nuclei and cytoplasm which are both rich in basic proteins. However, the origin and role of this pool of cytoplasmic ribonucleoprotein are obscure since it is not known if nucleohistone is transferred between the nucleus of the phialide and the cytoplasm during each mitotic division. Further work would be necessary to determine if these basic proteins operate in the regulation of nuclear function and RNA synthesis in the manner suggested by Huang & Bonner (1962). REFERENCES
ALFERT, M. & GESCHWIND, I. L. (1953). A selective staining method for the basic protein of cell nuclei. Proc. natn. Acad. Sci. U.S.A. 39, 991-999. ALPERT, M. & GOLDSTEIN, N. O. (1955). Cytochemical properties of nucleoproteins in Tetrahymena pyriformis; a difference in protein composition between macro- and micronuclei.]. expo Zool. 130,403-419. BERTALANFFY, L. VON & BERTALANFFY, F. D. (1960). A new method for cytological diagnosis of pulmonary cancer. Ann. N.r. Acad. Sci. 84, 225-238. BUTLER, J. A. V., COHN, P. & SIMSON, P. (1960). The presence of basic proteins in microsomes, Biochim. biophys. Acta 38, 386-388. BYERS, T.]., PLATT, D. B. & GOLDSTEIN, L. (1963). The cytonucleoproteins of amoebae. II. Some aspects of cytonucleoprotein behaviour and synthesis. ]. Cell Bioi. 19,
467-475.
DANIELLI,]. F. (1947). A study of the techniques for the cytochemical demonstration of nucleic acids and some components of proteins. Symp. Soc. expo Biol. I, 101-113. DAVENPORT, R. & DAVENPORT,]. C. (1965)' A cytochemical study of cytoplasmic basic proteins in the ascidian oocyte. ]. Cell Biol. 25, 319-326. HADLEY, G. & HARROLD, C. E. (1958). The sporulation of Penicillium notatum Westling in submerged culture. II. The initial sporulation phase. ]. expo Bot. 9, 418-425. HORN, E. C. & WARD, C. L. (1957). The localization of basic proteins in the nuclei of larval Drosophila salivary glands. Proc, natn. Acad. Sci. U.S.A. 43, 776-779. HUANG, R. C. & BONNER,]. (1962). Histone a suppressor of chromosomal RNA synthesis. Proc, natn. Acad. Sci. U.S.A. 48, 1216-1222. LINDSAY, D. T. (1966). Electrophoretically identical histones from ribosomes and chromosomes of chicken livers. Arch. Biochem. Biophys. 113,687-694. PRESCOTT, D. M. & BENDER, M. (1963). Synthesis and behaviour of nuclear proteins during the cell life cycle.]. cell. comp, Physiol. 62 (Suppl, I), 175-194. Trans. Br. mycol. Soc. 53 (2), (1969). Printed in Great Britain
Trans. Br. mycol. Soc.
Vol. 53. Plate 23
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RIGHELATO, R. C., TRINCI, A. P.]., PIRT, S.]. & PEAT, A. (1968). The influence of maintenance energy and growth rate on the metabolic activity, morphology and conidiation of Penicillium chrysogenum.]. gen. Microbiol. 50, 399-412. SHEPHERD, G. R. & NOLAND, B.]. (1968). The intracellular distribution of basic proteins in the chinese hamster ovary cell. Expl Cell Res. 49, 238-250. D. PITT,
Department of Botany, The University, Exeter, Devon
EXPLANATION OF PLATE
23
Staining of basic proteins during phialide maturation in Penicillium notatum Fig. I. Young phialide from 3-day-old culture, first spores forming; intense cytoplasmic fast green staining. Red filter. x 1800. Fig. 2. Mature phialide in which fast green staining of the cytoplasm of the phialide is still pronounced, but lessthan in Fig. I. Cytoplasmic and nuclear staining in spores. Nuclear staining in the mycelium (n, nuclei). Red filter. x 1800. Fig. 3. Penicillus with mature pbialides in which sporing has ceased, only nuclear fast green staining remains in the phialides (n, nucleus); nuclear and cytoplasmic staining of spores. Red filter. x 1800.
PENICILLIUM ARGILLACEUM SP.NOV., A THERMOTOLERANT PENICILLIUM
In the course of an investigation of fungi occurring in mine tips in Staffordshire, a brown-coloured, thermotolerant species of Penicillium was encountered. Three strains of the same species, sent to the CBS for identification, were isolated from wood chips by Dr T. Nilsson, Skogshogskolan, Stockholm. The species proved to be sufficiently different from all described species of Penicillium (Raper & Thorn, 194-9; Kulik, 1968) to warrant its description as a new species. Cultures of this species as well as dried type material are deposited at the Centraalbureau voor Schimmelcultures, Baarn, the Netherlands. Penicillium argUlaceum sp.nov. (Fig. I) Coloniae in agaro maltoso celeriter expandunt temperatura 30°C, velutinae, hyphis laxe intertextis surgentibus, avellaneae; reversum incoloratum vel avellaneum. Hyphae vegetativae hyalinae, leves, 1'5-3 pm diam, Conidiophori oriundi vel ipso e substrato vel ex hyphis aeriis, 60-400 x 2-3(4) pm, hyalini, septati, parietibus vulgo asperulatis vel nonnumquam levibus. Penicilli biverticillati asymmetrici, plerumque consistunt e verticillis duarum ad quinque metularum capitulum phialidum portantium, nonnumquam etiam maiores uno vel duobus ramis additis, Penicilli monoverticillati quoque adsunt. Omnes partes penicilli modice compressae, hyalinae, plerumque asperulatae, sed nonnumquam leves, Rami 12-35 x 1'5-3 pm. Metulae 12-25 x 1'53 pm, sursum expansae ad 4 pm diam. Phialides ad decenas in verticillo, plus minusve abrupte convergentes ad tubulum angustum, plerumque 9-14 x 1'5-2 pm, tubulus conidiiferus 1-2 x I pm. Conidia cylindracea, ellipsoidea vel ovoidea, hyalina, levia, 2'5-4'5 pm longa, plerumque 3-4 x I '2-2 pm; catenae conidiorum intermixtae vel in columnis laxis tortuosis cohaerentes. Typus: CBS 101.69, isolatus ex cumulo deiecto carbonariorum, Staffordshire, Anglia, ab H. C. Evans, Maio 1967.
Colonies on Czapek agar attaining a diameter of 4- to 4-'5 em within weeks at 30 DC; with surface growth almost velvety; somewhat loosetextured, consisting of a very thin network of hyphae either above or 2
Trans. Br. mycol. Soc. 53 (2), (1969). Printed in Great Britain 20-2
ofBiochemistry and Chemistry, Imperial College, London, S. W. 7
P. G. MANTLE and E. s. WAIGHT, Departments
DISTRIBUTION AND PROPERTIES OF BASIC PROTEINS IN THE PHIALIDE OF PENICILLIUM NOTATUM DURING CONIDIATION In a study of the cytochemical changes in the phialide of Penicillium notatum Westling during conidial development intense cytoplasmic staining in alkaline fast green was observed in the early stages of phialide differentiation which declined on maturation of the penicillus. Since it was also observed that cytoplasmic ribonucleic acid (RNA) distribution closely paralleled that of the basic proteins, the possible relationship between the two was followed throughout development. Penicillium notatum was grown on malt extract agar disks on coverslips for 3-5 days at 20 DC which gave a range of coverslip cultures consisting primarily ofyoung phialides after 3 days' growth to predominantly mature or senescent penicilli after 4 and 5 days' growth respectively. Coverslips were removed from the cultures and the adhering colonies were fixed in either 95 % alcohol, or acetic: alcohol 3: I, or 10 % neutral buffered formalin. Since the methyl green-pyronin and Feulgen techniques are not generally suitable for fungal material and gave inconsistent results in P. notatum, nucleic acids were stained with toluidine blue or trypan blue or by the acridine orange fluorescence method of Bertalanffy & Bertalanffy (1960). General protein staining was achieved by the mercury-bromophenol blue method at low pH and the coupled tetrazolium reaction (Danielli, 1947). Basic proteins were selectively stained by alkaline fast green FCF (pH 8· I) by the method of Alfert & Geschwind (1953), but since fading in permanent mounts was rapid, temporary preparations were made in glycerine jelly. Nucleic acids were removed, as applicable, with 5 % trichloroacetic acid (TCA) for 15 min at 900, with ribonuclease (RNase) or cold perchloric acid, or with deoxyribonuclease (DNase). Deamination was carried out by treatment with a mixture of equal volumes of 5 % TCA and 5 % sodium nitrite solutions at 20° for 30 min, a treatment known to destroy the e-amino groups of lysine but not the guanidino groups of arginine. Amino groups were blocked by fixation or post-fixation in 10 % neutral buffered formalin for 3 h at 20 0 • Arginine was detected by the Sakaguchi reaction. RNA and total protein methods revealed high concentrations of these substances in the young phialides with decreased amounts in the older structures. Removal of nucleic acids with TCA followed by alkaline fast Trans. Br. "tyeol. Soc. 53 (2), (1969). Printed in Great Britain
Notes and Brief Articles
305
green staining gave a strong reaction in the cytoplasm of young phialides (PI. 23, fig. I). Mature phialides in which spore production was at a peak also showed relatively strong fast green staining (PI. 23, fig. 2), whereas the proportion of phialides showing strong staining decreased in older cultures (PI. 23, fig. 3). At all stages of phialide development the cytoplasm and nuclei of the conidia stained intensely. Selective removal of nucleic acids with RNase or perchloric acid or with DNase before fast green staining revealed that the nuclei of the mycelium and the phialides contained typical DNA-bound histone, but the cytoplasmic staining of the young phialides was due primarily to RNA-associated basic proteins. When tissues were deaminated before RNA removal, alkaline fast green staining was reduced but it was still appreciable. However, deamination after removal of RNA drastically reduced cytoplasmic staining, thus confirming the existence of RNAassociated basic proteins and indicating that they were rich in lysine. Arginine staining showed low activity in the mycelium, phialides and spores, strengthening the view that the alkaline fast green staining was due primarily to lysine-rich proteins. Formalin-fixed tissues subsequently subjected to RNA extraction with perchloric acid and then stained by the alkaline fast green procedure showed reduced cytoplasmic staining compared with the controls which were TCA-extracted and stained, indicating that at least a part of the staining by the latter method was due to basic proteins not associated with RNA. If formalin-fixed tissues were subjected to RNA extraction and then the exposed amino groups were subsequently blocked by a further period of formalin fixation, cytoplasmic fast green staining was abolished. Such procedures carried out on tissues originally fixed in acetic: alcohol gave similar results, but the general level of staining was appreciably reduced, indicating that the fixative had probably removed some of the basic protein. The use of 95 % alcohol as a fixative overcame this difficulty to some extent, but as this is a poor fixative for fungi it resulted in distorted preparations. It was difficult to reconcile the original observations of cytoplasmic basic proteins in Tetrahymena made by Alfert & Goldstein (1955) with the absence of appreciable Feulgen-stainable DNA in the cytoplasm. However, it is now well established cytochemically (Horn & Ward, 1957; Davenport & Davenport, 1965) and biochemically (Butler, Cohn & Simson, 1960; Lindsay, 1966; Shepherd & Noland, 1968) that such proteins can occur in association with cytoplasmic RNA. The present work clearly shows a decline in lysine-rich cytoplasmic ribonucleoprotein of the phialide during the phase of rapid mitotic division accompanying conidial production. At the end of conidial production only nuclear DNA-bound histone could be demonstrated by the fast green procedure at pH 8,1. It is apparent from the RNA and total protein observations that a rough parallel exists between the distribution of these and the cytoplasmic ribonucleoproteins during phialide development. Such observations are particularly interesting in the light of the Trans. Br, mycol. Soc. 53 (2), (1969). Printed in Great Britain 20
Myc·53
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Transactions British Mycological Society
findings of Righelato, Trinci, Pirt & Peat (1968) that there is no net synthesis of protein or RNA in chemostat cultures of P. chrysogenum after induction of conidiation in mycelial cultures. It seems that the induction mechanism alters the metabolism of the mycelium in such a manner that existing protoplasmic components are translocated to the phialides where materials appropriate to conidial formation are produced and accumulate. Transfer of proteins between the cytoplasm and the nucleus is now well established (Byers, Platt & Goldstein, 1963), and it is also known in Amoeba (Prescott & Bender, 1963) that migration to the cytoplasm of nuclear proteins bearing labelled basic amino acids occurs before mitosis and that these return to the nucleus at a late stage in cell division. The work of Hadley & Harrold (1958) indicates that spore production is exceedingly rapid once conidiation commences in P. notatum, and the accumulation of high concentrations of cytoplasmic ribonucleoproteins in the young phialide could be a means of providing an adequate pool of available basic proteins for subsequent transfer to daughter conidial nuclei and cytoplasm which are both rich in basic proteins. However, the origin and role of this pool of cytoplasmic ribonucleoprotein are obscure since it is not known if nucleohistone is transferred between the nucleus of the phialide and the cytoplasm during each mitotic division. Further work would be necessary to determine if these basic proteins operate in the regulation of nuclear function and RNA synthesis in the manner suggested by Huang & Bonner (1962). REFERENCES
ALFERT, M. & GESCHWIND, I. L. (1953). A selective staining method for the basic protein of cell nuclei. Proc. natn. Acad. Sci. U.S.A. 39, 991-999. ALPERT, M. & GOLDSTEIN, N. O. (1955). Cytochemical properties of nucleoproteins in Tetrahymena pyriformis; a difference in protein composition between macro- and micronuclei.]. expo Zool. 130,403-419. BERTALANFFY, L. VON & BERTALANFFY, F. D. (1960). A new method for cytological diagnosis of pulmonary cancer. Ann. N.r. Acad. Sci. 84, 225-238. BUTLER, J. A. V., COHN, P. & SIMSON, P. (1960). The presence of basic proteins in microsomes, Biochim. biophys. Acta 38, 386-388. BYERS, T.]., PLATT, D. B. & GOLDSTEIN, L. (1963). The cytonucleoproteins of amoebae. II. Some aspects of cytonucleoprotein behaviour and synthesis. ]. Cell Bioi. 19,
467-475.
DANIELLI,]. F. (1947). A study of the techniques for the cytochemical demonstration of nucleic acids and some components of proteins. Symp. Soc. expo Biol. I, 101-113. DAVENPORT, R. & DAVENPORT,]. C. (1965)' A cytochemical study of cytoplasmic basic proteins in the ascidian oocyte. ]. Cell Biol. 25, 319-326. HADLEY, G. & HARROLD, C. E. (1958). The sporulation of Penicillium notatum Westling in submerged culture. II. The initial sporulation phase. ]. expo Bot. 9, 418-425. HORN, E. C. & WARD, C. L. (1957). The localization of basic proteins in the nuclei of larval Drosophila salivary glands. Proc, natn. Acad. Sci. U.S.A. 43, 776-779. HUANG, R. C. & BONNER,]. (1962). Histone a suppressor of chromosomal RNA synthesis. Proc, natn. Acad. Sci. U.S.A. 48, 1216-1222. LINDSAY, D. T. (1966). Electrophoretically identical histones from ribosomes and chromosomes of chicken livers. Arch. Biochem. Biophys. 113,687-694. PRESCOTT, D. M. & BENDER, M. (1963). Synthesis and behaviour of nuclear proteins during the cell life cycle.]. cell. comp, Physiol. 62 (Suppl, I), 175-194. Trans. Br. mycol. Soc. 53 (2), (1969). Printed in Great Britain
Trans. Br. mycol. Soc.
Vol. 53. Plate 23
• •
t--- n
_
•
n
2
.:
J
...
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Notes and Brief Articles
3°7
RIGHELATO, R. C., TRINCI, A. P.]., PIRT, S.]. & PEAT, A. (1968). The influence of maintenance energy and growth rate on the metabolic activity, morphology and conidiation of Penicillium chrysogenum.]. gen. Microbiol. 50, 399-412. SHEPHERD, G. R. & NOLAND, B.]. (1968). The intracellular distribution of basic proteins in the chinese hamster ovary cell. Expl Cell Res. 49, 238-250. D. PITT,
Department of Botany, The University, Exeter, Devon
EXPLANATION OF PLATE
23
Staining of basic proteins during phialide maturation in Penicillium notatum Fig. I. Young phialide from 3-day-old culture, first spores forming; intense cytoplasmic fast green staining. Red filter. x 1800. Fig. 2. Mature phialide in which fast green staining of the cytoplasm of the phialide is still pronounced, but lessthan in Fig. I. Cytoplasmic and nuclear staining in spores. Nuclear staining in the mycelium (n, nuclei). Red filter. x 1800. Fig. 3. Penicillus with mature pbialides in which sporing has ceased, only nuclear fast green staining remains in the phialides (n, nucleus); nuclear and cytoplasmic staining of spores. Red filter. x 1800.
PENICILLIUM ARGILLACEUM SP.NOV., A THERMOTOLERANT PENICILLIUM
In the course of an investigation of fungi occurring in mine tips in Staffordshire, a brown-coloured, thermotolerant species of Penicillium was encountered. Three strains of the same species, sent to the CBS for identification, were isolated from wood chips by Dr T. Nilsson, Skogshogskolan, Stockholm. The species proved to be sufficiently different from all described species of Penicillium (Raper & Thorn, 194-9; Kulik, 1968) to warrant its description as a new species. Cultures of this species as well as dried type material are deposited at the Centraalbureau voor Schimmelcultures, Baarn, the Netherlands. Penicillium argUlaceum sp.nov. (Fig. I) Coloniae in agaro maltoso celeriter expandunt temperatura 30°C, velutinae, hyphis laxe intertextis surgentibus, avellaneae; reversum incoloratum vel avellaneum. Hyphae vegetativae hyalinae, leves, 1'5-3 pm diam, Conidiophori oriundi vel ipso e substrato vel ex hyphis aeriis, 60-400 x 2-3(4) pm, hyalini, septati, parietibus vulgo asperulatis vel nonnumquam levibus. Penicilli biverticillati asymmetrici, plerumque consistunt e verticillis duarum ad quinque metularum capitulum phialidum portantium, nonnumquam etiam maiores uno vel duobus ramis additis, Penicilli monoverticillati quoque adsunt. Omnes partes penicilli modice compressae, hyalinae, plerumque asperulatae, sed nonnumquam leves, Rami 12-35 x 1'5-3 pm. Metulae 12-25 x 1'53 pm, sursum expansae ad 4 pm diam. Phialides ad decenas in verticillo, plus minusve abrupte convergentes ad tubulum angustum, plerumque 9-14 x 1'5-2 pm, tubulus conidiiferus 1-2 x I pm. Conidia cylindracea, ellipsoidea vel ovoidea, hyalina, levia, 2'5-4'5 pm longa, plerumque 3-4 x I '2-2 pm; catenae conidiorum intermixtae vel in columnis laxis tortuosis cohaerentes. Typus: CBS 101.69, isolatus ex cumulo deiecto carbonariorum, Staffordshire, Anglia, ab H. C. Evans, Maio 1967.
Colonies on Czapek agar attaining a diameter of 4- to 4-'5 em within weeks at 30 DC; with surface growth almost velvety; somewhat loosetextured, consisting of a very thin network of hyphae either above or 2
Trans. Br. mycol. Soc. 53 (2), (1969). Printed in Great Britain 20-2