CURRICULUM VITAE

Actualizado al ao 1984

CARLOS RAMIREZ GOMEZ

La publicacin del presente libro cuenta con la autorizacin de Da. Conchita Ramrez Gomez, hermana del ilustre profesor autor de este libro.Como podremos comprobar a travs de su curriculum y de las reseas a las que se hace referncia, as como en el contenido del presente documento ha sido uno de los grandes cientficos y miclogos del siglo XX, habiendo sido declarado el Miclogo nmero 1 en 1985 en el congreso internacional de Cientficos celebrado en Blgica.

Los honores todos para un hombre que naci en el pas y en la poca equivocada. El resto de los pases con inters en su rama de investigacin se disputaron su sabidura e intervencin hasta los ltimos das de su vida.

Dedicado a Da. Conchita Ramrez, mi mejor amiga. Cuya esperanza es que el esfuerzo y saber de su hermano estuviera al servicio de toda la humanidad. Motivacin que gui el trabajo y todo el esfuerzo del sabio a lo largo de los aos de dedicacin absoluta a la investigacin.

Realizado por l mismo en vida.

1978 - CARLOS RAMIREZ GOMEZ

Profesor de Investigacin del Instituto Jaime Ferrn de Microbiologa del C.S.I.C.

CURRICULUM VITAE 1948 - LICENCIADO EN CIENCIAS NATURALES, Universidad Comptutense

1950 - DOCTOR EN CIENCIAS NATURALES, Universidad Complutense

HISTORIAL CIENTIFICO EN EL CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS. 1948.- Contratado por el Instituto "Jose Celestino Mutis de Farma -cognosia para dirigir el

Laboratorio de Histologa Vegetal para el estudio de plantas cauchferas, hasta octubre de 1950.

1950.- En octubre pasa al Instituto "Jaime Ferrn" de Microbiologa, realizando trabajos de identificacin

de mohos y leva -duras en esputos de tuberculosos, en calidad de Becario. En Diciembre de dicho ao es

nombrado Jefe de la Subseccin de Micologa General de dicho instituto.

1952.- Se traslada a Holanda pensionado por el Consejo para ampliar estudios sobre Mohos y Levaduras en

el Centraalbureau voor Schimmelcultures en Delft y Baarn.

1954.- Es nombrado Colaborador Cientfico en Ciencias Biolgicas.

1956.- Pensionado por el Consejo, se traslada a Paris para ampliacin

de estudios sobre Hongos superiores y sus toxinas en el Laboratoire de Cryptogamie del Musum National

d'Histoire Naturelle de Paris. Alli realiza un trabajo sobre la ecologa de las levaduras que viven sobre

hongos superiores.

1957-58.- Se traslada a Ginebra con la consideracin de pensionado

por dos aos, en cuya Universidad y en el Laboratoire de Botanique

Gnerale, con el Prof. F. Chodat, realiza investigaciones sobre Fisiologa de hongos, descubriendo los

Ritmos Circadianos en la respiracion de las levaduras.

1959- Con la misma consideracin de Pensionado, se traslada por un ao a la Universidad de Zurich, al

Laboratorium von Allegemeine Botabik,

con el Prof. U. Leopold, realizando investigaciones sobre Gentica de hongos,

principalmente sobre la estructura intragentica de los cromosomas de la levadura Schizosaccharomyces

pompe, trabajo que fue objeto de una comunicacin en 1963 en XI International Congress of Genetics,

celebrado en la Haya en Septierabre de 1963.

1959.- Es nombrado Investigador Cientifico del C.S.I.C.. del Patronato "Alonso de Herrera"

1960-63.- Habiendo obtenido un Postdoctorate Fellowship del National Research Council of Canada, se

traslada a la McMaster University de Hamilton, Ontario, Canada, en el Laboratorio de Botnica, con el

Prof. J.J. Miller, donde por el espacio de cuatro aos realiza investigaciones sobre la esporulacin,

su fisiologa, su cariologa en las levaduras, llegando a fotografiar por primera vez en estos hongos

los cromosomas y las divisiones

mitticas.

1964-67.- Con un contrato del ejrcito de los E.E.U.U., se traslada a Ridgefield, Connecticut, al New

England Institute for Medical Research, donde durante cuatro aos realiza investigaciones sobre las

interaccines entre las bacterias patgenas y los microhongos en sistemas abiertos y los mecanismos de

resistencia y susceptibilidad a las enfermedades. En dicho Instituto fue nombrado Jefe del Laboratorio

de Micologa.

1968 - Regresa al Instituto "Jaime Ferrn", donde vuelve a ocupar su puesto de Jefe del Laboratorio de

Micologa General y Aplicada.

En el verano de dicho ao se traslada a Hamilton, a la MacMaster University, para familiarizarse con las

tcnicas de Autoradiografa, obteniendo el Titulo de usuario de Sustancias Radioactivas de la Comisin

de Energa Nuclear de Espaa.

1970 - En octubre de dicho ao es invitado oficialmente por el Prof. Scott del Departamento de Qumica

de la Universidad de Yale, New Haven, Connecticut (USA), para participar en un proyecto sobre produccin

de vitamina B12 por

microorganismos.

1971 - Nombrado Profesor de Investigacin del C.S.I.C.

1972 - se traslada a Lille, donde participa a un curso de un mes en el Instituto Pasteur dirigido por

el Prof. Buttiaux, sobre Microbiologa de los Alimentos.

Seguidamente y por tres meses, se traslada al Institituto de Microbiologa Tcnica y de los Alimentos

(TNO) de Holanda en Zeist, para realizar estudios sobre micotoxinas y su deteccin en los alimentos y

piensos.

(Para aos siguientes vanse las pginas de publlcaciones, etc.

RESUMEN DEL HISTORIAL

Contratado dos aos (1948-1950)

Becario del C.S.I.C: cuatro aos (1950-1953)

Colaborador Cientfico: cinco aos (1953-1957)

Investigador Cientfico: Doce aos (1957-1968)

Profesor de Investigacin: Once aos (1968-1984)

TOTAL : Treinta y cuatro aos,

COLECCIN PERSONAL DE HONGOS (MOHOS Y LEVADURAS) EN EL LAB0RAT0RI0 DE MIC0L0GIA GENERAL Y APLICADA. Desde el ao 1950 en que ingreso en el Instituto "Jaime Ferrn" emprend la tarea de conservar en coleccin

permanente los mohos y levaduras aisladas e identificadas, procedentes de diversos sustratos. Esta

coleccin alcanzo en 1956 la cifra de 2.000 cepas entre mohos y levaduras. Desgraciadamente, desapareci

al ausentarme al extranjero, pues fue llevada a la Universidad de Salamanca sin mi autorizacin.

Al regresar al Instituto en 1968, reanude la tarea de reconstruir una nueva coleccin de hongos, que pasa

en la actualidad la cifra de 7.000 cepas, entre mohos y levaduras procedentes de toda clase de sustratos

(aditivos de alimentos, especias, comerciales, tierras, aire, agua, hospitales, etc.), tanto de Espaa,

como del extranjero (U.S.A., Chile, Argentina, Tierra del Fuego, Sahara, etc.)

Para evitar las continuas resiembras las conservo liofilizadas y en tierra esteril.

TRABAJ0S PUBLICAD0S CLASIFICADOS POR MATERIAS SOBRE ECOLOGA MICOLOGICA 47 trabajos publicados: 9 en Microbiologa Espaola y los restantes en revistas

extranjeras.

SOBRE FISIOLOGA DE HONGO. 3 trabajos en Microbiologa Espaola y 7 en revistas extranjeras.

SOBRE GENETICA DE LEVADURAS Un trabajo en los Proceedings del IX INTERNATIONAL CONGRESS OF GENETICS. La Haya

, 1963,

SOBRE CARIOLOGA DE MOHOS. Uno en revista extranjera y 3 en Microbiologa Espaola.

SOBRE TECNICAS MICROFUNGICAS. Uno en Microbiologa Espaola y cuatro en revistas extranjeras

SOBRE FERMENTACIONES. Uno en Microbiologa Espaola.

SOBRE METABOLISMO DE HONGOS. Cuatro trabajos: 2 en Microbiologa Espaola y dos en revistas extranjeras.

SOBRE INTERACCINES LEVADURA-BACTERIA EN CULTIVO CONTINUO. Dos trabajos en Microbiologa Espaola.

SOBRE MICOTOXINAS Cinco trabajos: dos en revistas espaolas y tres en extranjeras.

SOBRE RADIACIONES GAMAS Y SU INFLUENCIA EN LA ESPORULACION Un trabajo en revista extranjero

80 TOTAL DE TRABAJOS PUBLICADOS HASTA LA 1984: 27 en revistas espaolas, el resto en revistas extranjeras.

TRES MONOGRAFIAS

"COMPILATION OF DESCRIPTIONS OF NEW CANDIDA SPECIES WITH KEYS TO ALL SPECIES OF THE GENUS DESCRIBED UP TO DATE" 1974. Micr. Esp. : 27: 15-80.

LOS PSILIDOS DE ESPAA" Bol. Real. Soc. Esp. Hist. Nat. Vo. 55, 56, y 57, 210 pg. 1955, 1956, 1959.

"MANUAL AND ATLAS OF THE PENICILLIA" 1982. Elsevier PuB. Co Amsterdam. XV + 874 pgs.T

TRABAJOS EN COLABORACIN

X) Sa colaboraciSn con LA SECCIN DE MICRQBIOLOGA TECNICA, se estaa reali-zaado estuciios sobre

el pool de amiaoScidos eh los HALOBACTSRIUM, con el fia de tener mas datos para peraiitir situar

estos microorgaaismos ek el lugar que les corresponda taxonomicamente*

2) Coa la SEC-ION DE FIJACION DEL NITROGENO se esta estudiando el papel que

desempenaa LQ5 HQKGQS SUPERIORES en la utilizacion total de las ligainas, prod

ducto dc desehcho de las papeleras*

3) Ea colaboracion coa un medico Alerg6logo de las Palmas de Gran Canaria, se

esta 11 vando ua ESTUDIO ECOLOGICO DEL MEDIO AMBIENTE de esta Capital, en

cuaato a la MICOFLO.U CONTAMINANTS DEL AI3E Y SU INFLUENCIA p LAS ALS3fi*AS |

7/ Referencias de publicaclones o Tesis doctorales en relacin con el Proyecto:

Sobre tazonoma de mohos y ecologa mlcolgca ha pubilcado veinte trabajos. Sobre fisiologa

da mohos ha pubilcado diez trabajos. Sobre accin de las radiaccines gamma sbre la esporulacin

pubilcado un trabajo. Sobre metabolismo de Mohos ha pubilcado cuatro trabajos.Sobre

Micotoxinas ha presentado dos trabajos en el Congreso Nacional de Mlcrobiologa da Granada

(1973), dos en el de Salamanca (1975), uno en el International Symposium on Mycotoxins in

Foodstuffs da Paris (1976). De los trabajos pubilcados, 44 tratan de temas de Micologa General

y Aplicada. Dos Tesis doctorales presentadas y una on preparacin

tratan de la micoflora micotoxignica contaminante de produccin destinados a la alimentacin

humana.

Repercusin

Trataremos do reunir en este apartado, aunque no exaustivamente la repercusn Internacfonal

de nuestros trabajos, que se hace bien patento

en las numerosas menciones en revistas extranjeas y en tratados de nuestra especialidad:

1954,* J.Bofdn et F,Abadie.Bull.Soc.Mycol.France,70: 353-383;1957, J.Boidin et J.M. Adzet,

Bui|iSec, MymlFrwice, 73:331*342; 1958, "The Chemistry and Biology of Yeasts, A.H. Cook

(editor); 1959 Van der Walt,

J.P. Antony Van Leewenhoek 25: 458-464; 1962, Artagaveitia-Allende, R.C.

y C. Leganani.

Micopathologa et Mycologa Appl. 18: 140-146; 1962, L. de mONTEMAYOR

y B. Heredia de Ganero, Mycopatholga et Mycologa Appl. 18: 1-52; 1962,

Artagaveita-Allende, B.C. Mycopathologa et Mycologa Appl. 25: 299-302; 1962, M.

W.Miller, H. J. Phaff and H.e.Synder. Mycopatologa Appl. 16: 1-18; 1965 J. Boldin

J. Abadie et Y. Lehoday. Bull. Soc. Mycol. France 81: 5-23 1965, M.C.Pignal et J. Boldfn, Dull.

Soc.Mycol,France SI; 197-226; 1965, D, Yarrow..Ant, van ' oe Leewenhoek 3It 405-467;

f965,R*C#Artagaceftfe>AMende,Myoop*thologa et Mycologfa Appl* 2S;237*273; 196> J.Bofdfn,J,

Plgnal et M.Besson, Bull, Soc.Mycol.France 8tj566*60@; 1966 "The Fungi", An advanced Treatise,

Ed,G, C *Afnsworth and A#S#Sueeman,Vot* I pag 135-I4tj 1966, G,ffiurtan en "The Fungi", Vol,

II pgs.367; 1967* J. J, Miller, Bull,Sec,Hist, Natur.Toulouse,Francef 103;3a7*339; 1964,

J.Bo!dln,F.Abadle et Y, Leho* dey et M.C Filial,Buff.Soc,MycoUFrence, 80*397*438j W7,R.Moniro*

cher, Bvtil Soc.Mycol.Framce 83*641-730 J 1968, F. Abedle,My wathologle et Mycologfa Appl.

36i81*t3j 1968, MlNor^M* liter notional Atomic Ertorgy Agency,Vlnna pg. 181-10*1 J* J* Milter

and G^offman-Ostenhoff, ZeftachrM fur AHgemelne Mfkroblofogfe, 44t63*294f If 69, J,J. Ml Her

on "The Yeasts", EoiA^H.Rdfi and J, S.HarHaon, Aeectemlc Press* Vol I Pfl. 93-104*334*381} 1970,

"The Yeasta* A Teconomic Study, Lodde*, J.Ed.pgs ||fiIM,)7fi.861l1Pti.lofri 13SU

LISTA DE ESPECIES NUEVAS DE MICROHC )NGOS PUBLICADAa

17:214-218, 1082 19: 08-102, 1984 19 98*102. 1984

(Paris) Mycol. rparia)

Pichia pseudopolymorpha*, Pichia rhodanensis

1952.- Sporotrichum sanguineum p-.., Mycol# (Pmrim) 1954.- Saccharomyces sociasi Rv,

Myeol. Candida boidinii Rav.

1953

Pichia strasburgensis

Endomycopsis balearica Debaryomyces toletanus Pichia chambardii

Myeal. (P,ri). 18il49-l56. 1983 Rsv. Mycol. (Paris), is, 149-186. 1983 Rev. Mycol.

(P.ria). 18t 149-156. 1963

1954

Bull

Mir. Ep. 7:107T110^".1954 Mler. Ep, 7illllX4# 1954

Men*. Soo. Una, Lyon. 23:151.152

1957.-Geotrichum zambettakesii

Sporobolomyces albidus Candida anomala

Rsv. Mycol. (Paris).25 433-436, 1954 Mler. Esp. 10: 215-247, 1957 Mlcr. Esp. 10:

215-847. 1957

Sporobolomyces boleticola Micr. Eap. 10: 215-247, 1957 Torulopsis buffoni Mlcr. Eap.

10: 215-247,'1957

37-41, 1986

191-200, 1956

Mlcr. Eap. 9j 147-162, 1956

1956.- Sporobolomyces hispanicus Mier. Esp. 9; Endomycopsis ovetensis Mlcr. Esp. 9;

Torulopsis holmii, var. acidi-lactici

1972.- Candida iberica

Candida levantina

Can. J. Microbiol. 18: 1778-1780, 197S Rav.

Mycol. (Paris) 40: 108-110, 1972

1978.- Torulopsis navarrensis Mycopatholoffia 63s 81-63, 1978

Penicillium hispanicum

Torulopsis pampelonensis *J ,Can. J. Micron. 24: 433-435, lira Blastobotrys navarrensis

Myeopatholojia. 63: 41-45. Penicillium fagi Mycopathologi*. 63: 57-59, 1978

(an pranaa) (on pranaa)

(an prensa)

Mycopathologa

Penicillium grancanariae Myeopathologa Penicillium palmensis Mycopathologa

1978.- Halobacterium vallismortis Bacteria extricta hal6ila.

Can. J. Microbiol. (*n prem

En total 26 especies nuevas de hongos (22 levadura-y 4 mohos) y una nueva especie de bacteria. A estas

especies nuevas hay que afla4

dir las siguientes especies nuevas de Penicillium qu estan en prensa: P. turolense, P. murcianum, P.

ovetense, P. asturjanum, P. onobense, p. castellonense, P. aragonense, P. aurantio-flammiferum, P.

gallaicum p. granatense, "Penicillium ilerdanum, P. gerundense, P. valentinum, P. alicantinurn, P.

malacaen.se, P. tarraconense, P. vasconiae y P. novae-caledoniae Smith, var. album, nov. var.

lo que suman en total 40 especies nuevas de microhongos.

1984

CARLOS RAMIREZ PUQLICACIONES

1949.- Ramirez C. Nota sobre hall

.azgo de "Acarapis woodit" (Acarioais de las abejas) en la isla de Malloroa. Bol. Real. Soc.

Hist. Nat, Madrid. - Ramirez, C, Socias, A, e

|^ 1955

/^1956. W 1956.

/ 1956

I 1952

j 1952 4 1953

1953.

C 1953. 1 1953.-? 1954.-^ 1954.-

< W.9 54.-

I 1954.-

)Vl955.-

47 611-118. Gil. Presencia de Aspergillus y Penicillium en esputos y orina de tuberculoses.

Micro- biologa Esp. 5: 113-121.

1

- Ramirez, C. Une nouvelle espece de Sporotriohum. le Sporo-

trichum sanguineum. Revue de Mycologie. Paris. 17j 214-218

Trois nouvelles especes de levures isolees de liqueur tan-nantes. Revue de Mycol. Paris. 18'!

149-158.

Ramirez C. Introduccin de una modificacin al metodo de Wickerham para determinar el poder

fermentativo de la rafi-nosa por las levaduras. Estratto degli atti del VI Congresso

Internazionale di Microbiologa. Roma. Septiembre. I: 872-873 Ramirez, C. Contribucion a la

tincin de ascosporas y clami-dosporas de las levaduras. Microbiol. Esp. 6: 239-246.

Estudio de nuevas especies de levaduras ai&ladas de distintos sustratos. Microbiol. Esp. 6:

249-253.

Deux nouvelles especes de levures isolees de divers substrats. A Revue de Mycologie (Paris).

19: 98-102.

- Ramirez, C., et J. Boidin. Saccharomyces chambardii, nouvelle J

espece de levure isolee de liqueur tannante. Bull. Mens. Soc.

Linn. Lyon. 23: 151-152.

Ramirez, C. Socias, A, y Pelaez Campomanes, F. Una nueva eg- i pecie de Debaryomyces. El

Debaryomyces toletanus. Microbiol. Esp. 7: 111-114.

Ramirez, C, Genestar, R., y Socias A. Una nueva especie de "7 Endomycopsis: Endomycopsis balearica

aislada de concentrado de tanino de encina. Microbiol. Esp. 7: 107-110. Ramirez, C. "Los Psilidos

de Espafia". I. Bol. Real. Soc. Esp. Hist. Nat. 53: 151-218.

Ramirez, C. y Socias A. Estudio cariologico de algunas levaduras ascosporogenas y

anscosporogenas. Microbiol. Esp. 8: 389-

417.

Ramirez, C. "Los Psilidos de Espafia". II. Bol. Real Soc. Hist.

Nat. 54: 63-106.

Ramirez C, Pelaez Campomanes, F. El Sporobolomyces hispani^jis

so. nov., nueva especie de Sporobolomyces aislado de extracto

de corteza de encina. Microbiol. Esp. 9: 37:41.

Ramirez, C. y Sierra, G. Una nueva variedad de Torulopsis hol-

mii (Jorgensen) y estudio de sus productos metabolicos.

Microbiol. Esp. 9: 147-162.

II 1956 /? 1957

tf 1957. 7" 1957.

$1 1959.

^ 1959.

-?3 1959.

Zh 1960.-

2;,-1960.-

1963. -Jl 1964.-

^^T 1964.-4^1965.-%t> 1968.-}( 1968.-3 ^1969.-

13 1970.-3^1972.-

.- Ramirez, C, y Pelaez Campomanes, F. Una nueva especie dP

Endomyces: Endomyces ovetensis. MierobioX. Esp. 9: 3.91-200 ' *

. - Ramirez, C. Contribucin al estudio de la ecologa de las

levaduras. I. Levaduras aisladas de hongos carnosos. Microbiol ,.

Esp. 10: 215-247. "-"-croDiol. )|

elemen-Microbiol.

Real Soc.

Ramirez, C. Nota sobre algunas modifieacione* introducldas a la tincin de nucleos de

hongos. Microbiol. Esp, 10: 201-203

Ramirez, C. y Socias, A. Cromosomas ligados y Mitosis tales en los nucleos de Aspergillus

y Penicillimjj Esp. 10: 259-286.

Ramirez, C. "LOS PSILID0S DE ESPANA". III. Bol Esp. Hist. Nat. 57: 5-87.

Ramirez, C, et Chodat, F. Etudes sur la periodicite des echan-ges gazeux chez une levure:

Kloeckera apiculata. Microbiol. Esp. 12: 265-296.

- Ramirez, C. Methode rapide et simple pour determiner le degre

de fermentation du raffinose par les levures. Ant. van Leeu-

wenhoek. 25: 445-448.

Ramirez, C. Une nouvelle diagnose d'une levure isolee d'Amanita JTJ livido-palescens:

Geotrichum zambettakesii (Ramirez) comb nov. Revue de Mycol. (Paris). 25: 433-436.

Ramirez, C, and Miller, J. J. Observations on vegetative nuclear division inScJccharomyces

cerevisiae. Can. J. Microbiol. 8: 603-608.

Ramirez, C, and Miller, J. J. Accumulation of free proline during yeast sporo^enesis.

Nature. 197: 722-723. Ramirez, C. Studies on the causes of reciprocal inhibition of

pseudomycelium formation between parallel colonies of yeasts of the genus Candida. Can.

J. Microbiol. 10: 931-933. Ramirez, C, and Miller, J. J. The metabolism of yeast

spo-rulation. VI. The changes in amino acid content during sporo-genesis. Can. J.

Microbiol. 10: 223-231.

Miller, J. J., Kingsley, V., and Ramirez, C. Comparative sensitivity to Gamma radiation

of yeast mitosis and meiosis. Radiation Bot. 5: 384-390.

Ramirez, C, and Ransom, J. P. The growth of Candida albicans and Salmonella typhimurium

in mixed cultures. Microbiol. Esp.

21: 77-88.

Ramirez, C, Ransom, J. P., and Pitkin, D. R. The in Vitro Interaction of Candida albicans

and Salmonella typhimurium. Microbiol. Esp. 21: 88-91.

Ramirez C. Antibacterial action of non-volatile substances extracted from Artemisia

tridentata Nutt. ssp. tridentata. Can. J. Microbiol. 15: 1341.

'Uamivez, C, Rodriguez, D., and Lanzuela, F. Rapid diagnosis of Candida albicans. Mycopath.

et Mycol. Appl. 40: 10?-111. Ramirez C, and Gonzalez, C. Candida iberlca sp nov. A new

U species of yeast Isolated from Spanish sausages* Can. J. Micro** biol. 18: 1778-1780.

3 ^ 1974 Si 1974 311976 3^1976.

j^ 1977. It (? 1978.

|i? 1978.

Ijl 1978.

Jj> 1978.

4j If 19 78.-^ 1978.-

4

4

b 1979.-

Martinez, A.T., and Ramirez, | Ecological importance of antibiosis in soil. Production of antibiotic

by species of Penicillium. Anales Edaf. Agrobiol 3s-159-166.

!T t 1980.-

Torres, J., Guarro, Suarez, G, Sufie , N., C^lvo, M.A.

and Ramirez, C. Morphological changes in strains of'Lper-

gilluB flavus Link ex Fries, and A. parasiticus SpeaTeT

related with aflatoxin production. Mycopathologa: 72*'

171-174.

Tl 1980

i'^> 1980.

| r 1980.

2T & 1980.

Calvo, M.A., Guarro, J., Su&rez, G., and Ramirez, C. Airborne Fungi in Barcelona City (Spain). I.

A two-year study (1976-1978). Mycopathologa. 71: 89-93. S'3 1980.- Calvo, M.A., Guarro, J. Suarez,

G., and Ramirez, C. Airborne Fungi in the air of Barcelona, Spain. V. Studies of the spore content

of air in dwellings. Ann. Allergy. 44; 228-234.

Ramirez, C, and Martinez. A.T., and Berenguer, J. Four ^ ^ new species of Penicillium isolated from

the air. Mycopathologa 72: 27-34. Ramirez, C., and Martinez, A.T. Some new species of Penici- ^

Ilium recovered from the atmosphere in Madrid and from

other substrata. Mycopathologa. 72: 181-191.

Guarro, J., Calvo, M.A., and Ramirez, C. Soil Ascomycetes

(Sphaeriales) from Catalonia, Spain. I. The genus Chaeto-mium Kunze ex Fresenius. Nova Hedwigia.

28: 191-216.

iT 7 1980.- Calvo, M.A. , Guarro, J., Suarez, G., and Ramirez, C. Airborne Fungi in the air of Barcelona

(Spain). 111= The ge-nusAspergillus Link ex Fries. Mycopathologa. 71: 41-43.

$

&H 1983.

if 1983.

U 1984.

61 1984.

xpensive method for>

Ramirez, C. A convenient and ine preparing fungal conidia for scanning

electron Mycopathologa 83: 17-18. m

Martinez, A.T. and Ramirez, C. Rhizosphaera oudemansii (Sphaeropsidales)

associated with needle cast of Spanish

Abies- pinsapo. Mycopathologa. 83: 175-182.

Ramirez, C, and Gonzalez, C. Penicillium flavido-stipj-tatum sp. nov., isolated from sand dunes at

the Arches National Monument, Utah, U.S.A. Mycopathologa 88:3-7

Ramirez, C, and Gonzalez, A. Rhodotorula grinbergsii I sp. nov. isolated from decayed wood in the

evergreen rainy Valdivian forest of southern Chile. Mycopathologa 88: 51-53.

i^ 1984. 1 1984.

1984.- Ramirez, C, and Gonzalez, A.. Two new species and one variety of nitrate-utilizing,

mycelial Candida isolated from decayed wood in the evergreen rainy Valdivian forest of

bfrSoutsMign Chilpa I M$j{fcop&thologa& 88 : 55-60.

3^

Ramirez, C., and Muntanola-Cvetovic, M. Penicillium ju-goslavicum sp. nov., isolated from sun

flower seeds (Helianthus annuus L.). Mycopathologa 88: 65-69 Ramirez, C., and Gonzalez, A.

Pichia scaptomyzae sp. nov. isolated from Scaptomyza multispinosa Malloch (Diptera) in southern

Chile. Mycopathologa. 88: 79-81.

If 1984.- Ramirez, C, and Gonzalez, A. Five new filamentous, glucose-fermenting Candida

isolated from decayed wood in the evergreen rainy Valdivian forest of southern Chile. Myco-

pathologa. 88: 83-92.

73 1984

71 1984.- Ramirez, C, and Gonzalez, A. Two new, amycelial Candida isolated from decayed wood

in the evergreen Valdivian forest of southern Chile. Mycopathologa. 88: 99-103. Ramirez, C. Validation

of Candida citrica sp. nov. (Furu-kuwa, Matsuyoshi, Minoda & Yamada). Mycopathologa. 86: 125.

7 4 1984.

Ramirez, C., and Gonzalez, A. Candida santjacobensist and

Candida ancudensis, two new species of yeasts isolated

from decayed wood in the evergreen rainy Valdivian forest

of southern Chile. Mycopathologa 88: 105-109.

Jf 1984.-

Ramirez, C, and Gonzalez, A. Two new filamentous, non

fermenting Candida and a new Schizoblastosporion isolated

isolated from decayed wood in the evergreen Valdivian

forest of southern Chile. Mycopathologa 88: 165-171.

li 1984.

Ramirez, C, and Gonzalez, A. Two new filamentous, fermenting ^

Candida isolated from decayed wood in the evergreen Valdivian

forest of southern Chile. Mycopathologa. 88: 185-189.

1984.- Ramirez, C, and Gonzalez, A

S, r*om dec of southern

Candida ous yeast anamorph

: "- isolated from decayed

Three new, non-fermenting and nitrate-negative Candida

1 / 184

wood. Antony van Leeuwenhoek.

wood in the evergreen rainy Valdivian forest Chile. Mycopatholo&ia 87: 175-180, Dill, Ingrid, Ramirez, C,

and Gonzalez, A.. lignophila sp. nov., a new basidiomycet from south Chilean rotting 50: 219-225.

1 i 1985.

C

Mycopathologi;

Ramirez, C, Dill, Ingrid, and Gonzalez, A. Emendation ^ Candida shehatae (Buckley & Van Uden). 89: 119-121.

$0 1985

Ramirez, C, and Gonzalez, A. Two new species of Apiotri-

CONGRESOS .wINTERNACIONALES

1953.- Ramirez, C. Introduction de una modificacin al metodo de

Wickerham para determinar el poder fermentativo de la rafino-sa por las levaduras. Estratto degli

Atti dei VI Congresso Internazionale di Microbiologa. ROMA. Sep. I: 872-873

1963.- Ramirez, C, Friis, J., and U. Leupold. Allelic recombination and complementation in

adenine-requiring mutants of Schizosaccharomyces pombe. Proceedings of the XI Internal Congress

of Genetics. THE HAGUE, The Netherlands. Sept. I: 7.

1973.- Gonzalez, C., Gutierrez, C., and Ramirez, C. Effet du pH, du NaCl et de la temperature sur

la synthese et sur l1action des esterases chez Halobacterium. Proceedings of the 1st International

Congress of Bacteriology. JERUSALEM. ISRAEL. Sept.

1974.- Ramirez, C., Gutierrez, C, and G onzalez C. , The G^enus Detail

ryomyces: Revision of the species includec in the genus under t the light of new criteria. IV

International Symposium on Yeasts. VIENNA, AUSTRIA. June. I: E-8

1974.- Ramirez, C., and Moriyon I. New sppcies osf yeast isolated from an acid washed brown soil.

IV International Symposium on Yeasts. VIENNA, AUSTRIA. July. I: E-5. 8-12 JULY.

1976.- Ramirez, C, and Guergue, J. M. Incidence of Aflatoxin potential contamination in Spanish

sausages. Ill International Symposium on Mycotoxins in Foodstuffs. PARIS, FRANCIA. 16-18 Sept.

I: 10.

1977.- Ramirez, C, and Martinez, A.T. A new species of yeast

isolated from beech forest soil. V. International Specialized Symposium on Yeasts. KEZTHELY,

HUNGRIA. Sept. 12-15.

1978.- Ramirez, C, Martinez, A. T., and Ferrer Soledad. Three

new species of Penicillium. XII INTERNATIONAL C0NGR OF MICROBIOLOGY. MUNICH, ALEMANIA FEDERAL.

3-8 Sep. J; C*Z1

1981.- (Rarxtibtteztf JCU;, and Martinet, A. T. Contribution to the

Preparation Techniques of conidia for scanning electron microscopy. IV INTERNATIONAL CONFERENCE

ON CULTURE

19-25. P

C. adn Ramirez, C. The THE HAGUE. 15-17 Sept.

COLLECTIONS. BRNO, CHECOSLOVAQUIA. July

1981.- Gonzalez, C., Gutierrez of indicators organism*

TIONAL SYMPOSIUM OF THE FEMS

siSnificane

I INTERNA-

1983.- Ramirez, C. amd Gonzalez, A. New species of yeasts isolated from Chilean "palo podrido"

in the rainy VaXdivian Forest IX INTERNATIONAL SPECIALIZED SYMPOSIUM ON YEASTS. SMOLEMICE

CHECOSLOVAQUIA. Abril 18-22. Abstracts: 25-26, -

CONGRESOS NACIONALES 9

1971.- Solana, A, , Mo ran. C Mnfin? r D' -

, XT.. , "Ulan, u, iviunoz, c, Ramirez, C, y Gonzalez, C. "Aislamiento e identificacion

de levadu-ras de muestras de salchichones curados", in CON GRESO NACIONAL DE

MICROBIOLOGA. Barcelona, 2-3 Junio. Resumenes. I; 15.

1971.- Munoz, C., Moran, C, Solana, A,, Gonzalez, C, y Ramirez, C. "Poblacin bacteriana

en distintas muestras de salchichones curados". Ill CONGRESO NACIONAL DE MICROBIOLOGA.

Barcelona, 2-3 Junio. Resumenes. I: 129.

1973.- R amirez, C., Echarri, L., y Sanz, J. "Aislamiento y clasificacion de la micoflora

de especias comer-ciales". IV CONGRESO NACIONAL DE MICROBIOLOGA. Granada. Septiembre.

Resumenes. I: 7

1973.- Sesma, B., Ramirez, C., y Rodriguez-Burgos, A. "Estudio de las diferentes

especies microbianas que condicionan la maduracion de algunos embuti-dos." IV CONGRESO

NACIONAL DE MICROBIOLOGA. Granada. 8 de Septiembre. Resumenes. I: 20.

1975.- Gutierrez, C, Gonzalez, C, y Ramirez, C. "Nuevas aportaciones a la fisiologa

y bioquimica de la fa-milia Halobacteriaceae. V CONGRESO NACIONAL DE MICROBIOLOGA.

1-4 Octubre. Salamanca. Resumenes. I: 54.

1975.- Martinez, A. T. y Ramirez, C, "Estudio de la Comu-nidad microfungica de un suelo

Andico. Conslflflera-ciones generales. Estudio taxonomico y ecologico del genero

Penicillium. V CONGRESO NACIONAL DE MICROBIOLOGA, Salamanca, 1-4 Octubre. ResumenesI:

C-69

1976.- Gutierrez, C, Gonzalez, C. y Ramirez, C. "Asimila-cion de hidratos de carbono

por bacteri&s halofilas estrictas valoradas por auxanogramas". Ill REUNION CIENTIFICA

DE LA SECCIN REGIONAL DEL NOROESTE DE LA SOCIEDAD ESPANOLA DE MICROBIOLOGA. Oviedo.

4-5 Junio. Resiimenes. I: 124.

1977 _ Martinez, A. T. y Ramirez, C. Importancia ecologica en los medios naturales del

fenomeno de la antibiosis: produccin de antibi6ticos por cepas de Peni~

AU t^ I ^A lAAA-''^ * fff iff;

UHL

un Andofuelo

1 argo *A,

Santia-

1977. -

iium. VI CONGRESO NACIONAT nt, . _____ go de Compostela. 6-9 j^8 MIC*BI0L0GIA. SanJ

Martinez, A.T. y Ramirez, C mMM

dad de los hongos microsc6picos en ** l* Com"**~

Variaciones a lo largo del perfil "" 7M~w,*,aei0' Wm aficrf, VI CONGRESO NACIONAL DE MICROBIOLO^*0 ^ go

de Compostela: 6-9 Julio.

1977. -

nov

gfierrez, C, Gonzalez, C. y Ramirez, I vi CONGRESO NACIONAL DE MICROBIOLoga. Santiago de Compostela 6-9 Julio.

"Halobacterium vallismorti SD

bacteria halofila extrema, asimiladora de hidratos de carbono". Resumenes. I: 111.

1979 Martinez, A.JT., y Ramirez, C. "Tipo de ornamenta cion superficial de los conidios de Penicilliunu"

VII CONGRESO NACIONAL DE MICROBIOLoga. Cadiz, 24 Septierobre al 1 de Octubre .

1984.- Gonzalez, A., Ramirez, C. y Grinbergs, J. "Palo po drido". Una transformacion biologica de la madera en alimento

animal, i II REUNION CONJUNTA DE MICO-LOGA. 31 Mayo-2 Junio, Bellaterra (Barcelona).

TESINAS DIRIGIDA3 Y PHmam^n^ ||

1) Aislaraiento y Clasificaci^n H I ~ il

,y ittbuxcacion de la flora contarolnante de espe

cias comerciales (Canela y plmienta negra), 1973. ECHARRI PRIM, LUIS. Universidad de

Navarra.

Estudio de la Micoflora de una tierra parda bajo robledal. 1973. MORIYON URIA, IGNACIO.

tfjiverslclad de Navarra.

Estudio de la Micoflora presente en un Andosuelo Espaol .

1973. MARTINEZ FERRER, ANGEL, T, Universidad de Navarra,

4) Estudio de la Micoflora presente en el piment6n picante,

1974. FERRER ORTEGA, MARIA SOLEDAD. Universidad Complutense

Madrid.

5) Aislamiento y clasificacion de mohos y levaduras aislados de

piraenton comercial.

1974. GOMEZ CEA, MANUEL. Universidad de Navarra.

6) Produccin de aflatoxinas por la micoflora contaminante de

especias comerciales.

1974. GUERGUE HUALDE, JUAN MANUEL. Universidad de Navarra.

7) Levaduras de la fermentacion de aceitunas: Negra de Aragon,

Ojiblanco, Manzanilla fina y Manzanilla Carrasquefia.

1975. RUBIO RABANERA, ROSARIO. Universidad de Navarra.

+ + + TESIS DIRIGIDAS

1) Contribucion al estudio de las levaduras de taninos.

1955. GENESTAR SERRA, RAFAEL. Universidad Complutense, Madrid

Contribucion al estudio de las levaduras de vinos de Jerez. 1962. ZAJARA LOPEZ, JUAN.

Facultad de Farmacia, Universidad de Sevilla.

Levaduras en la fermentacin de aceitunas verdes. 1965. GONZALEZ CANCHO, FERNANDO.

Facultad de Farmacia. Universidad de Sevilla.

Estudio de la comunidad microbiana durante la maduracion del salchichon elaborado en

Pamplona.

1975. SESMA VEA, BEGOftA. Universidad de Navarra.

r

/tV*/^- -*** S^A. f*^A\*~C4+

ah-

Xi^i /?6

sU

4L

CM. C*> *-H.

TESTS DIRIGIDAS (continuacin)

5) Estudio de la taxocenosis microfungica de una . ,,,,,, ,,.,

bajo robledal. ra P^rda

1976. MORIYON URIA, IGNACIO. Universidad de Navarra.

6). Estudio ecologico de la micocenosxs de un Andosuelo bale bosque de haya. 1976. MARTINEZ

FERRER, ANGEL TOMAS, Universidad de Navarra.

7) Contribucion al estudio de la micoflora atmosferica de la ciudad de Barcelona.

1978. CALVO TORRAS, MARIA DE LOS ANGELES. Facultad de Farmacia. Universidad de Barcelona.

8) Estudio taxonomico y toxicologico de la micoflora presente

en los almidones de uso farmaceutico.

1977. GUARRO ARTIGAS, JOSE. Facultad de Farmacia, Universidad de Barcelona.

9) . .

Micoflora contaminante y micotoxmas en especias comerciales.

1984. BERENGUER SOLER, JOAQUIN. Universidad Complutense, Madrid,

10) Flora micologica levaduriforme de las pudriciones de la madera

denominadas "palo podrido" de los bosques de la pluviselva

valdiviana del sur de Chile.

1984. GONZALEZ BERCERRA, ALDO ENRIQUE. Facultad de Ciencias Biologicas. Universidad

Autonoma de Madrid.

MC.

au &*&%*- ^vt-^

' I/A**? lf-*> gC-V **> H /&U. -U. f\-~'&'7

SL*-4A-

^L^M &^> ht *-k & %

&\^

CONSIDERACIONES ESTADISTICAS

sobre mi dedicacion exclusiva y total a i Tnwo tigacion durante el periodo de 15 aos (1968-iqa/n

Horas de trabajo obligatorias al afio: Horas de trabajo obligatorio

en 15 aos:

Horas que trabajo por ano:

Horas que he trabajado en 15 aos:

Horas extras por ano: Horas extras en 15 aos:

^Cuantos aos se necesitarian para traba-jar 51.960 horas de modo

normal?

Labor real en aos que he realizado hasta

ahora:

1.880 horas 28.200 1

5.344 i

80.160 u

3.464 u

51.960 t !

27 aos y 6 meses

42 aos.

80.160

2.8

Ritmo de trabajo que supone

28.200 Lo que supone un ritmo de casi tres veces el normal

2 7 aos y 6 meses.

Afios extras que he regalado al Consejo en 15 aos :

42 aos.

Afios oficiales de trabajo que fcubiesen sido necesarios para realizar la labor

efectuada en estos 15 aos:

2.010

;En que ano nos encontrariamos actualmente si toda esa labor hubiese sido hecha

de modo normal?

89 aos.

,Que edad tendria?

59 millones 400.000 pt

^Cuanto me deberia el Consejo por todas las horas extra en 15 aos?

Contents

Preface xi . . . . . . . . . . . . . . . . . . . . . . . xiiiAbbreviations of International Culture Collections

Part 1 General discussion Chapter 1

3Introduction 4Typification of the genus Penici/lium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cultural and morphological criteria employed in the delimitation and description of species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Morphology and terminology of conidiogenous structures . . . . . . . . . . 9 Chapter 2

Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Examination of cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Scanning electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Camera lucida drawings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Preservation of penicillia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Part 11 The manual proper Chapter 3 The Monoverticillata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Chapter 4 The Asymmetrica 201 Chapter 5 Asymmetrica-velutina sub-section 345 Chapter 6 Asymmetrica-Ianata sub-section . . . . . . . . . . . . . . . . . . . . 419 Chapter 7 Asymmetrica-funiculosa sub-section 437 Chapter 8 Asymmetrica-fasciculata sub-section . . . . . . . . . . . . . . . . . . . . 447 Chapter 9 The Biverticillata-symmetrica or Lanceolata . . . . . . . . . . . . . .. 507 Chapter 10 The Polyverticillata . . .. 609 Chapter 11 Addendum..... ......... .... ...... . . .. ... .. . .. . . .. ... 619

Chapter 1 Gonorol blbllogro phy .. , , . , . , . , , . , , , . , , , , . , .. , , , , . 631 Chapter 13 Ac copt od spoclos ond varlotlos , .. , , , . , , , 641 Chaptor 14 Species of dubious ident ity . 647 Chapter 15 Disposition of species accepted by Pitt (1979 b) . 649

Check Iist of synonymies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653

Index to the scanning electron micrographs of conidia of the different

Author index 861

Colour plates - explanation, legends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665 Colour piates 673 Alphabetic list of the colour plates of the Penlcllliurn species and varieties . 801 Scann ing electron micrographs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805

species and varieties of Penicillium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 845 Glossary 849

o.

Subject index 865o .

Preface

'1'111': PURPOSE OF THIS 'MANUAL AND ATLAS OF THE PENICILLIA ' IS TO SUMMARIZE

as cxha ustivcly as possibl e th e currc nt information regarding th e identification nnd characi erization of all rccognizabl e spec ies 01' Penicillium published to date, a lld lo supply the lat est tcchnical adva nces av ailable as additional criteria in lhe idcntifica tion of the speci es , in orde r to facilitate this difficult task 1'01' those who are not highly specialized in my cological taxonomy, applying new ap-proachcs whi ch rest fundamentally on the principIe that a good illustration is worth more than a thousand words . 1'0 ac hieve this ende avou r, the book is de-sig'lIcd to provide the following.

(a) Full colour plates , reproduced from photographic transparencies, of the cultural characte r istics of fun gal colonies grown under sta nda rd conditions , (hu s providing an immediate ex am ple whi ch may be a first approach in identi-licalion Ior th e less specialized , inter ested , workers .

(b) Line drawings of th e most rel evant mi croscopic features of eac h species of Penicillium , represented by standard scale camera lucida drawings , whi ch may be uscd as templates for comparison with the strain under study .

(c) Scanning electron mi crographs of mature con id ia showing th e fine detail of the surface ornamentation of th ese structu re s.

(el) K eys and full descriptions of th e type species for th e ultimate identifica-tion and characte rization of all recognizable Penicillium species e1escribeel up to date .

This book is directeel mainly towards my cologists , microbiologists, bio chemists, soil scientists and organic che m ists in higher education , industrial laboratories (pharmaceutical , chem ical industries and food industries) and govern me nt research laboratories; it will als o interest undergraduate and post-graduat e students at universities a nd polytechnic schools.

The a u thor is indebted to Doctors Angel T. Martinez, Angeles Calvo, a nd Jos Guarro for their colla bo ration in preparing th e Penicillium con id ia for scanning mi croscop y ; to th e Department of Electron Microscopy of the U niv ersity of Barcelona , Spain, and to th e Department of Electron Microscopy

Xll

of the Institute of Soil Sciences of the National Research Council of Spain, Madrid, Spain, for their generous cooperation in the preparation of the scanning electron micrographs.

Very special thanks are extended to Dr Shun-ichi Udagawa for his informa-tion on Japanese Penicillium species, to Professor Lorenzo Vilas for kindly translating many Russian papers, and finally to Dr Carmen C . Gonzalez for her encouragement.

CARLOS RAMIREZ Madrid, May 1980

Abbreviations of International Culture Collections

AT e c : Am erican T ype Culture Collection , Ro ckville , MD, USA. III>UN : Botany Department , University of Nottingham , UK. 111 (; : Botanisches Institut, Greifswald , GDR. III1IM : Botanisches Institut , H ann .-Mnden , FRG. IIKM : Department of Type Cultures, Institute of M icrobiology, USSR

Academ y of Scien ces, M oscow , U SSR. IIIU . : Butterwick R esearch Laboratories , Welwyn , H erts , UK . C IIS : Centraalbureau voor Schimme lcultures, Baarn , The Ne the r

lands. CC M : C zechoslova k Collection of Micro-organisms, J .E. Purkyn e

University , Brno , C zechoslovaki a.

rcr: Imperial C hernical Industries, Wclwyn , Hcrts, U K . 11'0 : lnst itut c or Fcrmcnt aj io n, Osa ka, .Japan. lB S : Institut d 'Hygicn c , Srra sbo urg , Fru nce , IJFM : Instituto 'Jaime Ferrn' de Mi crobiologa , M adrid , Spai n. IMI : see C M I.

IMRU : In stitute of Microbiology , Rutgers , The State U niversity, N ew Brunswick , NJ , U SA.

IM UR : In stituto de Micologia , Universid ad e , R ecife , Brazil. IC C: Centro de Biologia , In stituto G ulbe nkian de C iencia, Oeiras,

Portugal . IOC: Instituto O swaldo Cruz, Rio de Janeiro, Brazil . IP: In stitut Pasteu r , Paris, France . IPO : In stituut voo r Planten ziek tenkundig O nde rzoek, W ageningen ,

The Netherl ands . ISP : International Streptomyces Project. KNC

& SF : G ist-Brocades N .V ., Delft , T he Ne the rla nds. LC: Laboratoire de C ryptogam ie, Paris, F rance. LCF: Laboratorio Central de Fit op atologa , Buen os Aires, Arge ntina . LM: Laboratorium voor Microbi ologie , Delft, The Ne the rla nds. LP : M ycotheque du Lab oratoire de Parasitologie de la Fac ult de

M decin e , Paris, Fra nce. LS H: London School of H ygien e and Tropi cal M edi cin e , London ,

U K. MN HN: Musum National d 'Histoi re Naturelle, Paris, Fra nce. M W: In stitut f r Spezielle Botanik , Abteilung M ycologie , Weimar,

GDR . NC I B : National Collec tion of Industri al Bacteri a , Ab erdeen , Scotla nd,

U K. NCTC: National Collec tion of T yp es C ultu res, Lo ndo n, U K . NCYC: Nation al Collection of Yeast C ultures, Brewing Industry

R esearch Foundation , Nutfield , U K . NHL: Nat ional In stitute of H ygienic Scie nce, T okyo , J apa n . NR RL: C ulture Collec tion U ni t, Fermen tati on Section, No rthe rn U til

izat ion R esearch Branch , U .S. Dep . Agric., Peor ia 5, IL , USA. NYSDH: New York Sta te Dep. H ealth , Alba ny, NY , U SA , OEC D : Organisati on for Econo mic Cooperation and Development ,

Paris, France. PC : Lab or at oire de C ry ptogam ie, Paris , France. PO: Planten ziekt enkundige D ienst , W agen in gen , The Ne therlands. P RL : Prairie R egion al Labor at ory , Sas katoon, Canada . QM: Quar termaster R esearch and Engineering Command, Natick,

M A , U SA .

IO,A : Ran cho Sa u ra Ana Borunic G a rdcns , C larc mon t , C A, USA . 'I'llTC: Department 01' Botany , Uuivcrsity 01' Toronto , Toronto ,

O nta rio , Cunada . I IAMII : Univcrsity 01' Albcrta, Mould H erbarium and C ulture Collec-

tion , Edmonton , Albcrta, Canada . un: Dep . Food Scicncc and T echnology , Univ. of California , Davi s,

C A, USA . l1SSR ,

A lU : USSR Antibiotics R escar ch In stitute , Moscow , USSR . WIl : U niv crsity of Wisconsin, Dep artment of Bacteriology , M adison ,

WI , USA .

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Introduction

'I'III{OUGH THE COOPERATIVE EFFORT OF THE NATIONAL RESEARCH COUNCIL OF Spain (Consejo Superior de Investigaciones Cientficas), a comprehensive rcstudy of the genus Penicillium sensu stricto has been conducted by the author al the Laboratory of General and Applied Mycology of the Instituto 'Jaime Fcrrn' de Microbiologa, Spain, during the past five years . This has led to the preparation of this volume covering this very difficult, but important genus of moulds .

A great number of Penicillium species and strains were examined in culture, microscopically and under the scanning electron microscope (SEM) by the author, with the technical assistance in the last task of Drs A .T. Martnez, Mara de los Angeles Calvo and J . Guarro.

The type species were obtained from the Centraalbureau voor Schimmel-cultures, Baarn, The Netherlands, the American Type Culture Collection, the Commonwealth Mycological Institute, Kew, UK, the Institute for Fermenta-tion, Osaka, Japan, and from our own fungus collection (IJFM).

The members of the genus Penicillium have always been an important factor in man's environment , but prior to the development of the microscope, they wcre brushed aside as green, blue-green, ycllow, whitc or brown moulds without serious attempt at interpretation. Green moulds were everywhere and names for them appeared in evcry enumeration of the fungi of a particular locality, or the spccies cncountered in the study of decomposng or fermenting substanccs . Saccardo (1882) and Wehmer (1894) reported Penicillia active in the destruction of citrus fruits. Wehmer (1893) reported certain of them to be active producers of citric acid. Sopp (1898) related them to the che ese industry. The development of culture laboratories during this period made possible their isolation and examination in pure culture; subsequent physiological studies led to increasing interest in thcir presence and significance.

By the 1940s , the Penicillia were being studied intensively in many laboratories in the United States and other countries . Their potential for the industrial production of certain organic substances was already well established; they were being used more and more as research tools for inves-tigating physiological processes and metabolic pathways in microorganisms .

Moulds belonging to this genus are among the most ubiquitous of all fungi .

' I'hcy coustitu to ih c norm al ami an nhundu nt purt 01' (he mycollu rn 01' nll soils, th cy play acti ve mi es in lllallY dccomposition a nd dct criorutiou pmcesscs , th cy attack man and his dornesti c a n irna ls , and they are arnong the most cornmo n 01' a ll labora to ry co nta m inan ts . Bcginning in the lat e lBOOs so rne 01' thcm wer c found to produce interestin g metabolic products a nd tod ay a cons ide ra ble number 01' spec ies are used in industry 1'01' th e production 01' food specialties , organ ic ac ids, enzymes, and a n tibiotics . T hus, many people trained in differ en t di sciplines a nd working in very di ver se fields , mu st conce rn th emselves with spec ies 01' Penicillium. For th ese peopl e , few 01' wh om a re spec ia lists in mycological taxonomy, th ere must be av ail able a means 01' identifying th eir specime n th at is under stable and easy to use , an d whi ch at the sa me time does not viola te th e basi c principIes a nd objectives 01' sound bot anical nomenclature .

Typification of the genus Penicillium

The ge nus was created by Link in 1809 1'01' moul ds producin g brushlike spo ru la ting structures , three species being recognized and described in ve ry gene ra l terms ; P. candidum, P. expansum and P. glaucum. Corda ( 1837- 1854), M ontagn e ( 1856 ), and othe r m ycologists 01' th e subse que nt 50 years described add itional species but gene rally without definite informati on. Since th e habitat 01' P. expansum was given by Link primarily as rotten fruit, this spec ies may be recogni zed a nd it is no w generally acce pte d as the Penicillium spec ies ca using th e rot 01' apples . In 1809 Link conside red P. glaucum to be relat ed to P. expansum and lat er (Link 1824) , wh en redescribing P. glaucum, he listed P. expansum as a synonym 01' it. In th e me antime Gray ( 1821) validated Link ' s P. expansum and P. candidum (but not P. glaucum) and thus the correc t cita t ion 01' the fir st species is P. expansum Link ex Gray .

An ascospo ric species was not reported until 1874 wh en Brefeld published his det ail ed report on P. glaucum Link , whe re in he described a nd illu strat ed scleroti oid ascocarps . A few m ycologists introduced othe r ge ne r ic names 1'01' thi s stage . Ludwig ( 1892) was th e first to propose suc h a separa tion , introducing th e name Eupenicillium 1'01' the mould stud ied by Brefeld . This was followed by Langeron ( 1922) , wh o, appa re n tly unaware 01' th e existence 01' the name Eupenicillium and without referring to Ludwig' s publication , prop osed th e gene r ic name 01' Carpenteles 1'01' the same mould . Benj am in (1955 ) created the genus Talaromyces 1'01' th ose ascospo ric species 01' Penicillium whi ch comprise th e P. luteum ser ies 01' R ap er and Thom (1949) . M ore recently Stolk and Samson (1971) cre ated the genus Hamigera 1'01' those species 01' Talaromyces characte r ized by asci developing singly from croziers, a nd th ey redefined th e genus Talaromyces by restricting it to species which produce asc i in chains.

Thom ( 1910, 1930), Cl ement a nd Shear ( 193 1) a nd R aper a nd Thom ( 1949)

;)

typificd th c gcnus Penicillium Link, liut su ch typifi cation is not in accordan cc with the l nt crn utional C ode 01' Bolanical Nom cu clutu rc (Stnllcu 1972) . According lo Articlc 1:1 1', thc designat ion 01' P. ctustaceum Linnacus ex Fries as I ype must be foll ow ed, because Fries (1832) in the Systema Myeologieum vol. 3 , aeeepted Link's genus Penicillium, hut designat ed P. erustaeeum Linnaeus ex Fries , based on Mueor crustaccus L. (with the syno ny ms P. glaueum Link and P. expansum Link) as the typ e spec ies of the genus . Linnaeu s based his Mu eor crustaceus on M ieheli ' s (1729) 'Botrytis non ramosa, alba, seminibus rotundis ' and referred to plate 9 1, lig . 3 01' Mieheli's iUustrations . This figure possibly illustrat es a Botrytis-like er us tose fungus whi eh shows sim ila rities to P. exponsum . Hawksworth and Pitt ( 1976) therefore proposed th e eonserva tion of Penieillium Link as the leetotype . They designat ed a neotyp e IMI 39761 = CRS :125 .48 = Thorn 4852 of P. expansum, whi eh becomes the neot yp e of th e conse rve d ge nus .

No perfeet state is known in P. expansum nor in relat ed spee ies. The gene rie llame Penieillium is based on an imperfect fungus and eonseque ntly it represen ts a ge nus of the H yphorn ycetes.

H owever, R ap er (19 57) contes ted th e desirability of applyin g dual nom en d ature to PeniciUia , emphas izing the unity 01' all PeniciUia and insisting that the genus Penicillium sho uld indude both ascogeno us and anascogenous spec ies. Eve n tho ug h induding both perfeet and imperfect spec ies has its advantages, in accordance with the International Code 01' Botanical Nomendature (Stafleu 1972) (A rt. 59) th e genus Penicillium sho uld be res tric ted to th e anascospo ric species.

The ' M anual of the Penieillia ' (Rap er and Thom 1949), whi ch describes 99 accepted sp ecies and varieti es of Penieillium sen su str icto and list s almost 700 spec ies, stiU remains nowadays the sta ndard and indispens abl e guide to the taxon orn y of th e genus, despite a pleth ora of new species described since its publica tion . R aper and Thorns grasp of relation ships with in th e genus was ex traordinary and, by reducing more th an 700 species lo synon ym y, and forrning the remainder into series, they made it possible for erudite workers to be reason ably confide nt of most identification s. H owever, beeause 01' the paucity of obv ious taxonomic characte rs, and th e arbitrary nature 01' many 01' the M anual's gro upings, th e genus Penieillium stiU remains taxonomically difficul t , even 1'01' expe rts .

M any new , add itional, species and varieties have been deseribed in the literature, representing sorne 140 Peni ciUia, and various new surveys have been published (Abe 1956 ; Kulik 1968 ; Pidoplicko 1972 ; Samson et al. 1976 ; Fassatiov 1977 ; Pitt 1979b) in the yea rs sin ce the ' Man ual' was published , and so it is ob viou sly now out 01' dat e.

The acc u mula tion of all this m at eri al seemed suffieient to th e au thor to suppo rt a systematic restudy 01' aU PeniciUia. As th e present syste m 01'

classifying thesc fun gi is hascd primarily on cultu ra l a nd morphological characte ristics, th e aim 01' ou r 'Manual and atlas 01' thc Peni cillia ' has bcen to review the gcnus as presentIy dernarcated taxonomically, in orde r lo provide a basis for th e practical c1assification of those Penic.:illia that are onl y kn own by their imperfe ct state. *

Cultural a nd morphologi cal cr ite r ia em p loyed in the delimitation and des cription 01' species

As has been outlined by R aper and Thom (1949), the taxonomy ofthe genus Penicillium is based primarily on the cultu ra l and morphological characteristics 01' colonies grown on standard ized agar medi a . The applicability and adequacy 01' th ese principies are reflected partly by th e fact th at th ese author s were able to reduce the number 01' spec ies ass igned to thi s genus from more th an 700 to 99 well-defin ed and ea sily recogni zed taxa , and fu rthe r, by the fact th at their procedures have now been universally accepted 1'01' th e study 01' the taxon omy 01' th e Penicill ia .

Abe (1956) suggested th at the use 01' cultu ral and morphological characte risti cs shou ld be supplemented by th e study 01' bioch emical prop erties 01' th e cultures, e .g . th eir ability to produce antib iotics, th e solubility of pigme n ts, th e pH changes effected in liquid medi a , th e colour reacti on 01' cu ltu re filtrates with ferric chloride, the production 01' fluorescent subs tances, and pectinase ac tivity . While such informati on may con tribute to a better knowledge 01' th e physiology 01' these fungi , it is doubtful whether the us e 01' such criteria will be 01' sign ifica nce in the taxonomy 01' th ese moulds, particul arl y as many 01' th ese proposed b iochemical properties have been found to be dep endent on strain.

Accordingly , th e cultural and morphological criteria introduced by Thom (1930) a nd exte nded by R aper and Thom (1949) have been adopted with littl e modifi cati ons and cons iste ntly applied in the present taxonomical treatment 01' th e genus Penicillium.

C ULTURAL CHARAC TERISTICS

The colony 01' a Penicillium exhib its certa in typical and striking characte ristics. T hese inc1ude colour and colour changes; texture whi ch may be velvety when the stipes arise from the subs tratum, lanose when th e stipes 01' the conidio

Note . Shortly after complet ion of the prese nt study, the book "The Genus Penicillium and its T eleom orph ic Sta tes, Eupenicillium and Talaromyces' by John I. Pitt (1979b) was published . Three species of Penicillium, P. erythromellis Hockin g, P. dendriticum, and P. loliense Pitt were describ ed in it as new in the world. These have bee n inc1uded here in an Addendurn , where they a re described and illust rated applying the sta ndard methods used throug hout the present work. O n th e other hand , a nd as a matter of conve nience, no acco unt had been made here of Pitt' s new class ificat ion ofthe genus Penicillium. For further information , the book itsel f(Pi tt 1979b) should be consu lted .

phorcs arisc from simple ncrial hyphnc, [unlculose when the stipc arisc 1'1'0111 prostrat c Iascicles 01' acri al hyphae, uvfasciculatc whcn the stipcs ari sc 1'1'0111 crcct lusciclcs 01' aerial hyphae or syuncma ta . 111 severa] spccics thc stipcs 01' the coni. liophores are both mononcmatous and loos ely synnematous, as in P. expansum. In P. claviforme and in P. concentricum the synnemata are differentiated into a d istin et stalk and an enlarged head composed of the sporulating structures . Another typical characteristic is the habit, which may be restricted to broadly spread ing, and range from plane to deeply furrowed 01' wrinkled . The suhstratu rn may 01' may not be dis coloured . Conspicuous drops 01' transpired lluid are charac te r istic of sorn e spec ies but not 01' othe rs . Odours may 01' may 1101 be produced, and if present may be charac te ristic 01' just 'mouldy ' .

Although th e gen eral appearance 01' su ch colonies is influen ced by the n-lative abundance 01' conidial structures and sclerotia , whi ch may vary from srrain to strain , th e cultu ral charac teristics 01' a species seem to depend on the use 01' a particular substratum .

Colon ies 01' most species and strains grow well on th e usual Czapek' s solu tion agar. Sorne species, however, are charac ter ized by a ver y pOOl' dcvelopment on this medium. In most cases su ch restricted growth may be attributed to th e in ability 01' th ese strains to synthe size the vit amins necessary or to utili ze nitrate as a source 01' nitrogen . On th e othe r hand , malt-extract agar usuall y suppo rts good growth . The composition 01' the medium has a marked effec t on the texture and

surface charac te r 01' colonies. On Czapek ' s agar medium the colonies are usuall y clos e textured , compa ratively thi ck and sometimes va r ious ly furrowed 01' wrinkled . On malt-extract ag ar medium th e co!onies are u su ally loose textured , comparatively thin and plane, a nd sometimes appear zanat e .

COLOUR OF CONID IA L AR EAS

Colony colour other th an shades 01' blue-green as well as colour changes are ver y us eful in the identification 01' strains and character iza tion 01' species.

Every description 0 1' Penicillium emphasize s sorn e colour 0 1' se ries 0 1' colours as charac te r ist ic 0 1' con id ial areas. When using several cultu re medi a , it is necessary to specify th e colou rs upon each by name, 0 1' numerical references to sorne al' th e recognized colou r standards. Ridgway 's 'Colour St andards and Nomenclature ' (191 2) has been used by Raper and Thom (1949) . Much of the European lit erature referred to Klincksieck and Valette ' s 'Code des Couleurs ' (1908) . Sgu y ' s 'Code Universel des Couleurs ' (1936) was published in Fr an ce . M ore recently , R ayn er (1970) published his 'Mycolog ical Colour Chart'. Reference to such manuals will undoubtedl y become necessary in th e future since both Ridgway 's , and Klincksi eck and Valette 's works are out al' print and consequently no longer eas ily available .

Colou r records show cons ide rable variations 1'01' particul ar strains as

int e rp ret ed by dilTe renl individunls , 01' eve n by th e sa me person working a t diff c rcnt times . For thi s rcason cita tion of co lours is nevc r a n absolu to gu ide. In gene ral, however, th e colours produced by a given strain upon a sta ndard ized subs tratum under standard cond itions, fall co nsisten tly within a narrow range of shades . H ence, th e cu ltu re medium must be spec ified if the co lours cited a re to have real value . O n the other hand , colou r photographic records 01' colonie s represent an excellen t means 1'01' di scarding personal interpret ation 0 1' colou rs and have been used by the au thor throughout the present study. The ae r ial mycel ium 0 1' th ese fun gi is gene rally colourless. Areas 01' yellow

and red hyphae commonly appe ar in th e one -stage branched Penicillia 0 1' R ap er and Thom' s ( 1949) Biver ticillat a -symmetrica sec tio n. In th is case th e colou ring substance is largel y deposited as granules on th e surface 0 1' the cell rathe r th an with in th e cell itself.

Submerged mycellium , on th e othe r hand , may show di stincti ve colours which are best apprec ia ted from below and a re routinc1y desi gnated as colour 0 1' reverse. Di rty white 01' dull buffs are sometimes produced , but th e commonest colours are var ious shades 0 1' ye llow, orange, red 0 1' purple . In sorn e cases th e pigment produced may diffuse into the surrou nding agar medium. T he di stincti ve and bright pigments a re parti cul arly ev ide nt in fresh isolat es.

E X UDA T E AND ODOUR

Dist incti vc odours and transpi ration fluids in th e colon ies may sometimes be 0 1' va lue as a means of identification . H owever , th ese characteristics a re 01'te n va riable and sorne isolat es tend to loose th eir odo u r and th eir ab ility to produce exudate on continued cultiva tion.

COLONY MARGIN

In a favourable subst ratum th e colonies are usu ally approximately circula r, a lthough marginal areas may be characte r istically even, ste lla te, 01' crenated .

Z ONATIO N

Surface growth ca n be observe d in many species. Sorne 0 1' th em are zonate when grown upon one subs tratum and not upon othe rs. Ullscheck (1928), in an a tte mpt to expla in th is phe nomeno n, stated th at colon ies growing rapidly secre te by produc ts 0 1' th eir me tabo lism in concentrations su fficie nt to suppress 0 1' reduce growth . The mycelium then advances into fresh medium , resuming rapid growth and producin g abundant fruit in g structures, only to be depressed again by th e excessive byp roducts 01' its renewcd ac tivity .

VAIUATION S ANI> M UTAT ION S T ite Peni cillia represc nt a variabl e gro llp 01' moulds. Sectorial m utation s,

loss (JI' ahi lity lo produce spores and t he rnany variati on s which often acc u r, nlles l lo the cha ngability 01' th cse fun gi . T hese variations may devel ap as a rcsu lt 01' progressive cha nges under cantinued labaratary cultivatian. When I he change cornrna nly rernains stablc thra ugh subscque nt subcultures it may he rcgarded as a true mutati on. When the change continues to sho w fur th er chunges in th e same, 01' in sorne other directi on , it is usually regarded as a vur iai ion .

Mutants characte rized by colou r cha nges or striking nutriti on al deficien cies ;11'(: sorne times isolated from nature . Several strains have been observed to dcvclop in a manner typi cal of well- recognized spec ies upon an enriched mcdiurn suc h as malt extract aga r, but to grow sparse ly and very atypically Il!l0n Czape k's agar medium .

Areas of sterile , or esse nt ially sterile overg rowth m ay a t times occur in o rhc rwise typi cal str a ins of almost any species of Penicillium. This overgrowth ra n be isolat ed and cult iva ted separately and th en exhibits littl e if any of the r ha racreristics of the par ental strain.

Morphology and terminology of the conidiogenous structures

T he developmen tal seque nce and morphological features of th e fru iting structu res are of prime impartance in th e identificati on of fungi. Dist inction and recagnition of species within th e genus Penicillium is based on th e study of conidial st ructu res .

T he terms used for the description of the eleme nts of the Penicillia are ada pted to th e modern terminology as esta blishe d at K ananaskis in Canada ( Kcndrick 1971 ) . The term conidiophore is used for th e en tire system af conidiogenous cells, together with the differentiated suppo rting struc tures. T he tcrrn can idiophore as used by R ap er and Thom (1949) is repl aced by stipe.

' r~1E STIPE Import ant fea tu res of the st ipes include th eir len gth , septa tion, di ameter and

or igin in relati on to the subs tratum and to each othe r. Althou gh va ria tion in stip e len gth usu ally occu rs in mo st cu ltures, th e majority of stipes are of a defin ite ave rage len gth which is characte ristic of the spec ies . They are often comparatively short, less th an 100 tut , espec ially whe n th ey arise from ae rial hyphae and bea r simple penicilli . When they arise di rectly from th e basal felt 01' subs tra tum they are usu ally longer but seldom exceed 250 p.m in len gth . When long , th ey are sometimes branched and ofte n beal' one- or two-stage branched penicilli . T he wa lls may be smooth and th in or m ay be various ly rou ghen ed , appea ring deli cately echinulate , gra nula r or asperula te. In sorne

10

cases, suc h as P. roqueforti , the wall s may be marked by conspicuo us conc re tions or warts . D iffer ences in th e st ipe wall are co ns iste n t in certain spec ies. In othe r species th is character m ay va ry markedly with th e culture medium . Separation of cons ide rable usefulness, however , ca n still be based upon thi s character since cer ta in spec ies appea r to be ca pable of develop in g rou ghness under all or ce rta in spec ific conditions, whe reas the stipes of other spec ies remain smooth under all conditio ns. Stipes are usu ally uniform in di ameter , but in sorne simple forms, R aper and Thom 's ( 1949) M onoverticillat a sec tion, th e apices are conspicuous ly swollen or infl at ed .

The stipe m ay be sim ple and unbranch ed or var ious ly branc hed. It m ay ar ise frorn vegeta tive hyphae within th e subs tratu m or fro m aerial vegetative hyphae va r ious ly branch ed . It may sta nd alone, or be more or less closely aggregated in to clus ter, fascicles , or definit e synne mata .

TH E P ENI CI L LUS

The branch ed part of th e conid iophore a bo ve th e stipe is refer red to as th e penicillus. An elabo rate description of all elements comprising the penicillus has been give n by R aper and Thom ( 1949). O nly th e sali ent features of th ese structu res that a re observed in spec ies of Penicillium a re di scu ssed here .

The typ e of penicillus characte r istic to th e spec ies can best be det ermined by studying preparations wh ich m ak e possibl e the rapid com parison of large numbers offruiting structu res . This can best be don e by making habit ske tches and drawings of conid ia l structures with th e a id of th e camera lucid a, at standard scale, sho wing th e relative dim ensions of penicilli , th e typ e of branching observed, and the arrangement of the conid ia l cha ins.

In describing species, or in the ide ntificat ion of unknown strains, the typ e of penicillus most commonly produced must be det ermin ed , since allocatio n to major sec tio ns is mad e upon thi s basis .

There is for eve ry species a gene ral typ e , number , ar rangement and size of elements which ca n be described in fairly tangibl e te rms. Camera lu cid a drawin gs sho w in de ta il the pattern and cellu lar str uc tures th at are regarde d as characte r istic of a parti cul a r species . The wri tten description , how ever , sho uld be broad enough to include a range of patterns and dimension s th at occu r.

TH E PHIALIDE

T he term phialide (V u illernin 1910) ac tually replaces th e term sterigmata incorrectIy a pplied by R aper and Thom (1949) to the terminal cells of the penicillus which bear the con id ia (see Cole and Kendrick 1969 ). It m ay be de-scr ibe d as a transformed a nd highly differentiated cell with a more or Iess cylind r ical body of definite Iength and diameter which is charac te r ist ica lly

lO. 1.1. A. Typica l simp le ponlclllus, os soen in P. lnutuontnns Westling,

nolstlng of a terminal vurtlcll of phialides only. B,

vplcul romig enous penlclllus, as seen in P. hnrlosl! Smith, cons ist ing of

pro otrote or ascending tipos, orising from the ubutratum or from creep ing

noriol hyphae, typically rumif ied, w ith rami either A e single celled (metula -like) or long and septate, usually unequal in length, simple or rebranched, not producing defi nite apical verticils of metulae or ramu li, w ith penicilli typ ically simple, borne terminally on the apices of the ramif ied st ipes. e, Typical divaricate penicillus, as seen in P. jenthine um Biourge, consist ing of a terminal group of irregularly arranged E rami and /or metulae bearing clust ers of phialides (one- o stage branched) . O, Typical branched) . E, Typical which bears a symmetrical biverticillate-asymmetric biverticillate-symmetric verti cil 01 closelv packed, ponicillus, as seen in P. penicillus, as seen in typically lanceolate phialides. expsnsum Link ex Gray Penici//ium variabile Sopp, (a =con idia; b =phialides; emand. Thom, showing consisting of a stipe c =metulae; d =rami; metulae and rami below the terminating in a simple e =st ipe of the phialides (two-staqe vert icil of metulae, each 01 conidiophore .)

Latin ampulla = flask) of the type show n in fig: 1.1 A and B. The one -stage branchcd forms, R ap er and Thom's (1949) Biverticilla ta - symmetrica sect ion, p roduce phi alides that are us ually acerose (from the Latin acerosus = needle-shaped) like leaves of Pinus , as shown in fig: 1. 1 E . A few species produce almost cylindrica l phi alides, lacking a definite terminal narrowing (see R ap er an d T hom's ' M anual of the Pen icill ia ' (1949) , page 49 , Fig. 12 A l and A2) .

T he phialide neck (the conid ium -bearing tube of R aper and Thom 1949) was tcr med collulum by Za leski (1927) , (from the Latin collulum = little neck) , it is usually long in sorne spec ies (see R aper and T hom's Manual 1949, page 47, Fig. 11 B) or short, and is also of taxon omical im portance .

lO. 1.1. A, Typ ical simple nlclllus, 8S 900n in P. quontnns Westling , 110lBtlno of a term inal

vortlcll of phialides only . B, vplcul rarnlqenous

ponlclllus , as seen in P. //IIrlus;; Smith, consist ing of prontrate or ascend ing tlpos, arising from the ubntratum or from creeping oriol hypha e, typically

Arurnlfiod, with rami either e single celled (metu la-Iike) or long and septate , usually unequal in length, simple or rebranched, not producing definite apical vert icils of metulae or ramuli , with penicilli typ ically simple , born e terminally on the apices of the ramified stipes . e, Typ ical divaricate penicillus, as seen in P. jenthinellum Biou rge, cons isting of a terminal

Egroup of irregularly arranged rami and /or metulae bearing oc1usters of phialides (one-stage branched) . O, Typ ical branched). E, Typical which bears a symme trical biverticillate -asymm etric biverticillate-symm etric vert icil of closelv packed, penicillus . as seen in P. penicillus, as seen in typi cally lanceolate phialides. expensum Link ex Gray Penicillium variabile Sopp, (a == conidia; b == phialides; emend . Thom, showing cons isting of a stipe c == metulae ; d == rami; metulae and rami below the term inating in a simple e == stipe of the phialides (two-staqe verti cil of metulae , each 01 con idiophore.)

Latin ampulla == flask) of the type show n in fig: 1.1 A and B. T he one-s tage bran ched forms, R aper and Thorns (1949) Bivert icilla ta- sym metr ica section, p rodu ce ph ialides that are us ually acerose (from the Latin acerosus == needle-shaped) like leaves of Pinus, as shown in fig: 1. 1 E. A few species produce almost cylindrical phialides, lacking a definite terminal narrowing (see R aper and T ho m's ' Manual of the Pe nic illia' (1949), page 49, Fig. 12 A l and A2) .

T he phialide neck (the conidium-bearing tube of R aper and T hom 1949) was tcr med collulum by Zaleski (1927) , (from the Lat in collulum == little neck), it is usually long in sorne species (see R aper and Thom 's Manual 1949, page 47 , Fig. 11 B) or sho rt, an d is also of taxono mical impor tance.

M I(TUJ.Al-: Thc ICI'IIl metula (We sllillg 1911) is maint uincd fill' (he pcnuh imutc hrnn ch

which bca rs the whorl 01' phialides . Thcy are charac te r ist icnlly borne in a te rminal vcrticil on the st ipc, or on it and cm one 0 1' more branch cs . Whenever the wa lls a l' th e stipe a re smooth, those of the metulae are also cons iste n tly smooth. W he n the walls 01' th e st ipe are pi tted or ro ug h, the walls 01' th e metulae may or may not be sim ilarly ro ughened .

BRA NCH E S

AII cells be tween metulae and stipes are refer red to as branc hes . 1'0 indicat e the type 01' branching, th e terms proposed by R aper and Thom ( 1949) are replaced by the te rms simple (not branch ed = monoverticill at e) , one-stage branched (b ive rticilla te, as in P. citrinum or species 01' th e Biverticillat a-sym metrica sec tion), two-stage branched ( = Biverti cill at a - symmetri ca , as in P. italicum W chmer, var. italicum Sa mson, Stolk et H adl ok 0 1' P. expansum), or three or more stage branched, as in P. griseoJulvum.

CON ID IA

T he con id ia are cndogenous and unicellular. Eve ry conidiu m ar ises th eoreti call y as a cylind rical bod y within and extrud ing from the phialide neck , as in m any other fungi with phialospores. The shape, size and sculp turing 01' conidia are ve ry import an t characteristics a l' species. Conid ia may be globose, ovoid or ellipsoidal, smooth or various ly ro ughe ne d and com parativc ly small or large , but usually not exce ed ing 4 .5- 5.0 uit: in diameter. Singly th ey appe a r hyaline under th e light mi croscope , but in a m ass they show various shades al' blue- green , yellow or brown . The conid ia are produced in parall el or di vergent chains . Bccause the phialides are m ostly ar ra nged parallel in th e sim ple pen icilli (monovertic illate) , th e conid ia l chains are 01'ten arranged in com pact to ill-defined colum ns. When pe n icilli a re di varicate or asy mmetr ica l, th e conidia l chains are usu all y tangled or divergent. In ce rtain spec ies colu m nar or tangled masses al' con id ia form cont inuous crusts covering large areas al' the colony. In any case th e general ar ra ngement al' conidial chains in the mass is u suall y recognizable in successive cultures a l' each species under study.

SCLEROTIA

Typically th ese cons ist 01' very hard masses a l' pse udo pare nc hy mato us tissue in which cell walls are very thick. Among th e sim ple Penicilli a, R ap er and Thom 's Monoverticillata section , th e production a l' ellip tica l to glob ose sclerotia is regularly characte r istic al' members a l' th e P. thomii series. In P. turbatum al' th e same series , small, soft m asses 01' thi ck-walled cells are usually encou nte red . These h ardly re present true sclerotia in the sense al' P. thomii and P. raistrickii, but ac tually th eir presence is st ill accepted as evi de nce al' a

1'('1111 ionship lo thcse spccic, hcncc thcir assi f.{IWI ion to theru. AItIIlJIlf.{h IIIt: naturc 01' th csc stI'IICtllJ'(;S is still ohscurc, th cir cxtrao rclina ry

rmH;l lIh la llcc lo 11'111.: nscomnta , in addit ion lo siudics ca rricd out by Scolt I tll)lI) , whi ch lcd lo thc discovcry 01' the pcrf cct Iorms 01' P. lapidosum Raper el

1;'(;lIl1dl, IJ, ochrosalmoneum Udagawa, P, hyrayamae Udagawa , and P. cinnamo-1)/1111/11'1'11111 Abe I!X Udagawa , prcviously known and described as forming only clcrotiu, indicntcd lo us that sclerotia may be regarded m erely as abortive \lll'llllla la , owinf.{ to a gendic block. Furthermore, to con firm our personal pedlll 01' vicw, the oppositc 01' Scott's findings , namely, a strain of previously dl's('J'il>cd perf cct form , producing abortive ascomata has recently been 111 1IH:l'vcd by the author of the present study. Hamigera avellanea Stolk et Samson, MIl'a ill I.JFM 7239 , isolated in Barcelona by DI' A . Calvo in April 1980, and Me' llt lo liS Ior idcntifi cation , was unable to produce as ci, and ye t produced the I ypica l nct work of yellow-encrusted hyphae which forms the envelope of the lIHl' llllla ta 01' this specics (appearing as sclerotium-like structurcs). With no d lllll>l , th aboye strain suffcrs a genetic block at th e pathway leading to the lill'llIalion 01' asci and as cospores. Thus, it will probably be a matler of time uutil sonicone reports as comata for P. thomii al' P, raistrickii, to m ention only I WII 01' thc best kn own sclerotium-producing species .

'haptcr 2

M aterial and methods

A I'i\Ri\MO UNT REQlJIREMENT OF DET ERMINATlVE TAXONOMIC METHODS IS T I-li\ T I hcy provide the maximum a mou nt of information from the fewest possible lelll s in the minimum time. First must come the cho ice ofmedia: Thom (1910) 1I 1led potato agar and bean agar; W estling (1911) used prune cxtract gclatine; ll iulIrge (1923) a nd Zaleski (1927) grew their cu ltures on Raulin' s solu tion plus cln t ine 0 1' agar. Raper and Thom (1949) em ployed three standard media:

( :zlIpck ' s agar , stee p agar , and malt-extract agar. In more recent studies stccp If.l lll' has sometimes been omitted because it is not widely ava ilable . Abe (1956)

IIIH:d , in ad d it ion , a Czapek's agar medium with nitrite subst itu ted for nitratc. 111 Il llld ies on Eupenicillium , Stolk and Scott (1967) added descriptions of gr owth 1111 co ru -meal ag ar. Pitt (1973) recommended that the three basic media uscd IIY Rnpcr a nd Thom (1949) for prep a ring co lonia l a nd morphologieal dl'llcl'ipli ons be reduced to two : Czap ek ' s yeas t-au tolysate agar (CYA) and ur ult -cxtract aga r (MEA) . In th e present work we adopted th e foIlowing threc Imll ic media , wh ose formula a re given below: Czapek's agar (CA), CYA and M EA. AIl species curre n tly described have been grown on th ese three standard cu l tu re media. AH th e forms grow well at room temperature (20 -25C) with d illusc d aylight. Hundreds of Penicillium strains , including th e typ e stra ins ohruined from the most important internation al cultu re coIlections, have becn xudi cd a nd com pared. The cultu res cite d in th e text by number, bearing the prcfix IJ FM , are accessioned with our own fungus culture coIlect ion .

S T i\ NDA RD IZE D CULTU RE MEDIA

Czapek '5 agar medium has the foIlow ing co m posit ion (Rape r and Thom 1949) :

3 .0 gNaN03 . K 2H P0 4 . 1.0 g M gS04 7H 20 . 0 .5 g KCl . 0. 5 g FeS04. 7H20 . 0 .01 g Su crose . 30 .0 g Agar (Difeo) . 20 .0 s Di still ed wat er . up to 1000 m

Tite pH is not udj ustcd and tite medium is autoc1aved la r 15 min at 121C (15 lb overprcssurc).

Preparation time Ior media bascd on Ozapek's solution muy be reduced by preparing the min eral ingrediente oth cr than ph osphate as 100-fold stock solution, and adding it to the medium at the rate of 10 mI per 890 mI of water used .

Czapek's eoneentrate has the fol1owing com posit ion per 1000 mI of solution:

300.0 gNaN03 . MgS04 7H20 . 50 .0 g KCI . 50 .0 g FeS04 ' 7H20 . 1.0 g

This stock solution will keep ind efinite1y ; the slight precipitate of ferric hydroxide does not affect the final medium.

Czapekyeast-extraet agar has the fol1owing composition :

1.0 gK 2H P0 4 Czapek's conce ntrate . 10.0 mI Yeast extract (Difeo) . 5 .0 g Sucrose . 30. 0 g Agar (Difeo) . 20 .0 g Distil1ed water . up to 1000 mI

To avoid precipitation of phosphates and carame1ization of the sugar, the potassium phosphate is dissolved separate1y in 100 mI of water and autoclaved for 15 min at 121"C (15 lb overpressure). The othe r constituents are dissolved in 890 mI distil1ed wat er and 10 mI Czapek 's concentrate are added to thi s solution . The solution is autoclaved for the same time at the same temperature. After sterilization the two portions are mixed asept ical1y, while stil1 hot.

Malt extract agar has the fol1owing com position (Raper and Thom 1949) : Malt extract (Difco) . . . . . . . . . . . . . . . . . . . . . . . . . . .. 20 .O g Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.0 g Bacto-peptone (Difco) . . . . . . . . . . . . . . . . . . . . . . . . . . 1.O g Agar (Difco) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 .0 g Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. up to 1000 mI

The pH is not adj us ted and the medium is autoclaved for 15 min a t 121C (15 lb overpressure) .

Media other than C zapek ' s agar were used on slide cultu res in certa in instances when conid ia l development on this medium wa s poor or absent: CYA, MEA and corn-meal agar. In such instances , whenever they were u sed , they are indicated in the tex t.

'om-meal agar (Di fco), T o rchydrat c thc mcdium , su spen d 17 g 01' Bacto -rurn-mca l agar (Difco) in 1000 mi 01' co ld d isti lled wat er and heat to boiling to d l"Plolve the mcdium com plc tcly. Distribute in tubes and autoclave for 15 min 1I I ~ I oC or 15 lb overpressure. Thc final pH of the medium wil1 be 6 .0 and is nol udjus ted .

lU I.'I' U RE GROWTH AND INOCULATION MEDIA

C ultures were routinely grown on MEA. Erratic results occasional1y f( ,lIowed the use of old cultures as inocula. It is recommended that cultures

q~ e d less than 3 weeks be used . Good results were obtained using sta ndard, glass, lO-cm Petri dishes

l'o llla in ing a pproxim ately 20 mi of medium. Three inoculum points on each 1'IIIIe provid ed replication . As Penicillium conid ia are hydrophobic , sm all nucu la were mo st sa tisfac torily applied as diluted suspensions in a 0 .01 % iq ucous solution 01' Tween 80 by means 01' a gla ss ball-pointed pipette (fig .

O , I C), which has previously been ste r ilized by immersion in 95 % alcoho l and 11 11 :11 l1amed . The number of conidia in the inoculum appa re ntly has little effect 1111 the res ults.

I':xamination of cultures

1rcscriptions 01' colon ial characte ris tics 01' th e va rio us species studied were I llad e from three-spot inocul at ed medi a in Petri di shes. The media rou tine ly used were C A, CYA, and MEA.

For micromorphological studies slide preparations were made using an IlI" e OU S solu tio n 01' 0.01 % Tween 80 as the mounting fluid. Small fra gments

of a 7-day-old colony were torn up into ve ry small pieces with ve ry fine d issecting needl es (insect pins fixed on wooden handles will do perfectly) , suspended in the abo ye mounting fluid in test tubes a nd shake n for 2-3 min in u vortex mi xer a t hi gh speed in orde r to wash ou t th e clou d 01' conid ia, which othcrwise wo uld obscure the m icroscopi c observat ion 01' th e penicilli.

I NC UBATI O N TIM E

1"01' colony m easu rement to be 01' most value , a standard incubation tim e is r-sscntia l. The author has chosen 14 days as standard for colony measu rement nnd colour pho tographs . H owever, because penicilli di sintegrat e with age, rc nde ring m icroscopi c exam ination diffi cult , a sta ndard in cu bation period that is as sho rt as possible is highl y desirable for th at purpose. 7 days a t 25 C is cons ide red optimal for slid e cultu res.

S UDE CULTU RES

Con id ial structures were exami ne d gene rally on slide cultures al'ter 7 days incubat ion a t 25C , while th e fu ngus is in an ac tive state 01' growt h . T he

--

Fig. 2.1 . Equipment. A, Reichter 'Neopan' microscope (Austria) provided with a drawing device or camera lucida Reichter, Model 21,391 . B, Low power 'Kyowa' binocular (Japan), with zoom lens, extremely useful for making preliminary observations of colony characteristics. C, 1 mi inoculation pipette with a closed and bent ball-pointed end.

Fig. 2.2 . Equipment. A, An 'exploded' view of Cole and Kendrick 's culture chamber, showing the drilled and slotted slide, the agar medium, and the coverslips wh ich seal the upper and lower surfaces. B, View of the Reiss microchamber, showing: (a) the slide, (b) strip of adhesive paper , (e) side parts of the chamber, (d) the coverslip, and (e) the inoculum. C, An 'exploded' view of the drawing device: (1), Camera lucida . (2), Fixing screw to affix the camera lucida to the microscope. (3), Ad justing ring for focusing . (4), Fixing screw, to affix the camera lucida eye-piece. (5), Camera lucida prism's casing. (6), Screen for cutting off the path of light through the camera lucida 's prism when the apparatus is not in use. D, Schematic diagram showing the basis of the camera lucida theory.

medium gcnernlly cmploycd was CA . Iowever, when couidiul dcvelopmcnt on thi s substra tum was (mor , OYA, M EA , 01 ' corn mcal agur wcre uscd instcad .

C U LT URE CHAM BERS

Other techniques may be used 1'01' th e study 01' organ ization and ontoge ny 01' ae rial struc tu res . M any 01' them have been employed 1'01' thi s study . In order to ensu re aeri al growth, th e fun gu s is usually grown on slides on a thin film 01' an appropriate substratum. The conidiophores will grow off the sides 01' the medium in to the surrou nding a ir and ca n thus be examined (see Vernon 1931 ; Riddell 1950 ; Anton y and Walkes 1962 ; C ochrane 1963; Zobe ri 1967) . Cole and Kendrick (1968) described a small cult ure chamber 01' glass (fig. 2.2 , A) which allows cultiva tion 01' th e fungus in such a m anner that the developmen t 01' fu ngal struc tures can be continuo usly observe d and even filmed under high magnifi cat ion . The constructio n 01' such a chambe r, however, requires great per sonal skil!. Conside ring that many conidiopho res are gene ra lly negati vely geo tropic

(Cochran 1963), an eas ily producibl e and inexp en sive culture cham ber was described by R eiss (1970) (see fig . 2.2, B) , which allows the study 01' the development 01' reproduct ive fungal struc tures in an undisturbed position. Such a chamber has been used in our lab oratory with success . On a clean slide, two small str ips 01' glass (the thi ckness 01' a slide) are glued with nontoxic mucilage (i.e . U.H.U from Fisher, B hl , FRG) at a dis ta nce 01' 14 mm from each othe r. This frame is ste rilized . A ste rile coverslip (18 x 18 mm) is placed ayer the gap between the two glass strips and fixed to them with two str ips 01' adhesive tap e . A small piece 01' a Petri di sh cultu re 01' th e fungu s is removed under ste rile conditio ns and placed on the slide in fro nt 01' the spa ce between the cover and the slide . With a small and sterile str ip 01' glass thi s inoculum is pushed between the slide and the cove rslip . One side 01' th e open chambe r is closed with the strip 01' glass, the othe r remaining open to ens ure a sufficient supply 01' a ir.

T o enable negative geo tropic growth 01' con id iophores, th e inocul at ed chambe r is put in a vertical posit ion into a ste rile tr ou gh 1'01' slides , th e bot tom and side s 01' whi ch are lined with sterile wetted filter papel'. U nde r these cond itions rep roductive structures can grow upright int o th e air. The tr ou gh is incubat ed at an appro pria te temperature (20 - 25C) under diffuse light. Al'ter varyi ng periods the chambe r is removed and examined under the m icro scope . When the microscop ical study has been fini shed , alco hol is dropped into th e chambe r , th e coverslip is removed , cleaned and sterili zed , together with the lower part of the chamber. U se ofthe stro ng illuminati on necessary 1'01' making microphot ogr aphs causes fine drops 01' conde nsation occasionally to arise on th e inner side 01' the coverslip, but these evaporate sorne time afterwards .

,' ('tl ll ll imr electron microscopy l'hc scnnn ing elec tron mi croscop e (SEM), as a n a id in th e idcntificntion 01' Ill ll ~ i, nllowiug' thc obscrva tion 01' su rfa cc ornamen ta l ion 01' va rious o rga ns, is IiI': t'cIIJlillf{ iuc rca sin gly available . T he SEM stud ies by C la rke a nd Griffiths 1\)70) a nd Locci a nd Q uaron i ( 1970, 1971 ) have dcmonstrat cd as cospo rc -wall

IJI'I IIlIIIC lIla t io ll in most spe cics 01' pcrfect Aspergilli iEurotium, Emcricella, 8rll fo'Ya) , As a rcsul t , sorne hitherto unknown mi cromorphological Ica turcs 01' IIU' . I ~ p ()rcs 01' :17 species a nd varie ties of th ese fun gi have becn rccord cd. T he 1'.' ",ilIS obt uincd suggest a need for so rne tax onorn ic revi si n in o thcr 1' .'lllIclIllica lly important grou ps. This is cc rta i