SOMMAIRE

MANACHERE G. - Regulation of sporophore differentiation in some

macromycetes, particularly in Coprini: an overview of some experi-

mental studies from fruiting initiation to sporogenesis .. 291

PAUL B. - Une nouvelle espèce de Pythium isolée d'une saline de l'ouest

algérien 325

ABDEL-HAFEZ A.LI. - Mycoflora of broad bean, chick-pea and lentil

seeds in Egypt 335

MICHELOT D. et TEBBETT LR. - Les intoxications par les Cortinaires 345

PANDEY R. and KUMAR V. - Effect of short term funpasud

programme on non-target phylloplane fungi of soybean 363

Analyses bibliographiques 373

Table du Tome 9 375

CONTENTS

MANACHERE G. - Regulation of sporophore differentiation in some

macromycetes, particularly in Coprini: an overview of some experi-

mental studies from fruiting initiation to sporogenesis .. on 201

PAUL B. - A new species of eur isolated from a salt-marsh in

western Algeria (In French) .. 325

ABDEL-HAFEZ A.LI. - Mytetlora of broad bean, chick-pea and lentil

seeds in Egypt .. 335

MICHELOT D. et TEBBETT LR. - Poisoning by Cortinarius

mushrooms (In French) ... 345

PANDEY R. and KUMAR V. - Effect of short term fongicidal

programme on non-target phylloplane fungi of soybean .... 363

Bibliography ... 373

Table of volume 9 .... 375

Source : MNHN. Paris

CRYPTOGAMIIE

MYCOLOGIE

TOME 9 Fascicule 4 1988

Ancienne Revue de Mycologie. Dirigée par Roger HEIM

DIRECTEUR SCIENTIFIQUE : Madame J. NICOT

SECRÉTAIRE DE RÉDACTION : Mme M.C. BOISSELIER. EDITEUR : A.D.A.C

Publié avec le concours du Muséum National d'Histoire Naturelle

CRYPTOGAMIE, MYCOLOGIE est indexé par : Biological Abstracts, Current Contents,

Publications bibliographiques du CDST (Pascal)

Bibliothèque Centrale Muséum

3 3001 Q9ggpe9-Q/NHN. Paris

Source : MNHN. Paris

Cryptogamie, Mycol. 1988, 9 (4): 291-323 291

REGULATION OF SPOROPHORE DIFFERENTIATION

IN SOME MACROMYCETES, PARTICULARLY

IN COPRINI:

AN OVERVIEW OF SOME EXPERIMENTAL STUDIES,

FROM FRUITING INITIATION TO SPOROGEN

by G. MANACHE

ABSTRACT - Differentiation and morphogenesis of fungi are considered as model systems

with regard to the general biology of organisms. That is particularly true at the level of

vegetative structures (hyphal differentiation, conidiation..) where observations and exper-

iments can be conduced at the cellular level

In differentiation of sporophores of macromycetes constitutes also a fundamental problem,

the complexity of organized structures formed by such fungi is much greater than in the

case of most popular models generally studied

Schizophyllum commune, Fammulina velutipes and Coprini are good models for the study of

differentiation and morphogenesis of macromycetes. Schematically, when “maturity of fruit-

ing” is reached by vegetative mycelia, successive phases can be distinguished in the forma-

tion of sporophores; and correlatively, studied from specific physiological points of view:

firstly, an initiation phase of fruiting release; secondly; a morphogenetic phase s.s., including

the differentiation of the hymenium, and achieved by the fundamental subphases of sporo-

genesis and sporulation.

Numerous studies have been conduced on the influence of external factors - chemical and

physical - on such successive fruiting phases of Coprini; biochemical informations are frag-

mentary. An overview of experimental studies about Coprini leads to a survey of some ac-

tual problems to solve when studying sporophore differentiation of macromycetes.

RÉSUMÉ - La différenciation et la morphogenèse des champignons sont considérées com-

me des systèmes modéles dans le cadre de la biologie des organismes en général. Ceci est

particulièrement vrai à l'échelle des structures végétatives (différenciation hyphale,

Conidiation...) où observations et expérimentations peuvent être conduites à l'échelle cellulai-

re. Si la différenciation des carpophores de macromycètes constitue aussi un probléme fon-

damental, la complexité des structures organisées correspondantes est supérieure à celle des

modèles évoqués et généralement étudiés de manière privilégiée. Schizophyllum commune,

Flammulina velutipes et divers Coprins constituent de bons modèles pour l'étude de la

différenciation et de la morphogenése des macromycétes. Schématiquement, une fois atteint

par les mycéliums végétatifs un stade de "maturité de fructification”, il est possible de distin-

guer des phases successives dans la formation des carpophores et, corrélativement, d'étudier

ces phases quant à leurs spécificités physiologiques: d'abord une phase d'induction

* Invited paper presented at the 4th International Fungal Spore Conference, Ist July 1987.

Stirling, Scotland.

** Université Lyon I - Claude Bernard, Laboratoire de Différenciation fongique, U.A.

Mycologie, CNRS 1127, 69622 Villeurbanne Cedex, France.

Source : MNHN. Paris

292 G. MANACHERE

fructifére; ensuite une phase morphogénétique s.s., incluant la différenciation de l'hyménium

et s'achevant par les sous-phases fondamentales de sporogenése et de sporulation.

De nombreux travaux ont été conduits sur l'influence de facteurs externes - chimiques et

physiques - sur les phases fructiféres successives de Coprins; les informations biochimiques

ont fragmentaires. Une revue de travaux expérimentaux sur les Coprins permet d'envisa-

ger quelques problèmes actuels à résoudre et relatifs à la différenciation des sporophores de

macromycetes,

KEY WORDS : sporophores, Macromycetes, Coprini, fruitbodies, fruiting initiation, pho-

tomorphogenesis, meiosis, sporogenesis, biological rythms.

“Ultimately, chemostructural differentiation of the several types of functional

hyphae shaping the elaboration of reproductive stromas in the higher Ascomycetes

and Basidiomycetes raises the most provoking and mostly unanswered questions

pertaining to the visual achievement of macrofungal morphogenesis in nature.”

(TURIAN, 1983).

Differentiation and morphogenesis of fungi are considered as model systems

with regard to the general biology of plants and, eventually, of others organisms.

That is particularly true at the level of vegetative structures (hyphal differen:

tiation, conidiation,...) where observations and experimentation can be conduced

at the cellular level. Such systems are eventually genetically controlled and ame-

nable to biochemical researches (cf. various papers in SMITH, 1983; LOVET

1985).

If differentiation of sporophores of macromycetes constitutes also a funda-

mental problem, it appears that the complexity of organised structures formed by

such fungi is much greater than in the case of most popular models generally stu-

died for: ".. the development of any organized fungal structure requires that hy-

phae grow toward one another and cooperate in formation of the differentiating

organ; this is the diametrically reversed character of the invasive undifferentiated

mycelium. We are almost totally ignorant of the factors which control this pecul-

iar reversal in behaviour” (REIJNDERS & MOORE, 1985). Indeed the influ-

ences of environmental - nutritional and physical - factors on primordia initiation

and general morphogenesis of sporophores are rather well known (MANACH-

ERE, 1978, 1980, 1985), but the biochemical events really induced and regulated

by such factors are unknown in most cases. The most precise results concern ac-

tually Schizophyllum commune (cf. various papers, in SCHWALB & MILES,

1978; WESSELS, 1985). In a such model species, general biochemical processes

were described in parallel with fruiting evolution and according to genetical char-

acteristics (WESSELS, 1978, 1985). Coprini are also considered as good models

for the studies of differentiation and morphogenesis of higher fungi (MANACH-

ERE, 1970, 1974; MOORE & al., 1979; MOORE, 1984a, b) particularly from a

photobiological point of view. It is actually interesting to notice that the principal

characteristics of photoinduction of fruiting and photomorphogenesis of sporo-

phores - including abortion of primordia under uninterrupted light, described

and precisely defined for Coprinus congregatus (MANACHERE, 1961, 1970,

1971) were recently described for Coprinus cinereus (BALLOU & HOLTON,

1985). That appears as a clear confirmation of the general value of our model.

Nevertheless, for Coprini, biochemical information remains fragmentary and es-

sential problems remain to be solved.

A true difficulty is that final differentiation of sporophores of macromycetes

results from the progressive integration of successive differentiation phases. Such

Source : MNHN. Paris

REGULATION OF SPOROPHORE DIFFERENTIATION 293

interdependant phases are often studied by different searchers acting - and think-

ing ! - as "specialists" rather than as “generalists”. This paper will try to give ...

the “hybrid point of view of a ... specialised generalist’!

The initiation and subsequent development of carpophores from a vegetative

mycelium depend firstly, of course, of the genetical ability to fruit of such a

mycelium (cf. MEINHARDT & ESSER, 1983; RAPER, 1983, CASSELTON &

ECONOMOU, 1985). They depend also of an adequate preliminary vegetative

growth: physiological conditions necessary to obtain “maturity of fruiting” at the

level of a fungal thallus remain to be defined for a majority of species. Of course,

in most cases mycelia capable of producing normal carpophores result from the

plasmogamy of two haploid mycelia, genetically compatible; according to a

scheme of either bipolarity or tetrapolarity; but, in reality, there are many vari-

ants which complicate these simple modes (KUHNER, 1977). It appears that a

majority of studies have been conducted on the influence of external factors -

chemical and physical - on successive fruiting phases of some basidiomycetes.

Schematically, when “maturity of fruiting” has been reached by vegetative myce-

lia; two successive phases can be theoretically distinguished in the formation of

basidiomycetes carpophores: firstly an initiation phase and fruiting release; sec-

ondly a morphogenetic phase including the differentiation of the hymenium and

achieved by the fundamental subphase of sporogenesis, itself ended by sporula-

tion.

INITIATION PHASE

The accomplishment of this phase is evidently simultaneously dependent on

various environmental factors and more or less well defined endogenous factors

(genetical and physiological factors) (reviewed in MANACHERE, 1978, 1980).

Only some fundamental aspects will be discussed here.

Photo-induction of fruiting

Initiation phase of fruiting is a process of fungal differentiation often photo-

controlled, particularly in the cases of various Coprini. For instance, illuminated

cultures of Coprinus congregatus produced a ring of primordia on a minimal liq-

uid medium; just behind the front of growing hyphae at the time of illumination,

and no subsequent part of mycelia could be induced (DURAND, 1983b, Fig. 1).

ROSS (1982, 1985) also reported the existence of an area of inducibility in a

“pale mushroom” phenotype of the same species, but no primordia developed on

solid medium until the mycelia had reached the edge of the plate. Similar results

have also been reported for S. commune (RAUDASKOSKI & YLI-MATTILA,

1985). In the ROSS’ experiments, photoinduction appears to be “memorized” un-

til vegetative growth is achieved. Yet, we have demonstrated that photo-induction

can be also “memorized” by fully developed mycelia (MANACHERE & BAS-

TOUILL - DESCOLLONGES, 1985, Fig. 2). So, fruiting in dark-grown cultures

of C. congregatus ("dark mushroom” phenotype, usually studied in our laborato-

ry) is induced by a 12 h light-break. After a few days, the majority of cultures

present characteristic etiolated primordia. Such primordia can show a normal

development if submitted to suitable photoperiodic regime before abortion.

Moreover, in some cases, it was established that photo-induction is “memorized”

without recognizable morphological effects, for a period of 8.5 to 9.5 days. In

parallel, the visible etiolated photoinduced primordia abort after about 4.5 to 7.5

days in darkness.

Source : MNHN, Paris

294 G. MANACHERE

Fig. 1 - Localization of light-induced primordial formation of Coprinus congregatus (after

DURAND, 1983b).

The fungus was cultured in partitioned plastic Petri dishes. The first compartment was

filled with 20 ml of a semi-synthetic malt-agar medium. The second compartment

received a salt liquid medium. After inoculation of the solid medium with a mycelial

plug from stock cultures, test cultures were placed in continuous darkness at 25°C

Cultures grown in darkness for 9 days were then exposed to a 24h white light period.

As a result of photo-induction, cultures produced a ring of primordia on liquid medium.

Photograph was taken at the end of the light period. Dish diameter: 9 cm.

N.B.: in full darkness, cultures produced sclerotia and no fruiting primordia

The action spectra for the initiation phase of C. congregatus showed

effectiveness at 260, 280, 370 and 440 nm (DURAND & FURUY.

MNHN, Paris

REGULATION OF SPOROPHORE DIFFERENTIATION 295

CO m—À— pe yo es so

t 403. REMEN

Light break

At the end of PSD 2 Final stages

primordium

absent etiolated 1

without with aborted etiolated

pseudorhiza primordium

Fig. 2 - "Memorization" of photo-induction in Coprinus congregarus (after MANACHERE

& BASTOUILL - DESCOLLONGES, 1985).

Experiments were conducted to determine the effect of short irradiations (for instance

12h x 300 mWm? , white light) on the initiation and differentiation of sporophore pri-

mordia and on the morphogenesis in darkness of such photo-induced primordia. Photo-

induction is characterized either by development in darkness of etiolated primordia, or

by a subthreshold state where no visible primordia are recognizable, but where inter-

action with a subsequent light pulse can be observed. The photo-induced primordia

abort after about 4,5 to 7,5 days in darkness. The photo-induction itself is memorized

for a longer period (8,5 to 9,5 days) in darkness.

N.B.: the figure indicates the fruiting pattern of typical experimental treatment (E) and

corresponding control cultures (C0, C7). The experimental procedure is represented be-

low. Size of primordia and final sporophores are not to scale. P.S.D.1 and P.S.D.2 =

successive pre-stays of cultures in darkness following inoculation; the cultures (10, series)

are then submitted to (12h, 12h) light regime.

3). A pratically identical action spectrum for fruit-body formation of Schizophyl-

lum commune was also established (YLI-MATTILA, 1985). The nature of the

hypothetical "cryptochrome(s)" photoreceptor(s) is unknown.

Nevertheless, according to the more recent action spectra, it seems to be flavin

photoreceptors rather than carotenoids (DURAND & FURUYA, 1985; DU-

Source : MNHN, Paris

296 G. MANACHERE

Inhibitor (M) Primordial — Sclerotial

production production

Phenylacetic acid 10? 26 E

5.107 50 25

10? 82 52

Sane 82 88

NaN, 17 40 0

10 75 38

102) 76 70

1076 105 101

KI 107? 102 -

5.107 107 -

10? 100 =

5-Fluorouracil 5.105 o 1

10? 26 35

5.106 80 59

10° 92 78

5.107 110 85

Cyclohexinide 5.102 o 6

2.5.10? 15 E

105 103 24

5.106 = 54

1076 110 94

Table 1 - Effects of various inhibitors on primordial and sclerotial formation in C. congre-

garus (after DURAND, 1983b, 1987).

Inhibitors were applied for 3.5h during primordial photoinduction (3h of blue light) and

for 6h during chemically induced sclerotial formation in darkness. Inhibitor solutions

were added to the liquid salt medim (cf. Fig. 1, and more details in DURAND, 1983b).

The results show potassium iodide did not inhibit blue protoinduced promordial forma-

tion. Phenylacetic acid and sodium azide inhibited the primordial and sclerotial forma-

tion. The activity of phenylacetic acid on the non photoinduced process of sclerotial for-

mation in C. congregatus leads to the conclusions that this drug does not act at the

photoreceptor level.

N.B.: primordial and sclerotial productions are expressed as percentage of the controls

without inhibitors.

RAND, 1985, 1987). DURAND ‘special culturing procedure on liquid medium:

offers major advantages: particularly the precise localisation of a predictable area

of differentiation of primordia of C. congregatus, and also the possibility for ap-

plying and removing chemicals to the growing mycelia at any time before or af-

ter photoinduction. But experiments using inhibitors known to react with illumi-

Source : MNHN. Paris

REGULATION OF SPOROPHORE DIFFERENTIATION 297

nated flavins (potassium iodide; phenylacetic acid...) demonstrated that these

inhibitors do not act - while having other effects - at the photoreceptor level and

can no longer be regarded as specific inhibitors of this blue-U.V. light response

(DURAND, 1985, Table 1). According to DURAND (1987), the most promis-

ing approach to elucidate the nature of "cryptochrome" is to use a system in

which the photoreceptor level can be perturbated. Experiments are being carried

out to isolate riboflavin requiring mutants in Coprinus congregatus. So, bi

chemical and spectroscopic analysis of such strains should lead not only to eluci-

dation of “cryptochrome” and of the primary events of the phototransduction

chain but, also, to a better comprehension of fruiting initiation from a general

point of view. For instance, the light-induced primordia formation of Coprinus

congregatus was inhibited by various inhibitors of nucleic acid and protein syn-

thesis (DURAND, 1983b, Table 2). Cyclohemixide and a phenylalanine ana-

logue (4 fluophenylalanine) prevented the formation of primordia, and the incor-

poration of leucine during photoinduction was substantially reduced in the

presence of cycloheximide. Results suggest that cycloheximide inhibited protein

synthesis but not the incorporation of precursors of nucleic acid synthesis. Actual

results suggest the involvement of RNA and protein synthesis in light-induced

primordial formation in C. congregatus.

Inhibitor Before During After

photoinduction photoinduction photoinduction

5-Fluorouracil 77.9 26 85.4

(10724)

cycloheximide 103.6 102.7 44.7

(10)

Table 2 - Effect of time application of inhibitors on photoinduced primordial formation of

C. congregatus (after DURAND, 1983b).

The duration of inhibitor application was 3.5h and the length of light-induction (blue

light) period was 3h. IL appears that fluorouracil inhibited to a greater extent primordial

formation when added in liquid salt medium (cf. Fig. 1) during the photoinduction peri-

od, whereas cycloheximide inhibited primordial formation only when added after the

photoinduction period. These results presented evidence for the requirement of RNA

synthesis de novo followed by protein synthesis in primordial photoinduction.

N.B.: primordial formation is expressed as percentage of the controls without inhibitors.

Gene expression and induction of fruiting

More generally, if actually induction of fruiting of numerous lower and higher

fungi is rather easily controlled, very little is known about the mechanisms of

transduction of external stimuli into a differentiation response such as fruiting ini-

tiation, and of course, following events (sporophore formation,.. sporogenes-

is...). Studying gene expression during basidiocarp formation in Schizophyllum

commune, WESSELS and co-workers (cf. WESSELS, 1985) detected ^... few dif-

ferences in proteins between monokaryons and the derived dikaryon. Further-

more, it was observed a regulation of RNA sequences... confined to the period of

Source : MNHN, Paris

298 G. MANACHERE

basidiocarp formation in the dikaryon. The regulation involved a limited number

of abundant mRNAs for a number of which cloned cDNA sequences were ob-

tained and organized”. By molecular cloning of RNAs differentially expressed in

monokaryons and dikaryons of S. commune, a role of the detected dikaryon-spe-

cific RNAs in fruiting was recently suggested by MULDER & WESSELS (1986).

Between several observations, it was noticed that nine different RNAs abundant-

ly present in the fruiting dikaryon are present in very low or undetectable levels

in vegetative growing monokaryons or the dikaryon. In spite of major difficulties,

such studies about gene expression during basidiocarp formation have to be de-

veloped not only in the particular case of S. commune but also in the case of

more classical basidiomycetes, particularly Coprini. Indeed, morphogenesis of

such agaricales is more complex than morphogenesis of S. commune. Neverthe-

less it would be useful to study gene expression during basidiocarp formation in

Coprini not only by a comparison of monakaryons and the derived dikaryon,

but also by a comparison of dikaryons induced and non-induced to fruiting

(photo-induced or not, for instance) or by a comparison of strains from the same

species but more or less sensitive to light.

The problem of “fruiting-inducing substances”

For several decennies, monokaryotic fruiting has been observed in the case of

some normally heterothallic species (cf. STAHL & ESSER, 1976) and also in

monokaryotic cultures when wounded (ex: S. commune, RAPER & KRON-

GELB, 1958; LEONARD & DICK, 1973): models relating the various genes

implicated in monokaryotic sporophore production in S. commune have been

proposed and evaluated on their ability to explain the observed data (LESLIE &

LEONARD, 1979). At last, “abnormal” monokaryotic fruiting can equally be in-

duced from a monokaryotic mycelium of Coprinus macrorhizus (alias C. ciner-

eus according to VERRINDER GIBBINS & LU, 1984) by acellular extracts of

carpophores of a dikaryotic mycelia of this mushroom (UNO & ISHIKAWA,

1971). An identical effect can be obtained with acellular extracts of other basi-

diomycetes (e.g. Lentinus edodes, Pleurotus ostreatus, Flammulina velutipes.

Carpophores produced in these conditions are not as well developed as basidio-

carps produced by dikaryotic mycelia: nevertheless, the basidiospores which

they form are viable, germinate, and are of the parental type. EGER (1965a, b,

1968) indicated that the production of carpophores from mycelia of Flammulina

velutipes can be stimulated by the addition of basidiocarp fragments of this spe-

cies or other species. This is the case for Pleurorus "Florida. UNO & ISHIKA-

WA (1971) attributed the inducing power of extracts to a fruiting-inducing sub-

stance (FILS. They identified this as cyclic AMP and/or cAMP-binding

proteins (UNO & ISHIKAWA, 1973, 1976, 1982). However, SCHWALB (1974)

with Schizophyllum commune, WOOD (1976, 1979) with Agaricus bisporus and

von NETZER (1977) with Pleurotus ostreatus could not find any evidence that

cAMP would acts as a fruiting inducer. Conversely, a "F.IS. analog” was iso-

lated from A. bisporus sporophores and this induced haploid fruit-body forma-

tion in S. commune (RUSMIN & LEONARD, 1975, 1978): it does not appear

to be cAMP, but it may be a peptide. Some of these observations are compara-

ble to others (reviewed by MANACHERE, 1980) showing the stimulating frui

ing power of carpophore fragments placed on a vegetative mycelium (for in-

stance C. congregatus, MANACHERE, 1976, 1977; ROBERT, 1978).

Stimulating substances do not appear to be specific (cf. EGER, 1965b, 1968).

In parallel, there are also examples of microorganisms stimulating the fruiting

of higher fungi. For instance, SALAS & HANCOCK (1972) pointed out that

Source : MNHN. Paris

REGULATION OF SPOROPHORE DIFFERENTIATION 299

Penicillium oxalicum brings about a spectacular fruiting initiation and basidio-

carp production in Mycena citricolor when the two species are co-cultivated. One

or more unidentified basidiocarp-stimulating-substances (B.S.S.) were involved.

Mushroom growers consider actually that some more or less specific microor-

ganism have a positive effect on the release of fruiting of cultivated fungi, partic-

ularly Agaricus bisporus (COUVY, 1973). WOOD (1976) thought that such mi-

croorganisms do not release fruiting-stimulating metabolites but that they

contribute to the suppression of metabolites produced by the Agaricus vegetative

mycelium which could prevent fruiting. This last hypothesis is considered the

most plausible, as fruiting was not stimulated by culture filtrates not by suspen-

sions of Pseudomonas putida or other bacteria generally present in various Agari-

cus cultures. Moreover, fruiting of such mushrooms in axenic cultures is pro-

moted by the presence of active charcoal (EGER, 1961; COUVY, 1974; LONG

& JACOBS, 1974). Thus, the role of the microflora accompanying A. bisporus

mycelium might to be to eliminate fruiting inhibitors... rather than to produced

inducing compounds !

Finally, it seems that, as for “florigen” for higher plants, the hypothetical

substance(s) supposed to cause floral initiation (cf. WILKINS, 1969), F.LS. re-

main “a physiological concept rather than a chemical reality”. It seems also that

the concept of F.LS. covers a large spectra of molecules having a more or less

defined role in general metabolism rather than a specific power in reproductive

processes in fungi (ex: cerebrosides and ceramides from mycelia of S. commune

acting as F.LS. on the same species, KAWAI & IKEDA, 1982; sphingolipids

from wheat grain acting also as F.1.S. on S. commune , KAWAI & al., 1986;

anthranilic acid from a strain of Actinomycetess inducing the formation of the

stipe of the fruiting bodies of Favolus arcularius “even under dark conditions”,

MURAO & al., 1984; ... or ammonia inducing fruiting of Coprinus cinereus in

darkness, MORIMOTO & al., 1981, etc).

As mentioned above; monokaryotic fruit-bodies are generally more or less ab-

normalous. Nevertheless, recently, it was observed that originally monokaryotic

cultures of Coprinus cinereus developed normal fruiting bodies when subjected to

part cular nutritional stress from periods ranging from 3 weeks to several months

(VERRINDER GIBBINS & LU, 1984). The fruiting bodies arose on dikaryotic

tissue complet with clamps connexions, and underwent a normal meiosis with

four spores resulting from each basidium. IT appears, in this particular case,

that a stress can induce a rearrangement of the genome at the level of incompat-

ibility factors. The mechanism for such a transformation of monokaryotic into

dikaryotic tissue without mating is actually difficult to explain.

Some metabolic aspects of fruiting initiation

Nutritional researches on fruiting initiation are not reviewed here (MA-

NACHERE, 1980). Nevertheless, such researches naturally lead to the problem

of metabolic bases of the phenomenon in basidiomycetes. Fragmentary data are

known relative essentially to Agaricus bisporus, Schizophyllum commune, Copri-

nus cinereus (alias “lagopus” ) and Coprinus congregatus, but practically, they do

not allow us to suggest precisely those pathways implicated in initiation of fruit-

ing. Metabolic bases of the initiation phase of fruiting of macromycetes (partic-

ularly glucose effect on fruiting) were reviewed (MANACHERE & al., 1983).

Here will be evocated only some fundamental results.

When studying phenoloxidase activities in relation to fruiting of A. bisporus,

TURNER (1974) showed that laccase activity characterizes the vegetative phase,

Source : MNHN, Paris

300 G. MANACHERE

100

Relative quantum effectiveness

550 650

Wavelength (nm)

Fig. 3 - Action spectra in fruiting of Coprinus congregatus (after DURAND & FURUYA,

1985)

a) primordial photoinduction; b) photo-inhibition of development of “dark sensitiv? pri

mordia* (cf. Fig. 4). The ordinate value for the most effective wavelength was set ai 100.

whereas tyrosinase activity increases at the time of initiation and development of

sporophore buds. TURNER considers that the increase of extracellular laccase

in the compost (usual substratum of A. bisporus formed by fermented straw)

during the colonization phase, and then its disappearance at the time when the

first carpophore flush is developed, is connected to an attack of the substrate lig-

nin by the mycelium during the course of its growth. Activation of tyrosinase

during the formation of reproductive structures could results from the impover-

ishment of the medium at the end of the vegetative phase. WOOD & GOODE-

NOUGH (1977) observed with the same mushroom in axenic cultures an inverse

relation between the activity of carboxymethyl-cellulase and laccase as fructifica-

tion progressively advances; they did not note any significant variation in the ac-

livity of other enzymes (xylanase, laminarase, acid and alkaline phosphatases.

proteases). An increase of cellulase could contribute to the liberation of sugars

which can be assimilated by the mycelia and therefore participate in carpophore

formation. It can be noticed that, recently, DE VRIES & al. (1986) have ob-

served that ".. growth at 30°C in darkness prevented fruit-body formation of

Schizophyllum commune and induced the dikaryon, but not the progenitor mo-

nokaryons, to excrete laccase into the medium in amounts up to 3% of the ex-

tracellular proteins. The competence of the dikaryon of the same species to prod-

uce laccase activity, in contrast to the component monokaryons, was also noted

Source : MNHN. Paris

REGULATION OF SPOROPHORE DIFFERENTIATION 301

by LEONARD & PHILLIPS (1973). A major difference appears with observa-

tions on A. bisporus : the decline in laccase activity from dikaryon of S. com-

mune occurred in the absence of fruit-body formation, after glucose exhaustion in

the medium, and on the opposite, extracellular laccase activity remained practi-

cally unvariable, at a maximal level, during numerous successive days, when

non-fruiting mycelia of A. bisporus were submitted to analysis (WOOD &

GOODENOUGH, 1977).

Although they are fragmentary, other results show that the passage from the

vegetative phase of mycelial growth to the initiation phase of primordia is accom-

panied by important metabolic changes. WESSELS (1965) showed that the me-

tabolic activity of a whole culture of S. commune, as determined by gaseous ex-

changes, is firstly fermentative and, when the initiation phase of primordia

commences, becomes oxidative. WESSELS thus established for S. commune a

pattern which seems of general relevance by those who have studied the metabol-

ic aspects of fungal reproduction (cf. TURIAN, 1969). In fact, this metabolic

change appears to be related to variations in amounts of various metabolites,

particularly carbohydrates, firstly in the medium and later in the vegetative myce-

lium. Thus, WESSELS showed that fruiting initiation of S. commune can only

take place if exogenous carbon and nitrogen sources are available, but, over and

above the initial stage, these requirements can differ. STEWART & MOORE

(1974) showed that the quantity of reducing sugar and nitrogen in the form of

a-amines in the medium diminished between the beginning of the culture and the

observation of the first carpophore buds of Coprinus lagopus. ROBERT (1977b)

showed that the fruiting initiation of C. congregatus occurs when glucose from

the medium has been almost totally removed. Generally the pH medium of the

culture has become more alkaline when primordia appear (C. lagopus, STE-

WART & MOORE, 1974; C. congregatus ROBERT, 1977b, HORRIERE,

1977) but this may be also related to senescence of the culture (ROBERT,

19770)

Independently from the problematic question of F.LS. previously mentioned,

one can notice recent observations of increase in the level of cAMP in relation

with primordia light-induction of Schizophyllum commune (YLI-MATTILA,

1987): it seems possible that cAMP could control the breakdown of reserve poly-

saccharides; for, in the evocated studies, the sharp increase observed in the nu-

cleotide level took place earlier than a recognized decrease of reserve polysac-

charides (cf. RAUDASKOSKI & SALONEN, 1984). Experiments are to be

conducted to study this hypothesis which concerns various fungi, macromycetes

and others, where cAMP has been suggested to be involved in morphogenetic

and developmental processes.

MORPHOGENETIC PHASE

Some metabolic aspects of morphogenesis

Metabolic aspects of the morphogenetic phase of macromycetes (particularly

identity and mobilisation of reserves, enzymatic activities and metabolic

pathways) were recently reviewed (MANACHERE & al., 1983). Only some

fundamental results will be discussed here.

From fruiting initiation, the growth of basidiocarps implies the transfer of wa-

ter and various materials from the original mycelium ( Agaricus bisporus, BON-

NER & al.,1956; Coprinus lagopus, MADELIN, 1956a; Polyporus brumalis,

Source : MNHN, Paris

302 G. MANACHERE

PLUNKETT, 1958; Schizophyllum commune, WESSELS, 1965). MADELIN

(1960) found, at the time of release of fruiting of C. lagopus, that food reserves -

probably glycogen and proteins - from inflated cells of the vegetative mycelium

disappeared.

Once the initiation phase is accomplished, nutritional and metabolic aspects

are too intimately interwined to be distinguished from each other. In order to

simplify matters, only the principal species taken as examples earlier will be con-

sidered. As far as Schizophyllum commune is concerned, WESSELS (1963) es-

tablished that the respiratory ratio is above 1 until the primordia are formed,

then decreases and moves towards 1, The consumption of oxygen remains high

as long as exogenous glucose has not been totally exhausted and then falls sharp-

ly with a further decrease during carpophore maturation. During growth of pri-

mordia, while exogenous glucose must be present, an exogenous nitrogen source

is unused; the needs of primordia can be met from nitrogen compounds coming

from the vegetative mycelium. Subsequently, during the final phase of pileus for-

mation, there is no need for exogenous carbon or nitrogen sources; indeed, the

supply of the exogenous carbon source obstructs formation of pilei. WESSELS

showed that the completion of pileus formation of carpophores depends on a low

but continued supply of glucose, resulting from the hydrolysis of glucan following

the breakdown of arborted primordia and mycelium cell walls. In fact, on a

vegetative thallus of S. commune or other basidiomycetes, not all carpophore

buds accomplish their development, and only some buds reach the terminal stage

of maturity. The few data given above show the complexity of this problem. The

progression of basidiocarp morphogenesis depends on complex nutritional and

metabolic interactions: medium nutrients-vegetative mycelium, vegetative myceli-

um-primordia and dominating primordia-dominated primordia. In the case of C.

congregatus when studying the behaviour of cultures on liquid media; one can

notice that primordia formation takes place only when the mycelial growth de-

clines strongly, this being correlated with carbohydrate depletion of the medium.

Thereafter growth of primordia is dependent only on nutrient supply from the

basal medium until the fruit-bodies reach maturity: carbohydrates reserves are

used during fruiting and strongly metabolized during the last 48 to 24 h of fruit-

bodies maturation (i.e. during stipe elongtion and cap expansion) It has been

established that a hot-water-soluble polysaccharide is particularly used during

these processes (ROBERT, 1977b, 1978). Rhythmic production is succesive

flushes ends with depletion’ of this carbohydrate reserve which constitutes the

fruiting potential of the cultures.

On the other hand, basidiocarp morphogenesis is also a function of inter-rela-

tions between the stipe and the pileus. Thus, the terminal phase of carpophore

development of C. congregatus depends, according to ROBERT (1977a), on the

accumulation of carbohydrates and proteins apparently translocated from the

stipe to the maturing pileus. The final stage of morphogenesis of this Coprinus is

not however autonomous: as excised carpophores cannot reach normal size even

when excision is done during the final hours of development (BRET, 1977b). Si-

milarly, carpophores excised from Agaricus bisporus do not reach normal size

(TURNER, 1977). However, GOODAY (1974, 1975, 1982) showed that the ter-

minal and rapid elongation phase of Coprinus cinereus basidiocarps is an auton-

omous endotrophic process. HAMMOND & NICHOLS (1976) have shown that

mannitol accumulates in basidiocarps of Agaricus bisporus at the same time as

the level of trehalose diminishes. However, at the end of development, the quan-

tity of mannitol also decreases in the carphores and this is converted to another

material used by the mycelium or the basidiocarp. According to the observations

of BORRISS (1934a), the glycogen which first accumulates in the lower and pe-

Source : MNHN, Paris

REGULATION OF SPOROPHORE DIFFERENTIATION 303

ripheral parts of Coprinus “lagopus” stipes progressively moves towards the up-

per parts to be finally utilized during basidiocarp maturation. RAO & NIEDER-

PRUEM (1969) noted the predominance of trehalose in the stipe of C. “lagopus”

and glucose in the caps, but made no measurement of changes in the amounts of

these components during successive stages of development. Electron microscop-

ical observations (WATTHEWS & NIEDERPRUEM, 1973) showed that, at a

juvenile stage of primordia development of the same species; polysaccharides

(probably glycogen) accumulate more in the lower cells of the stipe or those cells

destined to give the future hymenium. According to KITAMOTO & GRUEN

(1976), the largest carpophores produced by a culture of Flammulina velutipes

utilize, during the course of their development, not only the residual glucose from

the substrate but also, partially, the constituents of the mycelium and of those

small primordia which do not develop fully. Trehalose, mannitol and arabitol are

produced from glycogen and transferred to the stipes and pilei of carpophores

which develop to completion.

Develop. Control Experiment Inhib.

stage n bie AL n LÁ. ^L 1

- I9 5 35 |36 | 5.6 * 0.2| 4.8 € 0.5| 57 | 6.1 € 0.2| 3.8 * 19.9

-17h45 |34 | 6.3 € 0.2|20,8 + 1.9) 47] 6.5 + 0.2) 8.6 + 0.8| 58.3

- a5 h40 |42 | 7.1 * 0.2|47.1 + 1.3] 65 | 7.2 + 0.2/21.9 + 1.3] 53.5

= 13h 50 |51 8.2 * 0.2 48.9 * 0.9| 57 | 8.7 + 0.2 31.9 + 1.2] 34.8

Table 3 - Role of the cap on stipe elongation of C. congregatus: inhibitory effect of caps

from photoinhibited primordia on elongation of decapitated stipes from primordia

whose maturation is induced (after ROBERT & BRET, 1987).

Development stage: hours before autolysis, control: decapitated stipes only; experiment

caps from primordia developed during 5 days in continuous light were then applied, fol-

lowing decapitation; n: number of treated stipes.

N.B.: other informations cf. Fig. 4.

MOORE & EWAZE (1976), state that measurement of the specific activities

of representative enzymes of the pentose phosphate cycle, Embden-Meyerhof-

Parnas pathway and the tricarboxylic acid cycle in Coprinus cinereus sporo-

phores at different stages of the development indicates that glycolysis is the major

route of carbohydrate catabolism through sporophore development. Enzymes of

the pentose phosphate cycle were always at lower specific activities than the en-

zyme of the EMP pathway, and the activities of the pentose phosphate cycle en-

zymes declined drastically as development proceeded. This conflicts with the

findings for Agaricus bisporus (LE ROUX, 1965), but the changes in some en-

zymes were qualitatively similar to those occurring in the development of Schizo-

phyllum commune (SCHWALB, 1974).

, ln practice, much remains yet to be done in order to understand the metabol-

ism associated with fruiting of higher fungi. The few reports referred to above,

essentially concerning the metabolism. of carbohydrates in connection with the

basidiocarp morphogenesis, are evidence of this. Other examples could have

Source : MNHN, Paris

304 G. MANACHERE

been cited. Thus, STEWART & MOORE (1974) established that GDHsan

(NAD-linked glutamate dehydrogenase) activity could be a component of the

imal vegetative metabolism of Coprinus lagopus sensu. LEWIS ( C. cinereus. )

but activity of GDI lxqpp increases in parallel with certain important changes in

terminal development of carpophores. The authors showed that GDHxap activ:

ity is subject to catabolite repression and derepression by urea; GDI Ixape activ-

ity being, inversely, subject to catabolite derepression and repression by urea-

Thus GDH could be the enzyme normally implicated in ammonia assim-

lation. GDI ips being reserved of specific functions associated with metabolic

iterations in connexion with the phenomena accompanying terminal develop-

ANE Cof the basidiocarp. More precisely, GDH yp specific activity increases rel-

atively more in the tissues of the cap than in the stipe and the basidiospores whe-

DUX remains low. However, such a level of GDHyapp activity has not been

found in the caps of numerous other species of Basidiomycetes (MOORE & AL

GHARAWI, 1976).

Finally, it appears that maturation of the cap of C. cinereus is accompanied

by a specific pattern of changes in enzyme activities and metabolite levels. The

most significant changes result in amplification of activity in tricarboxylic acid cy-

Cie and the urea cycle. “The system exemplifies different sorts of regulati

from substrate level to the gene level, and is an ideal model for study of the cau-

sative events that give rise to metabolic shifts which direct differentiation proc-

esses” (MOORE, 1984a). Nevertheless, generalization of particular results re-

mains problematic. For instance, if, in C. cinereus, urease was found in the

stipes, its absence from the pileus was thought to account for the accumulation of

urea and arginine in the pilei (EWAZE & al., 1978). On the opposite, Flammuli-

na velutipes seems to lack such mechanism, the concentration of urea remaining.

very low in both pilei and stipes during their growth (GRUEN & WONG,

19812).

Among the histochemical studies, KOMAGATA & OKUNISIII (1969)

showed that, in Coprinus kimurae, the activities of cytochrome oxidase, succinic

dehydrogenase and acid and alkaline phosphatases are localized at the carpe”

phore growth zone; essentially the upper part of the stipe and the edge of the pi-

pret’ Etnilar observations have been made for Polyporus brumalis (OKUNISHI

& KOMAGATA, 1975). Studies on Favolus arcularius have confirmed the local"

ization of respiratory enzymes (cytochrome oxydase and succinic dehydrogenase)

ai the level of active growth zones which are the terminal part of the stipe; the

entire pileus and the young hymenia (HORIKOSHI & al., 1973).

Regulation of fruit body morphogenesis at the level of individual sporophores

The general development of sporophores (stipe elongation, cap maturation)

requires a continuous supply of water and nutrients from the vegetative myceli-

um during most of their growth period. The identity of the translocated nutrients

has not been determined. Nevertheless, the elongation of isolated whole fruit

bodies of Flammulina velutipes was promoted by glucose and other low molecu-

lar weight carbohydrates (GRUEN & WU, 1972).

Many experiments show that, when a factor prevents pileus development, ex-

cessive elongation of the stipe follows. In fact, complex correlations regulate the

individual development of the two parts of sporophores. HAGIMOTO & KO-

NISHI (1959, 1960), HAGIMOTO (1963a, b) and GRUEN (1963) showed that

one or more growth substances elaborated at the level of the hymenial lamellae

probably control the elongation of carpophore stipes of the cultivated Agaricus

Source : MNHN, Paris.

REGULATION OF SPOROPHORE DIFFERENTIATION 305

-Xh. om uU ^

(a) — hi A ah

Fig. 4 - Light conditions and method used to show the presence of an inhibitor of stipe el-

ongation in the cap of primordia of Coprinus congregatus developed under continuous

light (original figure by ROBET, after ROBERT & BRET, 1987).

Decap.: decapitation = beginning of the experiment; - X h: stages of development of

operated primordia (cf. table 3) = X hours before autolysis ( @ ); C.L.: abortive primor-

dia produced under continuous light (b) ; L.i: initial length, measured just after decapi-

tation; Lf: final length, measured just after a minimum of 24h in light and then, cap re-

moval when necessary: A L: residual growth of only decapitated stipes ( control ) or

receiving caps from C.L. primordia ( experiment ); disc.: discarded parts.

N.B.: hatched periods are periods where darkness is not needed for normal develop-

ment; the black one correspond to an absolute requirement at 25°C; this last temper-

ature was maintained during all manipulations.

CONTROL

so that the unilateral elimination of lamellae from a young carpophore leads to a

preferential elongation at the side where the lamellae were conserved such that a

curvature is produced. Similar observations have been made with Flammulina

velutipes (GRUEN, 1976) and Coprinus congregatus (BRET, 1977a, b). The ac-

tive substance has not yet been identified. Moreover, the cap plays an essential

role in the determination of stipe elongation. So, in A. bisporus and F. velutipes,

stipe elongation depends on pileus during most of the growth period; growth

controlling activity originates in the lamellae (GRUEN, 1963, 1969, 1982). In C.

congregatus, the presence of the cap is also necessary until a late stage for com-

plete stipe elongation in developing fruit bodies (ROBERT & BRET, 1987).

It may appear that the stipe-pileus relationships are one-way, the pileus cont-

rolling the stipe elongation. This is however not the case as the stipes eventually

Source : MNHN, Paris.

306 G. MANACHERE

control the translocation of various nutrients from the vegetative mycelium to the

different parts of the basidiocarps and the latter remains dependent on the ori-

ginal thallus until the final hours of their development.

Going from the information given above and by the analogy, of our know-

ledge on the intervention of growth substances in iropisms of higher plants, it

WoStd seem reasonable to think that the positive phototropism (young spore

phores) and negative geotropism (terminal. elongation phase of mature sporo-

phores) characterizing certain phases of basidiocarp morphogenesis could be de-

formined by “hormones”. Such hormonal substances, released asymmetrically by

the hymenial lamellae of carpophores would determine the curvature of stipes by

bringing out the elongation of the upper cells of the later. However, in the case

or Harimulina velutipes, the stimulatory effect of stipe elongation by an extract

or lamellae is significantly increased if it is transmitted by nutritive agar, ez. po-

tato dextrose agar, rather by a simple agar (GRUEN, 1976).

Little is known about the mechanism of action of lamellae on stipe elongar

tion- A. bisporus lamellae produce a diffusate in agar which, when applied unila-

terally, causes a stipe curvature, indicative of growth promotion (HAGIMOTO

terale ENISHI, 1959, 1960; GRUEN, 1967). No curvature is observed when

TAA is applied instead of diffusate. In C. congregatus, removal of half the cap re.

sulted in negative curvature of the stipe when the operation Was performed more

than LD h before the end of the fruit body development (BRET, 1977b). Exper”

ian ts consisting of joining caps and stipes of sporophores of different ages have

shown the pattern of growth substances production in C. congregatiis. Stimu-

Mas Maximal when the deposited cap came from a fruit body that was 16

Flore the end of its development (BRET, 1977b). In parallel, sensibility of

stipe to substances produced by hymenial lamellae was alse maximal when such

a stipe was 16 to 18 h before the end of its development (ROBERT & BRET,

TOTT “Table 3): it is noticeable that the “minus 16 h stage” is characterized by the

beginning of meiotic divisions following karyogamy at 23 C (MANACHERE,

COURT MANACHERE & BASTOUILL - DESCOLLONGES, 1982). In the

same species, it was possible to inhibit elongation of the stipe when this process

becomes quite independent of the presence of caps (i.e. after the - 16 h stage pre-

viously mentioned): this was done by using caps from non-elongating and spore-

less aborting fruit bodies obtained in continuous light. It seems that light which

causes the primordia inhibition, and leads also to nuclear fusion in the young ba-

sidia (WANACHERE & BASTOUILL - DESCOLLONGES, 1982), may act by

Sümulating the synthesis - or only the accumulation - of an inhibitor in the cap

(ROBERT & BRET, 1987, Fig. 4). In continuous light, the presence of the inhi-

Sitor appears to prevent stipe growth in the primordium, but may also directly

arrest further development of meiosis.

All these results imply the production of growth- promoting or growth- inhib-

iting factors in the cap. However, as yet, there has been no successful biochemi-

iting feon ization of the factors involved. The question is also to determine if

such hypothetical factors are the same as those controlling the differentiation of

the nest flush, these substances being synthetized in the caps and diffusing

through the stipe to the basal mycelium (ROBERT, 1982).

In some isolate examples, the control of stipe elongation by the cap appears

similar to the phenomenon of apical dominance in higher plants: for instance,

the removal of the cap of a young sporophore of Asterophora parasitica is fol-

lowed. in full light, by the development of several lateral new primordia on the

remaining stipe (VIOT, 1970). In darkness, mycelia of the same species produced

Abnormal primordia with lateral ramifications, each ended by a reduced pileus:

Source : MNHN. Paris

REGULATION OF SPOROPHORE DIFFERENTIATION 307

First flush Second flush

d.wt. 13.48 mg

f.b. no. 2.94

(by fruiting culture)

fb. no... 1.04

(by fruiting culture)

First flush Second flush

1-23

Fig. 5 - Comparison of fruiting of Coprinus congregatus under 2 distinct photoperiodic

treatments: L D:12,12 regime (control series) and L/D: 1/23 regime (after MANACH-

ERE & BASTOUILL - DESCOLLONGES, 1983).

Number of cultures series: 25. Mean dry weight (d. wt.) and mean fruit body number

(fb, no.) by fruiting cultures and by flush are indicated. Culture temperature 25°C. Illu-

mination radiant flux density 300 mWm_ (white light).

such observations confirm previous hypothesis about physiological correlations

between pilei and stipes.

Regulation of fruit body morphogenesis at the whole culture level

Periodical fruiting characterizes several higher fungi (see MANACHERE,

1980; MANACHERE & al., 1983). In the case of C. congregatus, when cultures

are maintained for long periods under alternating daily periods of 12 h light and

12 h darkness, they produce mature sporophores according to an endogenous

Source : MNHN. Paris

308 G. MANACHERE

100%

75%

50%

25%

-36h -24h -20h -i6h-I2h

0 02°

dark sensitive

primordia i

; k a 2

D! 12-12

ips.

m . ne SS a

9 days 3 days ;48h E E 2h Oh

quee Ac ES PHASES

* oT

1st photostimulated photostimul OF SPOROPHORE

photoinhibited photoinditferent | DEVELOPMENT

Fig. 6 - Meiosis and sporogenesis evolution in Coprinus congregatus under photoperiodic

regime (L/D: 12/12; culture temperature: 25°C) (after MANACHERE & BASTOUILL

- DESCOLLONGES, 1982). Statistical representation of the evolution of meiosis from

the typical "dark sensitive stadium’ till the formation of the four meiotic nuclei; behind:

comparative study of cytological and morphological evolution of sporophores.

Source : MNHN. Paris

REGULATION OF SPOROPHORE DIFFERENTIATION 309

rhythm with a period which is dependent upon temperature: 4 to 5 days at a

temperature of 25°C, 6 to 9 days at 15°C, for instance (ROBERT & MANACH-

ERE, 1971; MANACHERE & ROBERT, 1972). A similar endogenous fruiting

rhythm is known in the case of several other higher species, particularly the culti-

vated mushroom Agaricus bisporus (COOKE & FLEGG, 1962; WOOD &

GOODENOUGH, 1977; HAMMOND, 1981).

In the case of C. congregatus, when mature sporophores of one or two succes-

sive flushes are picked, there is commonly a delay in the differentiation of the

next flush and a decrease in - and even a suppression of - the production of this

flush. Conversely, when exogenous sporophores (MANACHERE, 1977) or wa-

ter extracts (ROBERT, 1978) of picked sporophores are applied, one can ob-

serve a promotive effect, ic. an earlier differentiation of the following flush

(mainly with extracts from caps) and sometimes, a more abundant production of

sporophores (mainly with extracts from stipes).

When cultures of C. congregatus are maintained at 25°C under L D: 1/23 re-

gime (ie. | h of light per day), the first flush is delayed on the average of two

days in comparison with cultures under L D: 12:12 regime and this flush gener-

ally shows only I fruit body culture against 3 fruit bodies culture on average un

der the L D 12 12 regime. Nevertheless, if dry weight is considered rather than

number of fruit bodies, productivity is identical in the two series of cultures (VIA

NACHERE & BASTOUILL - DESCOLLONGES, 1985, Fig. 5). Similar results

were obtained when illumination was lowered from 300 to 10 mWm? , the cul-

tures remaining under a L D: 12 12 regime (ROBERT, 1982). It appears that

the reduction of the daily lighting (duration or energy level) is compensated by

internal regulation at the level of the whole culture. Such regulation controls the

global development of mature sporophores, particularly individual morphogenes-

is and rhythmical production. These observations confirm the physiological unity

of a culture of C. congregatus demonstrated by previous experiments (MA

NACHERE, 1976, 1977). A fundamental question remains unanswered in the

case of C. congregatus (MANA ERE & ROBERT, 1972) and all other spe

cies showing a fruiting rhythm: at what stage are the second and subsequent

flushes initiated ? All the flushes may be initiated at the outset, with the later

ones remaining cryptic in a stage not visible to the naked eye; or each might be

initiated just before or coincident with maturation of the preceding flush.

In the case of C. congregatus (but also in the cases of Sphaerobolus stellatus,

INGOLD & NAWAZ, 1967, and of Agaricus bisporus, COOKE & FLEGG,

1965) picking of the primordia (and not the mature sporophores as in the exper-

iments mentioned above) leads to an acceleration of the primordia development

of the following flushes, the acceleration being greater the earlier stage during

which primordia are picked. This could results from nutrient deprivation of dom-

inant primordia. Such hypothesis is coherent with past experimental demon-

stration of MADELIN (1956b) relative to. Coprinus lagopus or of WESSELS

(1965) relative to Schizophyllum commune and more recent observations of

GRUEN & WONG (1981b) showing that completely developed sporophores of

Flammulina velutipes derive their substrates indeed from the nutritive medium,

but also from material stored in the rest of the colony: loss of dry weight by

aborted primordia and stunted fruit-bodies parallels gains by large fruit-bodies.

Source : MNHN, Paris

310 G. MANACHERE

Physiological aspects of sporogenesis

For several decennies, works have been conduced to determine morphologi-

cal, histological and cytological characteristics of basidiocarps of numerous spe-

cies of Agaricales (cf. REUNDERS, 1963; KUHNER, 1926 and numerous pa-

pers mentioned in REUNDERS,1963; MOORE, 1984a, b). A particular

Attention has generally be given to the evolution of hymenial cells, from the

Sporogenesis

o +

dark sensitive

primordia

Irradiance

4 (a) at

+ (x) culture level

a6] CL 6h Ha Cuno ee. (mW m-2)

IW di — e]. er] 300

Ó—À— Me €

25°C 25

0 ,100

202109

25°C 2315 25°C

: 0 ,100

i Dm E

= 300

25'C IS 25°C

0 100

pong

(Saf eee ee | 24

25°C 17,5°C 25

i o 100

$ ld

[ence enr ENIM E 1-15

25°C 22,086: 25°C

Fig, 7 - Effects of light and temperature interactions on maturation of sporophores (includ-

ing sporogenesis) of Coprinus congregatus (after DURAND, 1983a). Cultures grown in

darkness for 10 days (P.S.D. = pre-stay in darkness) were then exposed to white light

(300 mWm? ) without interruption during 3.5 days. At the end of this period, they have

produced characteristic "dark sensitive primordia” (cf. MANACHERE, 1970, for in-

Stance), They are then submitted during 6h to various thermical and light regimen. Per-

centage of cultures with aborted primordia only (no basidiospores produced) and of cul-

tures with normal sporulating sporophores is evaluated 24h after the end of the

eventually maturating regimen.

N.Bs sp. +: sporophores with normal sporogenesis; sp. O: sporeless aborting primor-

ia.

Source : MNHN, Paris

REGULATION OF SPOROPHORE DIFFERENTIATION 311

young basidia stage to the “sporulating basidia” terminal stages (cf. HUGUE-

NEY, 1978: Coprinus congregatus; ROSIN & MOORE, 1985a, b: Coprinus

cinereus). Nevertheless few data concern the physiology of sporogenesis of such

macromycetes. In most cases, observations of ’normal” evolution of basidia are

given, including replication of DNA meiotic divisions and various ultrastructural

and cytochemical aspects (LU, 1982; THIELKE, 1982; Mc LAUGHLIN, 1982).

Beyond such useful reports, one can notice a lack about the control of meiotic

events and of sporogenesis by external and hypothetical internal factors: in other

words, the physiological aspects of basidial and basidiospore development s.s.

are practically unknown.

The morphological stages and phases of development of sporophores of C.

congregatus, and, correlatively, the nuclear behaviour of hymenial cells till com-

plete sporogenesis are well defined and determined essentially by daily light and

dark periods. Each stage is defined - at 25°C in most observations - by the num-

ber of hours before sporophore maturity, which is called 0 h stage (for instance, -

36 h stage represents 36 h before the Oh stage). With respect to the cytological

state, and particularly the development of meiosis, there is no karyogamy at the -

36 h stage. Only 50% of the basidia have diploid nuclei at the - 24 h stage. Usu-

ally, all basidia reach karyogamy between the - 20 and - 16 h stages, and the

meiotic divisions begin at the - 16 h stage and are completed at the - 12 h stage

(MANACHERE & BASTOUILL - DESCOLLONGES, 1982). On the other

hand, at 23 C there is a photoinhibition phase between - 36 and - 24 h stages,

light being necessary before and after this phase. In continuous light, after the -

36 h stage (stage of sensitivity to darkness) one can essentially observed an inhi-

bition of stipe elongation and an arrest of meiosis at the nuclear fusion stage

correlatively; one can observe no opening of pileus (MANACHERE, 1970, Fig.

6, 8).

A similar inhibitory effect of continuous light on stipe elongation and meiosis

(ie. arrest at the nuclear fusion stage) was then observed on primordia of various

other species of other Coprini: "C. /agopus at 35C (LU, 1972), dikaryotic spo-

rophores of C. macrorhizus at 28"C (KAMADA & al., 1978), monokaryotic spo-

rophores of a mutant strain of the same species at 25°C (MIYAKE & al., 1980).

In C. congregatus, the dark requirement observed at relatively elevated tem-

perature (20-25°C) is not found for temperatures less than 17.5°C. Normal devel-

opment of primordia in continuous light (300 mWm- ) was obtained by lowering

the basal temperature of the culture from 25 to 10°C for 6 h (ROBERT, 1971;

ROBERT & DURAND, 1979). More precisely, at low levels of irradiance (1-5

mWm- ) a slight decrease in temperature (25 to 22.5°C) was sufficient to release

primordia from photoinhibition (DURAND,1982, 1983a, Fig. 7). It was ob-

served that meiosis and consecutive sporogenesis are strictly dark dependent at

25°C, but could proceed in continuous light at 10°C (Fig. 8). It appears that the

dark requirement triggering meiosis and then besidiocarp maturation in several

Coprinus species, C. lagopus (LU, 1972), C. congregatus (MANACHERE &

BASTOUILL - DESCOLLONGES, 1982) could be temperature-dependent.

Furthermore, in certain higher and lower fungi the dark period which deter-

mines the terminal phase of development of sporophores, namely the phase of

maturation including sporogenesis, could be replaced by lowering the temper-

ature. For instance, some parasitic micromycetes, Alternaria solani and A. toma-

to, failed to develop conidia under continuous illumination at 25°C: however;

the inhibiting effect of light was no longer present at 15°C (ARAGAKI, 1961;

LUKENS, 1966).

Source : MNHN, Paris

312 G. MANACHERE

Dark sensitive

primordia

Fig. 8 - Effects of light temperature interactions on meiosis in Coprinus congregatus (after

MANACHERE & BASTOUILL - DESCOLLONGES, 1982).

Experimental conditions: cf. Fig. 7. Primordia reach “dark sensitive stadium” after 3.5

days under continuous white light. Meiosis, and consecutive sporogenesis, is normal

when such primordia are submitted to a dark period during 5 h, temperature: 25°C or à

dark period during 12 h, temperature: 25°C or full light during 6h, temperature lowered

from 25 to 10°C.

Source : MNHN. Paris

REGULATION OF SPOROPHORE DIFFERENTIATION 313

Such phenomena are observed in other groups of plants. Some of the devel-

opmental features cited for C. congregatus have their analogy in the photoperiod-

ic responses of some extremely sensitive short-day plants, particularly those

which can be induced by a single dark period such Xanthium strumariwn, Phar-

bitis nil, Lemna perpusilla (EVANS, 1969), Perilla ocymoides (DERONNE &

BLONDON, 1977) or strawberry (HEIDE, 1977). Therefore, floral initiation,

conidia formation or fruitbody maturation can be induced by a number of alter-

native pathways and are not strictly dependent upon a specific photoperiodic

process.

In parallel with studies on physiological correlations during morphogenesis

and sporogenesis of sporophores of C. congregatus, recent work was conducted

to determine the potential of fruiting of isolated hymenial lamellae used as inocu-

la, at various meiotic and sporogenetic stages (BASTOUILL - DESCOLLONG-

ES & MANACHERE, 1984, Fig. 9). When isolated lamellae were transplanted

at young stages (binucleate or karyogamy stages, i.e. -36 to -24 h stages), in most

cases young basidia did not develop beyond prophase 1 of meiosis. But inocu-

lation was followed, practically without exception, by a direct fruiting, no sporo-

phore of the first flush appearing on the vegetative mycelium developed around

the inoculum. The first fruiting flush was observed exclusively on the inoculum.

Conversely, when isolated lamellae were transplanted at older stages, meiosis and

sporogenesis proceeded normally and the potential for direct renewed fruiting di-

sappeared: the first fruiting flush was generally not observed on the inoculum but

on the surrounding mycelium. These results, among others, confirm that physio-

logical phases and stages of primordia of C. congregatus may be defined not only

by the usual morphological characters, but also by correlated cytological stages

of meiosis-sporogenesis at the level of hymenial cells.

From a biochemical point of view, one can notice interesting but isolated ex-

periments of RAUDASKOSKI & LU (1980) studying the effects of hydroxyurea

(HU) on meiosis and genetic recombination in Coprinus “lagopus”. The drug

was applied directly at the level of hymenial cells of primordia, at various devel-

opment stages. The effects of HU on the meiotic cell cycle suggest that the drug

can exert its effects by inhibiting the synthesis of deoxynucleotides: the two most

sensitive meiotic stages are mid-lat premeiotic S phase and pachytene-diplotene

period. Recently, MOORE & al. (1987) noticed that increase in GDHxapp yet

observed during terminal development of sporophores of Coprinus cinereus (cf.

STEWART & MOORE, 1974) ^... was initiated as karyogamy became evident;

enzyme activity stabilized for about 4 hours during meiosis, but resumed after

meiosis II and continued to increase until spore maturation... It is concluded that

expression of GDH app in the fruit-body cap of C. cinereus is either a compo-

nent part of the cellular programme involved in karyogamy, or is directly trig-

gered by that programme. Further study of this system will be an important con-

tribution to understanding of the immediate metabolic impact of nuclear fusion

events like fertilization”.

Cultures are then returned to classical photoperiodic regime (L/D: 12/12) where normal

meiosis and sporogenesis are observed. One can notice that basidia remain at “nuclear fu-

sion stage” when primordia remain under continuous light without lowering temperature.

Source : MNHN, Paris

314 G. MANACHERE

cultures

-36h -24 -22 2015.38

"a Î

a — re |

ig | sion

Le days #+-- 3,5 days -—*«- 5h. min. Pe ----—- 24h."L,D-12,12" or "Light"

PSD' or'Ligh" “Darkness”

Source : MNHN, Paris

REGULATION OF SPOROPHORE DIFFERENTIATION 315

CONCLUSION

There is an evident need for fundamental studies on different aspects of spo-

rophore differentiation in macromycetes, from primordia initiation to achieved

sporogenesis. Coprinus species remain useful models for such works, for in-

stance on metabolic processes, on nature of the photoreceptors, on hypothetical

fruiting-stimulating substances. The regulation of gene expression during some

fundamental events, particularly during primordia initiation merits a particular

attention. More than ever, the study of fungi - lower and higher - should contrib-

ute to a better understanding of various physiological general problems in plants,

such as photoperiodic control of reproduction in organisms, nature of blue and

U.V. photoinduced or photoinhibited reactions (cf. problem of "cryptochrome"),

interactions of external factors controlling morphogenesis and sporogenesis (par-

ticularly light and temperature) endogenous rhythmical processes of differen-

tiation of reproductive structures... In addition to such areas of prime interest,

there remain diverse correlations at all phases of growth and development, both

at the level of the individual sporophore and of the whole culture which consti-

tute a physiological unit analogous to a whole higher plant.

At last, from a strictly mycological point of view, most of the physiological as-

pects of meiosis and consecutive sporogenesis are still unknown. Researches in

this last field would indeed lead to a better understanding of the influence of vari-

ous external and internal factors on the reproduction of higher fungi, when act-

ing at the level of replication of DNA and successive meiotic events.

ACKNOWLEDGEMENTS

‘The author wishes to gratefully acknowledge Mrs Y. BASTOUILL - DESCOLLONG-

FS for valuable contribution to various researches mentioned in this paper and for prepara-

tion of most figures. Thanks are also due to Mr J.C. ROBERT for his help, particularly for

a critical review of this manuscript and preparation of figure 4, and to Mr R. DURAND

for useful complementary indications. Furthermore, particular thanks are due to Professor

G.W. GOODAY, University of Aberdeen, for a critical final review of this paper.

Fig. 9 - Influence of cytological and physiological stages of meiosis and sporogenesis on

“regeneration” potential of isolated lamellae of sporophores of Coprinus congregatus (af-

ter BASTOUILL - DESCOLLONGES & MANACHERE, 1984).

The drawing below the abscissa represent the degree of development reached by hyme-

nial cells when lamellae of the corresponding physiological stages (-36h, -24h, ... -10h)

are used as inocula (20 cultures/each physiological stage).

N.B.: at the top of the figure, illustration of direct [1 , mixed E3 and indirect E re-

generation.

Source

MNHN, Paris.

316 G. MANACHERE

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Cryptogamie, Mycol. 1988, 9(4): 325-333 325

UNE NOUVELLE ESPÈCE DE PYTHIUM

ISOLÉE D'UNE SALINE DE L'OUEST ALGÉRIEN

par Bernard PAUL *

RÉSUMÉ - Une nouvelle espèce, Pythium drechsleri présentant des sporanges contigus, des

oogones à paroi lisse et un système anthéridial complexe est décrite, Ce champignon à été

isolé de la saline d'Arzew (Sebkha d'Arzew) prés d'Oran. Les détails morphologiques de ce

champignon, sa tolérance au NaCl, ses relations taxonomiques avec d'autres espéces voisi-

nes ainsi que quelques facteurs écologiques influençant sa croissance sont étudiés.

ABSTRACT - A new species, Pyrhium drechsleri with contiguous sporangia, smooth walled

oogonia, and a complex antheridial system is described. The fungus was isolated from soil

samples taken from the salt-marsh of Arzew (Sebkha d’Arzew) near the city of Oran. Mor-

phological details of fungus, its tolerance of NaCl, its taxonomic relationship with other

species of Pythium, together with a note of some ecological factors influencing its growth

and reproduction, are discussed in this paper.

MOTS CLES : Pyrhium, taxonomie, tolerance NaCl, sporange contigu.

INTRODUCTION

Notre connaissance de la mycoflore des sols salés d'Algérie est très limitée.

DUBOST (1966) a isolé 74 souches de champignons des sols salins de l'ouest

Algérien mais aucune d'entre elles n'appartenait au genre Pythium. Au cours

d'une recherche sur les Pythium dans la saline d'Arzew, nous avons isolé une

nouvelle espèce. Ce champignom a été nommé Pythium drechsleri en hommage

de C. DRÉCHSLER dont la contribution à la connaissance relative au genre

Pythium et Phytophthora est considérable.

La Sebkha d'Arzew est située au nord-ouest de l'Algérie, à 8 kms de la mer

Méditerranée et à 20 kms de la ville d'Arzew. La superficie totale de la sebkha

est environ 250 hectares. L'apparition d'une sebkha est due à l'é aporation des

Caux phréatiques qui déposent en surface les sels dont elles sont chargées. Dans

les déflations, qui atteignent le niveau de la nappe phréatique, se forment des

mares d'eau salée permanentes ou temporaires dont les plus grandes constituent

les sebkhas (DUBOST, 1966). En hiver, la sebkha se remplit d'eau, atteignant

environ 1 mètre de profondeur, alors qu'en été l’eau s'évapore. Le sol de la

sebkha est argileux, ou limonoargileux et a un pH presque toujours supérieur a

8 et saturé en NaCl. Cette saturation provoque parfois la floculation des argiles,

MS d ons

Institut de Biologie, Université d'Oran, Es-senia, 31100 ORAN, Algérie.

Source : MNHN, Paris.

326 B. PAUL

donnant au sol une structure micropolyédrique qui rappelle celle d'un sable; par

contre en hiver et au printemps; quand la terre est gorgee d'eau, elle forme des

vases compactes et collantes particulièrement anaérobies (DUBOST, 1966).

Nous avons isolé le champignon 5 fois à partir de 40 échantillons de sol,

étudié sa morphologie, les caractères taxonomiques, sa tolérance au NaCl et les

fluctuations de concentration en sels totaux de la sebkha d'origine pendant une

année (Sept. 85 - Juin 86) pour bien démontrer les caractéres halophiles de

Pythium drechsleri.

MATÉRIEL ET MÉTHODES

Des échantillons de sol ont été prélevés superficiellement à la sebkha au lieu-

dit “Ferme du peuple". Il s'agit d'une surface rectangulaire de 300 x 30 m

légérement surélevée pár rapport au niveau de la sebkha proprement dite ct

peuplée par Halopeplis amplexicaulis Vahl. Le champignon a eté isolé par les

Phéthodes décrites pour Pythium capillosum et P. ornamentum (PAUL, 1987

ab). La purification des souches a été faite en utilisant des techniques simples

(PAUL, 1986 ab). Les cultures pures ont été transférées sur les milieux pomme

de terre - carotte agar(PCA), corn-meal agar (CMA) et sur eau gélosée.

Pour étudier ses températures cardinales, le champignon a été ensemencé

dans différentes boites de Pétri incubées à des températures allant de O0 à 50°C.

Pour estimer la tolérance de P. drechsleri au NaCl, ce sel a été incorpore à

différentes concentrations, entre 0 et 1000 mmol, dans le milieu PCA. L'étude a

été réalisée en boites de Pétri incubées à 25°C. Les mesures de croissance radiale

du champignon ont été effectuées quotidiennement.

Les mesures de la concentration en sels totaux ont été déterminées à partir de

mesures au conductimétre. La concentration en gl en sels solubles totaux est

calculée d'apres RICHARD & GOUNY (1965): à partir de la conductivité (sur

extrait aqueux du sol séché à l'air, rapport soleau = 1/5) exprimée en

milliohms em. Concentration en sels solubles totaux: C 0,64 x conductivité

OBSERVATIONS ET RÉSULTATS

La croissance de P. drechsleri est bonne sur PCA et CMA ainsi que sur les

graines de chanvre dans l'eau. Cependant, dans cette dernière condition il y a

fréquemment contamination par des bactéries et il est nécessaire d'ajouter des

antibiotiques pour conserver le champignon en milieu aqueux.

L'identification du champignon a été faite grace aux clefs données par

MIDDLETON (1943), MATTHEWS (1931), WATERHOUSE (1967) et

PLAATS-NITERINK (1981).

Pithium drechsleri sp. nov. (Pl. LIV).

Hyphae principales usque Sum diam. Sporangia contigua, zoosporae 18-20°C

formantur, zoosporae incapsulatae 6-8um diam. Oogonia laevia, globosa,

subglobosa, terminalia vel intercalaria, interdum catenata, 95-24um dian.

Antheridia unum vel plurima, interdum ramificata, monoclinata vel diclinata,

terminalia vel intercalaria; oogonium circumdantia, interdum constricta, raro

Source : MNHN, Paris

PYTHIUM 327

Planche I - 1-6: sporanges contigus, 7-8: vésicules avec zoospores; 9-16: appressoria

sphériques et en forme de faucille.

Plate | - 1-6: contiguous sporangia, 7-8: sporangial vesicles containing zoospores; 9-16:

spherical and sickle shaped appressoria.

Source : MNHN. Paris

328 B. PAUL

antheridia oriuntur ex appressorio; cellulae antheridiales inflatae. Oosporae

singulae, 9-20,8um diam. pleroticae | paries 1-2,5um crassus, globulosae.

sing entum radiale diurnim: 10,3 mm 25°C in agaro Solani tuberosi et carie

confecto. Temperaturae minima = 0-1°C, optima = 20-25°C, maxima = 40°C.

Holotypus in herbario universitati Oranensis conservatus (AS-23).

Le mycélium de Pythium drechsleri est hyalin, bien ramifié, jusqu'à Sum en

diamètre. Sur CMA et PCA les colonies sont submergées sans aucune formation

de mycélium aérien. Sur le dernier milieu elles ont un aspect composite mélant

rayons et rosettes. La croissance moyenne du champignon sur PCA a 25°C est

de 10,3 mm par jour. Les températures cardinales sont: minimum 0-1°, optimum

20-25° et maximum 40°C.

Les sporanges sont produits surtout dans l'eau ou bien sur eau pélosée. Ils

sont contigus avec plusieurs éléments sphériques et caténulés (Pl. I: 1-5; Pl. Hl:

7). ^ parür d'un de ces éléments nait un tube germinatif de dimension variable

portant sur son extrémité une vésicule (PI. I: 6-8; PI, Il: 6). Les zoospores sont

Por dans cette. vésicule à une température d'environ 20°C. Les zoospores

enkystées mesurent 6-8um de diamètre.

Les appressoria sont soit en forme de faucille, soit sphériques ei sont produits

en aoai ance sur PCA, CMA, ou eau gélosée. Parfois les appressoria en forme

Se abone se joignent bout à bout pour donner une structure de cordons (Pl. I:

12; PL. Il: 3-4). Il n'est pas rare de trouver des structures reproductives portées

par les appressoria (Pl. IIl: 19; PI. IV: 6)

Les oogones sont généralement petites, sphériques, terminales ou intercalaires,

parfois caténulées et toujours avec une paroi lisse. Elles mesurent entre 9,6 et

24um (moyenne 17,0) de diamètre.

La plupart des anthéridies sont monoclinales, parfois dic ales et même inter-

calaires, une à plusieurs par oogone. Une branche anthéridiale peut se ramifier,

entrer en contact avec plusieurs oogones et porter ensuite une oogone sur elle-

méme; parfois les anthéridies sont issues d'un appressorium. Le contact d'une

Dhheridie avec une oogone peut étre simple (Pl. Ill: 1, 2, 5. 11), ou alors

l'anthéridie entoure l'oogone (Pl. 111: 3, 9, 12; PI. IV: 4, 6) avec des constrictions

à plusieurs endroits (PI. III: 6; PI. IV: 3). Les cellules sont enflées et peuvent etre

1 à 5 par oogone.

Les oospores sont toujours plérotiques (PI. III: 13, 18) 9 à 20,8um de

diamaure (moyenne 16,9) avec une paroi lisse et mince mesurant de 0,93 à Aum

(moyenne 1,6) d'épaisseur.

Pythium drechsleri croit en présence de NaCl dans le milieu jusqu'à une

concentration de 800mmol. En effet sa croissance augmente avec Vincorporation

62050. 100, jusqu'à 200 mmol de NaCl, puis elle diminue brusquement à partir

de 300 mmol (Tabl. 1).

En ce qui concerne la reproduction, elle a été observée jusqu'à la concen-

tration de 100 mmol de NaCl. A 200 mmol il n'y a que trés peu de structures

reproductives, et à partir de 300 mmol, nous n'avons observé aucune anthéridie

où oogone. Cependant, le champignon continue à croitre jusqu'à une concen-

tration de 800 mmol (Tabl. 1).

Nous avons calculé la concentration en sels totaux de la terre dont provient

P. drechsleri et nous avons constate que celle-ci varie suivant l'accumulation ou

l'évaporation d'eau. L'évolution de la concentration en sels totaux de la terre de

Source : MNHN, Paris

PYTHIUM 329

Planche II - 1-2: appressoria portant des structures reproductives, 3-4: appressoria formant

des cordons, 5: appressorium en forme de faucille, 6: zoospores dans une vésicule juste

avant leur libération, 7: un sporange contigu. (échelle: 1-2 = 125um, 3 = 204m, 4, 5 et

7 = 16um, 6 =8um).

Plate 11 - 1-2: appressoria bearing reproductive structures, 3-4: appressoria forming rope

like structures, 5: sickle shaped appressorium, 6: zoospores in vesicle before liberation,

porum. sporangia. (scale: 1-2 = 125um, 3 = 20um, 4, 5 et 7 = l6um, 6

=8um).

la “Ferme du peuple” depuis le mois de Septembre 1985 jusqu'au mois de Juin

1986 est exprimée dans le tableau 2.

Source : MNHN. Paris

330 B. PAUL

30 yum

Planche III - 1-12: anthéridies et oogones, 13-18: oospores, 19: anthéridie et oogone portés

par un appressorium

Plate III - 1-12: antheridia and oogonia, 13-18: oospores, 19: appressoria bearing sexual

structures.

Source : MNHN, Paris

PYTHIUM 331

Concentration Croissance

de NaCl en mmol moyenne jour

en mm

0 10,3

50 120

100 136

200 133

300 80

400 50

500 32

600 0,5

700 027

800 0,14

Tableau 1: Croissance de Pythium drechsleri à différentes concentrations de NaCl à 25°C.

Table 1: Growth of Pythium drechsleri at different concentrations of NaCl at 25°C.

Mois sop oct nov dec jan fév mars avr mai juin

Concentration. 28,5 28 204 196 17 16 21 24.314 292

Tableau 2: Evolution de la concentration en sels totaux (g/l) au cours de l'année 1985:

Table 2: Seasonal fluctuation of total salt concentration (g/l) for the year 1985-86).

DISCUSSION

En même temps que Pythium drechsleri nous avons également trouvé une au-

tre espèce de Pythium (7 fois sur 40) qui a des caracteres trés proches de P.

drechsleri et quí fera l'objet d'une prochaine communication. La présence des

Pythium dans 12 des 40 échantillons nous améne à conclure que certaines

espèces de ce genre sont parfaitement adaptées aux dures conditions de la

sebkha d'Arzew.

In vitro notre champignon a pu croître jusqu'à une concentration de 800

mmol de NaCl. Cela représente 46,72 g de NaCllitre de solution. Dans la

sebkha d'Arzew pour l'année 1985-86 la plus forte salinité a été observée en Mai

1986 avec 31,4 g de sel litre (sels totaux). Nos résultats expérimentaux laissent

penser que P. drechsleri supporte une telle concentration de sel dans le sol et

qu'il pourra vivre pendant les années où la concentration de sel augmente

jusqu'à 46,72 g/1 (800 mmol).

Morphologiquement P. drechsleri est très different de toutes les espèces de

Pythium décrites jusqu’à ce jour. La combinaison des caractères présentés par P.

Source : MNHN. Paris

332 B. PAUL

30 um

Planche 1V - 1-6: reproduction sexuée - anthéridies et oogones de Pyrhium drechsleri.

Plate IV - 1-6: sexual reproduction - antheridia and oogonia of Pythium drechsleri.

drechsleri: sporanges contigus, oogones avec paroi lisse, anthéridies intercalaires,

est unique pour le genre. Les trois espèces munies de sporanges contigus sont Pe

oligandrum Drechsler, P. acanthicum Drechsler et P. ornamentum Paul, mais

Source : MNHN, Paris

PYTHIUM 333

chacune de ces trois espèces présente des oogones ornementées, tandis que à

drechsleri a des oogones à paroi lisse. P. vanterpooli Kouyeas & Kouyeas et P.

torulosum Coker & Paterson produisent des oogones à paroi lisse et sporanges

plus ou moins contigus, cependant aucune de ces deux espéces n'a d'anthéridies

Soit intercalaires soit avec des constrictions comme celles présentées par P.

drechsleri.

REMERCIEMENTS

Nous remercions le Pére Jean-Louis DECLAIS d'Ain-Temouchent pour la diagnose la-

tine, et Monsieur R. PERRIN de l'INRA de Dijon, France pour la révision du manuscrit

en français et pour ses conseils.

BIBLIOGRAPHIE

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Nat. Afrique N. 1: 9-28.

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Press, 136 p.

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genus Pythium. Mem. Torrey Bot. Club 20: 1-171.

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Kew, CMI.

Source : MNHN. Paris

Cryptogamie, Mycol. 1988, 9(4): 335-343 335

MYCOFLORA OF BROAD BEAN, CHICK-PEA

AND LENTIL SEEDS IN EGYPT

by A.LI. ABDEL-HAFEZ *

ABSTRACT - 69 species in addition to 4 varieties which belong to 22 genera were collected

from 32 samples of each of broad bean, chick-pea and lentil seeds gathered from eight

Governorates in Egypt on glucose-(18 genera and 59 species + 4 var.) and cellulose-(15

genera and 48 species + 2 var.) Czapek’s agar at 28°C. The results obtained from the 3

Types of seeds and on the 2 types of media were basically similar with the most frequent

genera were Aspergillus, Penicillium, Rhizopus, Mucor and Fusarium. From the preceding

genera A. niger, A. flavus, A. nidulans, A. terreus, A. flavus var. columnaris, P. chrysoge-

ium. P. clirinum. P. funiculosum, R. stolonifer, M. hiemalis and F. moniliforme were the

most prevalent, Also, several fungi were common or recovered only on cellulose agar plates

such as Chaetomium globosum, C. olivaceum, C. spirale, Acremonium strictum, Stachybotrys

chartarum, Microascus trigonosporus, Beauveria bassiana and Macrophomina phaesolina

RÉSUMÉ - A partir de graines de féves, de pois chiche et de lentilles (32 lots de chaque

provenant de 8 régions d Egypte), 69 espéces et 4 variétés de champignons, appartenant. à

52 genres, ont été isolées sur milieu Czapek contenant du glucose (18 genres et 59 espèces

4. 4 var.) ou de la cellulose (15 genres et 48 espèces + 2 var. à 28'C. Les résultats sont

équivalents pour les 3 types de graines et les 2 milieux avec, pour genres les plus fréquents,

Aspergillus, Penicillium, Rhizopus, Mucor et Fusarium. Parmi ces genres A. niger, A. flavus,

A. nidulans, A. terreus, A. flavus var. columnaris, P. chrysogenum, P. citrinum, P

funiculosum, R. stolonifer, M. hiemalis et F. moniliforme sont les espéces prépondérantes.

Plusieurs champignons sont également fréquents ou n'ont été trouvés que sur cellulose:

Chaetomium globosum, C. olvaceum, C. spirale, Acremonium strictum, Stachybotrys

chartarum, Microascus trigonosporus, Beauveria bassiana et Macrophomina phaseolina.

KEY WORDS : sced-borne fungi, broad bean, chick-pea, lentil, glucophilic fungi, cellu-

lose-decomposing fungi.

INTRODUCTION

Numerous investigations have been carried out on the seed- and grain- borne

fungi all over the world (ABDEL-HAFEZ, 1984a-b; CHRISTENSEN &

KAUFMANN, 1969; HANLIN, 1969; FLANNIGAN, 1978; LUTEY, 1963,

and several others) due to the deterioration of seeds and grains and production

of mycotoxins by associated fungi and their hazards to animal, bird and human

* Botany Department, Faculty of Science, Assiut University, Sohag, Egypt.

Source : MNHN, Paris

336 ALL ABDEL-HAFEZ

health (mycotoxicoses diseases) when used these seed and grain as feed and

foodstuffs.

In Egypt, information on the mycoflora of legumes crops is very limited and

most studies were focused on cereal grains (ABDEL-KADER & al., 1979; AS-

SAWAH & ELAROSI, 1960; EL-KADY & al., 1982; MOUBASHER & al.,

1972), Broad bean, chick-pea and lentil seeds are of the basic foods of people in

Egypt, production of the previous three types of seeds were about 310000, 20000

and 15000 tons in 1986 respectively, more than or sufficient for national con-

sumption. So that information on the composition, numbers and frequency of oc-

currence of glycophilic and cellulose-decomposing fungi of broad bean, chick-pea

and lentil seeds are of importance.

MATERIALS AND METHODS

Thirty-two samples of each of broad bean (Vicia faba L.), chick-pea (Cicer

arietinum L.) and lentil (Lens culinaris Medicus) seeds of the crop of 1986, of

0.5 kg each, were collected from eight Governorates in Egypt namely Cairo,

Giza, Beni-Suef, El-Minya, Assiut, Sohag, Qena and Aswan. The number of

samples from each Governorate and from each type of seeds was four. Each

sample was put in a sterile polyethylene bag sealed and put in another bag which

was also sealed. Storage of seeds in a double-bag container minimize the loss of

water content and yet gives sufficient aeration. Samples were kept in a cool place

during storage (3

Determination of moisture content of seeds

Replicate samples of seeds were milled and a weighed portion of their powder

was dried in an oven for 24 h at 105°C, then cooled in a desiccator and re-

weighed. The moisture content is expressed as percentage of the dry-weight.

Determination of germinability of seeds

100 seeds of each of broad-bean, chick-pea and lentil were incubated at 25°C

over a pad of moist sterile filter paper placed in a sterile petri-dish for 8-12 days

during which the seeds with healthy roots and plumules were counted and the

counts are expressed as percentages of the numbers of tested seeds.

Determination of seed-borne fungi

This was made by using the seed-plate method as employed by MOUBASH-

ER & al. (1972). Glucose-(10 g1) and cellulose-(19 g,1) Czapek’s agar were used

for isolation of glycophilic and cellulose-decomposing fungi, respectively. Rose

bengal (1/15000) was used as a bacteriostatic agent (SMITH & DAWSON,

1944). Five seeds of each of broad bean, chick-pea and lentil seeds were placed

on the surface of each of sterile glucose- and cellulose-Czapek’s agar medium.

Ten plates were used for each sample (5 for each medium). The plates were in-

cubated at 28°C for 7-10 days and the developing fungi were counted, identified

and calculated per 25 seeds. The colonies of slow growing fungi were transferred

to slants to ensure precise counting and then to plates for identification. Other

agar media were also used (Czapek’s + 0.05 yeast extract, malt agar and potato

dextrose agar) for identification of fungi.

Source : MNHN, Paris

MYCOFLORA OF LEGUMINOUS SEEDS 337

RESULTS AND DISCUSSION

The moisture content of broad bean, chick-pea and lentil samples (on oven-

dry basis) was considerably low and ranged between 5.6-7.9%, 6.4-8.7% and

4.9-6.5%, respectively. This means that the ability of associated fungi to grow,

penetrate and damage the embryos of seeds during storage is low since fungi

growth is dependent on high levels of moisture in seeds. Therefore, there is no

appreciable differences between the germinability of the three types of seeds test-

ed and the germination rate of the previous seeds ranged from 98% to 100%.

ycophilic fungi

The total count of fungi in seed samples tested fluctuated between 9-79, 30-84

and 5-56 colonies per 25 seeds of broad bean, chick-pea and lentil, respectively.

It is axiomatic that seed samples with high values of moisture contents contained

high numbers of fungi and vice versa. The highest numbers of glycophilic fungi

was recorded in chick-pea (1835 colonies per 25 seeds in every samples) followed

by broad bean (1570 colonies) and lentil seeds (975 colonies).

Fifty-nine species in addition to 4 varieties which belong to 18 genera were

collected from 32 samples of each of broad bean (15 genera and 45 species + 3

var.), chick-pea (13 genera and 45 species + 3 var.) and lentil seeds (12 genera

and 35 species + 2 var.) on glucose-Czapek’s agar at 28°C (Tabl. 1-2). Most of

these species were firstly isolated from Egyptian legumes seeds, but almost the

majority of them were recovered previously from cereal grains and peanut seeds

(ABDEL-KADER & al., 1979; ASSAWAH & ELAROSI, 1960; EL-HELALY

& al., 1968a-b; EL-KADY & al., 1982; MOUBASHER & al., 1972).

Aspergillus was the most common genus and was emerged from approximate-

ly 100 %, 97% and 94% of the samples contributing 55.7%, 54.2% and 43.6%

of total fungi in broad-bean, chick-pea and lentil seeds, respectively. It was re-

presented by 21 species and 3 varieties (1S + 2, 16 + 3 and 12 species + 2 var.

in broad bean, chick-pea and lentil seeds, respectively) of which A. niger, A.

flavus and A. nidulans were the most prevalent in the three types of seeds. They

occurred in about 50-97% of the samples comprising 4.2-59.9% of total

Aspergillus and 2.3-32.8% of total fungi in the 3 substrates tested. ABDEL-HA-

FEZ (1984b) isolated 16 species and 2 varieties of Aspergillus from 10 samples

of each bean, broad bean, lentil, lupine and pea seeds collected from Saudi Ara-

bia (Taif and EL-Baha regions) and the most common species were A. niger and

A. flavus on the 5 types of seeds. PARVEEN & DHIRENDRA (1981) isolated

A. niger, A. sydowi, A. terreus, A. flavus and A. nidulans from 4 different samples

of lentil seeds from India. It is worthmentioning that several members of

Aspergillus niger and A. flavus groups are widely distributed in soils and air and

other substrata including stored seeds and grains, spoiled fruits and vegetables,

animal and plant residues and various types of food product; cosmopolitan.

Also, several isolates of A. flavus, A. ochraceus and A. nidulans which isolated

from seeds and grains are well known that have the ability to produce mycotox-

ins (aflatoxins, ochratoxins and sterigmatocystins, respectively) as reported by

numerous workers (F.A.O., 1979; MISLIVEC & al., 1975; SCOTT & al., 1972;

SCHROEDER & BOLLER, 1973; EL-KADY & al., 1984). These compounds

(mycotoxins) have been implicated in diseases of human and as causing myco-

toxicoses in animals and fowls (DAVIS & DIENER, 1978; EL-ZAWAHRI &

al., 1977; SCOTT & al., 1970; SMALLEY & STRONG, 1974). A. terreus

(34.4% of the samples, 2.4% of total Aspergillus and 1.3% of total fungi) and A.

flavus var. columnaris (28.1%, 2.9% and 1.6%) in broad bean; A. terreus

Source : MNHN, Paris

338 A.LI. ABDEL-HAFEZ

GLUCOSE CELLULOSE

IRA Broad bean Chick-pea Lenti] Broad bean Chick-pes Lentil

ns xc ww ns nc us ic £e | NS ox [ns e NS x o

Aspergillus 15: 55.7 100 |16:3 54.2 96.9 |12«2 43,6 93,8 |12r2 59.2 100 [142 61.3 96.9 [1142 48.4 93.8

var. var. var. var. var. var.

Penioiilium 10° 28.7 96.9] 11. 29.1 91.8 | 8 36.9 95,6 | 9 22.3 4.4) 8 19.0 81.3] 7 18.5 62.5

Rhizopus 3 79 mal à 95 84 2 7766| 3 72 79| 3 9.3 75.0) 2 21.9 81.3

Mucor 3 21 49| 2. 15 314| 3. 4.7 43.8] 2 2.0 31.3) 2 11 21] 2 34 219

Fusarium 4 0.8 2.1| 5 1.0 31.3] 3 0.7 125] 2 1.6 18.8] 4 18 313] 1 19 94

Ce E A e A reper CES iac NN ine =

Cineinelta een ere MNT EM EEE CN

Cladosporiur d ese Lose ecce sen | ves epo t enis so ee oos SE LOCIS

Neurospora T Yost Calle EN A a ER

Trichoderma 22 ou DEMENS EE E ET I

Altemaria 1 Ce &i|i mz A E 0.3 ED A 6.5) 10 0.2 Bae - -

Botryotrichun cH iit. Gee SP E RE T

Chaetomium i oor eale re) ee 3950 | SR 2:9 28107 | 3,2 Sie Tani E e sg

Hurisola CUES eee se eller ar, ee, A o

var.

Aerementun DECEM d Rss MEO Seri tod

Drechslera MR ET re E A En ES ESS 1) PE En

| re EURO ANUS le ce 2 MT gle S

Pasoitoryces a AN A a ETE S B

Stachybotrys NN rE nals

Mioroasous EE EAL DAA

Boaweria eA a E a a an TRE

Maorophomina "onn secu Ex Er MUR EEE

Stavite mpceliun lets 04 la AE LS ee nest) 1005 0, tay 0B bs

Total 4g 10 — - Mes 100 — - age 00 — - Jie 100 - fage vo — - Jg v o -

‘Table 1 - Number of species (NS), percentage counts (%C, calculated per total counts of

fungi) and percentage frequency of occurence (%F, calculated per 32 samples) of vari-

ous genera recovered from broad bean, chick-pea and lentil seeds on glucose- and

cellulose-Czapek's agar at 28°C.

Tableau 1 - Nombres d'espèces (NS), pourcentages (%C) et fréquences d'apparition (%F)

des différents genres isolés de graines de fèves, de pois chiche et de lentilles sur milieu

Czapek (glucose et cellulose) à 28°C.

(31.3%, 2.2% and 1,2%) and A. fumigatus (25%, 1.2% and 0.65%) in chick-

pea; and A. terreus (25%, 3.1% and 1.3%) and A. ochraceus (25%, 3.3% and

1.4%) in lentil seeds were isolated in moderate frequency of occurrence. The pre-

vious Aspergillus species were isolated, but with variable numbers and frequency,

from Saudi Arabian leguminous crops (ABDEL-HAFEZ, 1984b).

ABDEL-HAFEZ & SHOREIT (1986a) recorded that 4. niger, A. flavus, A. ter-

reus, A. fumigatus and A. ochraceus or A. nidulans was the most common in

some Egyptian legumes crops. The remaining Aspergillus species were less fre-

quent and accounting collectively 7.9-11.8% of total Aspergillus and 4.4-6.2% of

total fungi in the previous three types of seeds (Tabl. 2).

Penicillium was the second most common genus and encountered in nearly

97%, 94% and 91% of the samples comprising 28.7%, 29.1% and 36.9% of tot-

Source : MNHN, Paris

MYCOFLORA OF LEGUMINOUS SEEDS 339

al fungi in broad bean, chick-pea and lentil seeds, respectively. From the genus

14 species (10, 11 and 8 species in the 3 types of seeds, respectively) were col-

lected of which P. chrysogenum, P. citrinum and P. funiculosum were the most

common; these occurred in about 13-91% of the samples giving rise to

6.9-66.7% of total Penicillium and 2.2-24.6% of total fungi in seeds tested. AB-

DEL-HAFEZ & SHOREIT (1986a) found that the previous species were preva-

lent, but with variable numbers and frequency, from some legumes seeds col-

lected from Upper Egypt. ABDEL-HAFEZ (1984b) identified 14 species of

Penicillium from bean, broad bean, lentil, lupine and pea seeds gathered from

Taif and EL-Baha regions at Saudi Arabia, and who found that P. citrinum in

bean; P. citrinum, P. corylophilum, P. frequentans, P. notatum and P. oxalicum

in broad bean; P. brevicompactum and P. citrinum in lentil; P. citrinum and P.

notatum in lupine; and P. chrysogenum, P. citrinum and P. funiculosum in pea

seeds were the most prevalent. The remaining Penicillium species were less fre-

quent and listed in Table 2.

Rhizopus, ranked third in the number of cases of isolation. It emerged in

about 78%, 84% and 66% of the samples contributing 7.9%, 9.5% and 7.7% of

total fungi in broad bean, chick-pea and lentil seeds, respectively. From the gen-

us 3 species were collected of which R. stolonifer was the most common in the 3

types of seeds. This species was also common in some seeds and grains collected

from Egypt (MOUBASHER & al, 1972; ABDEL-KADER & al, 1979;

EL-HELALY & al., 1968a-b; ABDEL-HAFEZ & SHOREIT, 1986a) and Saudi

Arabia (ABDEL-HAFEZ, 1984a-b). R. oryzae and R. arrhizus were less fre-

quent (Tabl. 2).

Mucor, ranked fourth in the number of cases of isolation and occurred in ap-

proximately 47%, 34% and 44% of the samples comprising 2.7%, 1.5% and

4.7% of total fungi in broad bean, chick-pea and lentil seeds, respectively. It was

represented by 3 species of which M. hiemalis was the most prevalent; M.

circinelloides and M. racemosus were less frequent. ABDEL-HAFEZ & SHO-

REIT (1986a) isolated M. hiemalis and M. circinelloides from Egyptian bean, lu-

pine and pea seeds. ABDEL-HAFEZ (1984b) collected 4 species of Mucor from

5 types of leguminous seeds of Saudi Arabia of which M. hiemalis in broad

bean, lentil and pea; and M. racemosus in bean and lupine seeds were the most

common. M. circinelloides and M. pusillus were less frequent and were isolated

only from broad bean and bean.

Fusarium was isolated in moderate or low frequency of occurrence from seeds

tested and occurred from nearly,28%, 31% and 13% of the samples comprising

0.8%, 1% and 0.7% of total fungi in broad bean, chick-pea and lentil seeds, re-

spectively. Five species were identified and these were F. moniliforme, F. grami-

nearum, oxysporum, F. solani and F. semitectum. ABDEL-HAFEZ (1984b)

isolated F. moniliforme, F. oxysporum and F. solani from legumes seeds at Saudi

Arabia. The remaining genera and species were of low or rare frequency and ac-

counting collectively for 4.2%, 4.6% and 6.4% of total fungi in the 3 types of

seeds, respectively (Tabl. 2). SUMMAR & HOWARD (1983) found that the

common microflora in dry peas, processing and dry beans, fababeans, lentils and

soybeans from Alberta (Canada) were species of field fungi: A/ternaria, Penicil-

lium, Rhizopus, Fusarium and Botrytis.

Source : MNHN, Paris

340 A.LI. ABDEL-HAFEZ

GLUCOSE CELLULOSE

Genera and species Broad bean] Chick-pea | Lentil [Broad bean [Chtck-pea | ..Lentil

ge mcr) tc Ner| tc nerf re nee nf nr

Total count 1570 - iss - | 975 - |1325 - fizo = | 785 -

Aspergillus gs 32 | 995 31 | 425 30 | 785 32 | 870 31 | 380 30

a. niger Van Tiegh. 515 31 | 520 30 |17 29 | 395 30 | 370 31 | 102 2

A. flavus (Link) Fes. 205 30 | 205 28 |146 24 | 175 29 | 249 28 | 75 26

A. nidulans Eidam 40 19 42 1 | 32 16 | 28 16 | 38 21 | 35 19

A. terreus Thom a u| 2210] 33, a a 9,4 1 3 9

A. flavus Vr. columaris Link æ 9| 4» 7| m 5| 50 13] 55 16 | 4 10

À: nidulans var. latus Thom & Raper Manet ess ss tss Et 23 ana e

4; ockraceus Wilhelm 335 8 eco» Rue hu CNT OURS

funigatuo res. d 4| 8| 2 3| 3 1| 45 m|ss 9

tamarii Kita e E 2h MO a}

l datus (Bain.) Thom 8 Church else d uas

L verateoler (Nuill.) Tirat smod Pas: res | mtis oid m d c

rugulosus Thom & Raper 4 2 = = LE aaa

clavatus Desmazieres 230559 EE cal

flavipes (Bain. b Sart.) Thoms Church RE RENE RE

parasitious Speare p T Raul sat =

aude (Bain. & Sart.) Thom & Church Fuera bouts uera reserare d Rasen:

candidus Link ex Fr. eR es emere RE RE e

sayptiaeus Moubasher & Moustafa esr den ur] EP ES rats

Senatus Kwon & Fennell pee ds ET aes LT

| oameus (Van Tiegh.) Blochwitz Eres epu adea ce Ls :

| quadrilineatus Thom & Raper eu Brent Ie

| ruber Thom & Church Re ea Te | Manes | eee | Feuer A ERI

| niveus Blochwitz Re EE Et

nidulans Var. acrtatatuo Fennell & Reper | - -| 2 1 ET ES

A. melleus Yukawa ze ans bou a t Re Eu RENE

4. oryzae Wehner ES | eae elem eee eral Gram te

Ponieiliiun aso 31 | 535 30 | 360 29 | 295 27 | 270 26 | MS 20

P. chryeogenim Thom 203 28 | 265 29 | 260 27 | 122 25 | 105 22 | 55 16

P. citrinum Thom a 12 |102 i| 57 7| $1 19 | 57 12 | 42 9

P. funiculooien Thom 6 3| 4) 7| 2% 4] 3 13| 3 8|2 5

‘ealiouen Thom a e| æ 6 | uw 3] 2 0 4 6) 4 4

jeeenii Zaleski fe weal EE d zw re x

nigricans Bainier BoA R E E AE H eE

brevi-corpactuy Dierckx | Re UE | URAI EE ees

oyolopium Westling FOE xp reete Sen pedcs enr m

frequantano Mestling de E CS ER EU

omy tophi tun Dierckx ed ete gt ee |e oli eos |e eS

rubrun Sopp t| aeos | RS PES

seeokit Zaleski - gera pte essc on RE”

7 duslauri Delacroix ENRE E E S

danthinellum Biourge zov €x DS ESS

albidum Sopp Ci OL

P. véridieatun Mestling Era | PRE

Bhincpus 124 25 | 175 27 | 75 21 | 96 23 | 132 24 | 172 26

R. stolonifer Ehren. ex Fr. Lindt 95 22 | 120 25 | 60 18 | 75 20 | 102 23 | 137 21

R. onyaae Went & Gerlings 31 6] 37 7| 4| 15 7] 9|393 8

R. arrhizua Fischer w ae E a ra E =

Mucor a3 16 | 2 u| æ aja aoj 9 | 27 7

M. hiemalis Wehmeyer a u| ie 7| 2 9| 19 6| 0 8] a $6

M. circinelloides Van Tieghem 35-08. id 74-14-83] 8-43 | 6 os 8

M. racemosus Fresenius KANE a a Eod eR co ens

usariwr wz 3| 1 1] 7 4] 2 6| % 9 1 3

P. monilifome Sheldon ate || Es e se ge E eral eat,

F. graminsarun Schwabe RE EL S p

F. ozjsporum Schlecht. zd aae d dS TERREA a E

P. solani (Mart.) Sacc. (SE EE ed USE

E. semiveotum Berk. & Rav. sey | eee an a | ae | ete ell Per oe

Syncephatlastrum racemosws (Cohn) Schroet. a rie eismod E MER a

Qireinella maces (Sorok.) Berl. & De Tont | 10 6| 5 4| 3 6| - sae :

Cladosporium herbarum (Pers.] Link ex Fr. FEBE rE A a =- 2

Neurospora crassa Shear à Dodge MA E E S a a ap ie:

Trichoderma viride Pers. ex Gray D A calomel TO gee A Oem nee lee aa

Source : MNHN. Paris

MYCOFLORA OF LEGUMINOUS SEEDS 341

Cellulose-decomposing fungi

The total count of putative cellulose-decomposing fungi in seed samples tested

fluctuated between 5-58, 17-63 and 3-42 colonies per 25 seeds of broad bean,

chick-pea and lentil, respectively. The highest counts of this group of fungi as in

case of glycophilic fungi was also obtained in samples with relatively high mois-

ture contents and vice versa. Forty-eight species and 2 varieties belonging to 15

genera were collected on celfulose-Czapek’s agar plates at 28°C, which means a

narrower spectrum of genera and species than on glucose agar and this is rea-

sonable since glucose is a more easily utilizable carbohydrate by fungi. The re-

sults obtained on cellulose agar plates were basically similar to those on glucose

with the most frequent genera being Aspergillus, Penicillium, Rhizopus, Chaeto-

mium, Fusarium, Mucor and Stachybotrys. They emerged from about 9-100% of

the samples comprising 1-61.3% of total fungi in seeds examined. From the pre-

ceding genera, Aspergillus niger, A. flavus, A. nidulans, A. terreus, A. nidulans

var. columnaris, P. chrysogenum, P. citrinum, R. stolonifer, Chaetomium

globosum, C. olivaceum, F. moniliforme, M. circinelloides and Stachybotrys

GLUCOSE CELLULOSE

Genera and species (Contd. ) Broad bean |Chick-pea | Lentil [Broad bean |Chick-pea | Lentil

To Ner| tc Net) tc Net] tc cr] tc ner] 1c nci

4 2

lternaria alternata (Fr.) Keissler

jotryotriehun atragriaeun Van Beyna

atomiin

2. globosum Kunze ex Fr,

x apinate Toph

4. grioea Traaen

Juscoatra Var. fuascatra Traaen

renontun etrioium W. Gams

Prechalena state of Cochliobolus ap

copulariopaie brevicaulta (Sacc.) Bainter

Pacctlomyoes vardotit Bainter |

Stachybotrys chartarum (Ehrenb. ex Link) Hugues

Woveasous trigonogporus Emmons à Dodge

Seaweria basotana (Bals.) Vuill.

Maovephoména phaseottna (Tassi) Goidy.

Sterile mycelium 4

son

52 x 12

Tcstotal count per 25 seeds in every sample; NCI=nunber of cases of isolation.

High occurrence: isolated more than 15 cases (out of 32 samples), moderate occurrence: from 8 to 15 cases, low

occurrence: from 4 to 7 cases, rare occurrence: less than 4 cases.

Table 2 - Total counts (calculated per 25 seeds of each type in every samples) and number

of cases of isolation (out of 32 samples) of fungal genera and species recovered from 32

samples from each of broad bean, chick pea and lentil seeds on glucose- and cellulose-

Czapek’s agar at 28°C.

Tableau 2 - Genres et espèces de champignons isolés à partir de graines de fèves, de pois

chiches et de lentilles (32 lots de chaque) sur milieu Czapek (glucose et cellulose) à 28°C.

Source : MNHN. Paris

342 ALI. ABDEL-HAFEZ

chartarum were the most prevalent. They occurred in about 13-94%, 9-97% and

9-88% of the samples comprising 0.6-29.8%, 0.4-26% and 0.8-17.5% of total

fungi in the 3 types of secds, respectively. Most of the preceding species were re-

covered previously, but with variable numbers and frequency, on cellulose agar

plates from some types of seeds and grains collected from Upper Egypt (AB-

DEL-HAFEZ & ABDEL-KADER, 1980; ABDEL-HAFEZ & SHOREIT,

1986b). Most of the fungal species recovered on cellulose agar plates were re-

ported to be cellulose-decomposing but with variable ability (FLANNIGAN,

1970; MAZEN, 1973, STEWART & WALSH, 1972; TRIBE, 1957, 1961,

1966). The remaining genera and species were less frequent and listed in Table 2.

In conclusion, the present results reveal that Aspergillus, Penicillium, Fusari-

um, Mucor and Rhizopus were consistently the most frequent genera in broad

bean, chick-pea and lentil seeds on the 2 isolation media. But, Chaeromium, Sta-

chybotrys, Beauveria, Microascus, Macrophomina and Acremonium were com-

mon only on cellulose agar plates. Also, comparison between the lists of fungi re-

covered in the present investigation and from some other legumes seeds in Egypt

(ABDEL-HAFEZ & SHOREIT, 1986b), Canada (SUMAR & HOWARD,

i ARAMA & SHARAMA, 1978; PARVEEN & DHIRENDRA,

Arabia (ABDEL-HAFEZ, 1984b) reveal that there is no fungal

flora characteristic of Egyptian leguminous crops, but these list may differ in the

numbers and in order of frequency of the component fungi.

BIBLIOGRAPHY

ABDEL-HAFEZ A.LI, and SHOREIT A.A.M., 1986a- Mycoflora of bean, lupine and pea

seeds in Egypt. Aswan Sci. Techn. Bull. 7: 121-142.

ABDEL-HAFEZ ALL and SHOREIT A.A.M., 1986b - Cellulose-decomposing fungi of

bean, lupine and pea seeds in Egypt. Aswan Sci. Techn. Bull. 7: 143-160.

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Source : MNHN, Paris

Cryptogamie, Mycol. 1988, 9(4): 345-361 345

LES INTOXICATIONS PAR LES CORTINAIRES

par Didier MICHELOT * et lan R. TEBBETT **

RÉSUMÉ - Les Cortinaires provoquent chaque année, tant en France qu'à l'étranger, des

intoxications graves conduisant les personnes atteintes à une insuffisance rénale aiguë. Les

espèces mycologiques principalement mises en cause sont Cortinarius orellanus, C.

speciosissimus et C. splendens, mais il n'est pas exclu que d'autres espèces du genre soient

toxiques,

Les toxines présumėes responsables sont l'orellanine et les cortinarines. Si leurs modes

d'action respectifs sont encore inconnus, ils ont fait l'objet de plusieurs hypothèses justifiées

par des analogies de structures et d'actions avec des produits chimiques ou pharmaceu-

tiques. Nous faisons ici le point sur les connaissances acquises concernant les intoxications,

les substances responsables et leurs mécanismes d action.

ABSTRACT - Every year, Cortinarius mushrooms are responsible for severe poisonings in

France and abroad leading’ intoxicated people to acute renal failures. The species of concern

are mainly Cortinarius orellanus, C. speciosissimus and C. splendens but it should not be ex-

cluded that other species within the genus may prove toxic as well.

The toxins in question are presumably orellanine and cortinarins; although their respective

modes of action are still unknown, several assumptions have been made, justified by struc-

tural and biological similarities to chemical or pharmaceutical substances. Our current

knowledge of the poisonings, the chemical material incriminated and their mechanisms is

being reviewed.

MOTS CLÉS : Cortinaires, Cortinarius orellanus, Cortinarius speciosissimus, Cortinarius

splendens, néphrotoxines, orellanine, cortinarines, intoxications.

INTRODUCTION

Au regard de la systématique, les Cortinaires représentent un ensemble com-

plexe dont certaines espèces, peu répandues en général mais souvent localement

abondantes, sont régulièrement la cause d’empoisonnements trés graves

(MOSER, 1983). Des intoxications rapportées à l'ingestion de Cortinarius

orellanus (Fries) Fries, Cortinarius speciosissimus (Kühner et Romagnesi) et

Cortinarius splendens Henry ont été signalées à plusieurs reprises et convena-

blement décrites; de surcroit, les connaissances actuelles issues des recherches

* UA 401 CNRS, Laboratoire de Chimie, Muséum National d'Histoire Naturelle, 63 rue

Buffon, 75005 Paris Cedex, France.

** Forensic Science Unit University of Strathclyde, 204 George Street, Glasgow G1 1XW,

Grande-Bretagne.

Source : MNHN, Paris

346 D. MICHELOT et I. R. TEBBETT

dans des domaines divers, laissent présager que nombre d'espèces au sein du

grand genre Cortinarius sont potentiellement très toxiques. La consommation de

ces champignons entraine une insuffisance rénale aiguë retardée. La nature des

substances chimiques incriminées a été partiellement élucidée : ce sont

l'orellanine, à structure bipyridinique, mais aussi les cortinarines à structure

cyclopeptidique; elles ont été décelées également dans des spécimens appartenant

à d'autres espéces du genre. Le mécanisme d'action de ces toxines reste

hypothétique; toutefois, au vu de certaines analogies de structure chimique, il a

été rapproché de celui de composés dont l'action pharmacologique est micux

cónnue.

Devant les dangers auxquels s'exposent les mycophages et pour répondre aux

demandes d'information émanant des services de toxicologie, nous tentons de fai-

re ici le point des connaissances acquises concernant, outre les intoxications, les

substances responsables et leurs mécanismes d'action.

HISTORIQUE DES INTOXICATIONS PAR LES CORTINAIRES

Le premier dénombrement systématique des cas d'intoxications occasionnées

par ces champignons a été fait en Pologne par GRZYMALA de 1953 à 1962

(GRZYMALA, 1964a, 1965). Il a recense 135 intoxications attribuees à C.

orellanus, a la suite desquelles 95,8% des personnes ayant consommé ce champi-

fron furent gravement atteintes. Parmi elles, 19 décéderent; le taux de mortalité

‘Meve donc a 14%. Par sa toxicité, cette espèce prend la deuxième place

f rière l’ Amanita phalloides (Vaillant ex Fries) Secrėtan. Par la suite, d'autres

T #dents dus à C. orellanus ont alors été signalés en Europe : en 1976, 3 intoxi-

_.LDMEIER, 1977) et plusieurs en France, dont 2 cas dans l'Est

{MARICHAL & al., 1977), et une intoxication collective en Bretagne (BROUS-

SE & al., 1981). Très récemment, une intoxication a été rapportée à Perpignan

en novembre 1987 (DELPECH, 1987); 3 mois auparavant, en septembre 1987,

26 militaires participant à un raid de survie dans le Morbihan ont été victimes

d'une intoxication collective; le complexe ore/lanus semble étre à l'origine de ces

derniers accidents (THOMAS, 1987).

Une espèce voisine, C. speciosissimus est, elle aussi, toxique (AZEMA, 1981).

En effet, 4 cas ont été mentionnés en Finlande en 1972 (HULMI & al., 1974),

d’autres en 1979 en Suéde (HOLMDAHL & al, 1980, 1984), en Ecosse

(SHORT & al., 1980; WATLING, 1982) et en Allemagne (NOLTE & al., 1987).

Une troisième espèce, C. splendens, sans rapport systématique étroit avec les

deux précédentes, a provoqué à son tour un empoisonnement en Suisse

(SCHLIESSBACH & al., 1983) et une intoxication collective en Haute-Savoie en

1979 (FINAZ DE VILLAINE, 1981; GERAULT, 1981; COLON & al., 1981).

Les manifestations cliniques sont analogues à celles des deux Cortinaires

précédents mais les empoisonnements moins sévéres.

La toxicité de ces trois espéces est donc indiscutable.

Bien que des cas d'intoxications par d'autres espéces du genre n'aient pas été

aussi catégoriquement signalés, celle liste n'est certainement pas exhaustive.

D'une part, les premiers symptômes apparaissent tardivement et donc l'identifi-

cation des espèces responsables repose sur une détermination a posteriori qui est

difficile et délicate de par la complexité systématique du genre. D'autre part les

éléments fournis par des expérimentations récentes sur l'animal et par des analy-

Source : MNHN, Paris

INTOXICATIONS PAR LES CORTINAIRES 347

ses chimiques laissent présager que d’autres espèces de ce genre puissent être tout

aussi toxiques. Les connaissances acquises sur l'image clinique des intoxications

par C. orellanus, C. speciosissimus et C. splendens suggérent que ces champi-

gnons et d'autres du genre, compte tenu de la longue periode de latence, ont été

responsables de quelques cas d'insuffisance rénale de causes alors inconnues.

SYMPTOMATOLOGIE ET CARACTERISTIQUES CLINIQUES DE

L'INTOXICATION PAR LES CORTINAIRES

Au travers de l'analyse des observations cliniques concernant les intoxications

aigués causées par C. orellanus, C. speciosissimus et C. splendens, il est possible

de dégager la trame d'un tableau symptomatologique commun et spécifique du

syndrome "cortinarien".

L'intoxication est caractérisée par un temps de ‘latence exceptionnellement

long, cette période d'incubation supérieure à 48 heures dépasse trés largement

celles communément observées lors d'intoxications graves par d'autres champi-

gnons, comme Amanita phalloides, par exemple. ll faut noter que cette

particularité rend possible des consommations répétées de champignons toxiques

avant l'apparition des premiers symptômes et peut aussi, vraisemblablement, en-

trainer des erreurs de diagnostic par oubli du rapprochement avec la consom-

mation de champignons sauvages. Les symptômes apparaissent 2 à 20 jours

aprés l'ingestion et plus la période d'incubation est courte, plus l'intoxication se

révèle être sévêre. Des nausées, vomissements et diarrhées accompagnés de dou-

leurs gastriques, abdominales et lombaires surviennent brusquement, puis ces

troubles digestifs disparaissent spontanément. Après un bref délai, une soif inten-

se avec sécheresse de la bouche, une sensation de froid, des frissons sans

température apparaissent, suivis d’anorexie, de fatigue musculaire et de

céphalées. Une insuffisance rénale aiguë s'installe progressivement avec oligurie

ou anurie. L'examen biologique des urines met en évidence une albuminurie,

leucocyturie et hématurie. Le bilan sanguin montre bien entendu une élévation

du taux d'urée et de créatinine, ainsi qu'une hyperleucocytose, A l'examen

histopathologique du tissu rénal, on observe dans la plupart des cas une néphrite

tubulo-interstitielle aiguë avec atteintes tubulaires pouvant aller jusqu'à la

nécrose et une infiltration cellulaire avec ademe et fibrose interstitielle. Si ces

lésions sont importantes, l'insuffisance rénale peut devenir chronique et définitiv

elle peut alors nécessiter des dialyses répétées et une transplantation devenue in-

dispensable pour restaurer la fonction rénale.

A ces manifestations rénales peuvent s'ajouter des troubles neurologiques :

somnolence, perte de conscience, convulsions, tremblements des muscles du visa-

ge, ainsi que des signes cutanés. L'atteinte hépatique éventuelle se réduit à une

faible cytolyse.

Le schéma 1 résume la chronologie et l'évolution des intoxications par ces

trois champignons.

Le traitement, effectué en milieu hospitalier, devrait être prioritairement

orienté vers l'élimination du composé toxique de la circulation sanguine, comme

l'ont suggéré HOLMDAHL & al. (1984), peutétre par hémoperfusion et

hémodialyse, et ce, méme si l'ingestion du champignon remonte à plusieurs

jours. Lors d'intoxications graves ou traitées tardivement, le traitement approprié

se réduit au traitement symptomatique de l'insuffisance rénale aiguë (LARCAN

& al., 1979; GARNIER, 1983).

Source : MNHN, Paris

348 D. MICHELOT et I. R. TEBBETT

Une particularité de l'intoxication par les Cortinaires est le caractère secon-

daire de l'atteinte hépatique; cet élément permet la distinction et l'élimination de

l'éventualité d’une intoxication par d'autres champignons vénéneux tels que I’

Amanita phalloides (WIELAND, 1986; MICHELOT & al, 1985) ou la

Gyromitra esculenta (MICHELOT & al., 1989).

Période de latence variable

Consommation unique

ou peu abondante

L—— — be Rétablissement # — Guérison

|Lombalgies, douleurs abdominales, Insuffisance rénale

vomissements, diarrhée, chronique.

asthénie (dialyses itératives

ou transplantation rénale)

Insuffisance rénale

aiguë

(Gonsommations répétées) Etre res

RE EEE ag Décès

Zjours purs 77 3 mois Emos > Temps

Schéma 1 - Chronologie et évolution des intoxications par les Cortinaires.

Scheme 1 - Chronology and development of the poisonings by Cortinarius mushrooms.

LES PRINCIPES TOXIQUES

En 1962, GRZYMALA a isolé de C. orellanus une substance qu'il désigna

sous le nom d'orellanine; celle-ci, expérimentée sur l'animal, produisait les mè-

mes effets toxiques que le champignon lui-même. TESTA, en 1970, suggéra que

cette substance était un mélange dont il nomma les quatre principaux composés

grzymaline, benzoine a et b et cortinarine.

Les premiers pas vers une analyse chimique réelle ont été accomplis en 1975

par ANTKOWIAK & GESSNER qui ont isolé un produit pur et, en 1979, la

structure de l'orellanine (1) a éterminée comme étant le N,N’-dioxyde de la

tétrahydroxy-3,3", 4,4 bipyridine-2:2° (ANTKOWIAK & GESSNER, 1979).

Après certaines incertitudes, ils ont confirmé la structure de cette molécule par la

synthèse chimique totale (1984). Par la suite cette méthode a été améliorée et

étendue à la synthèse d’autres produits de la même série (DEHMLOW &

SCHULZ, 1985, 1987; TIECCO, 1986; TIECCO & al., 1984, 1986, 1987).

L'orellanine a aussi été isolée de C. speciosissimus, la réduction de ce composé

par voie chimique, ou sa décomposition par la chaleur et par la lumière, condui-

sent à l'orellinine (Il), puis à l'orelline (III) non toxique (ANTKOWIAK &

GESSNER, 1985).

Source : MNHN, Paris

INTOXICATIONS PAR LES CORTINAIRES 349

A^ on Ls

aren WN AA oy,

Ho Ho

Sew Z^ "wn

HO. Z o^ c

1 i "

Orellanine. Orellinine Orelline

KURNSTEINER & MOSER (1981) ont purifié à leur tour, à partir de C.

orellanus, une toxine léthale, sensible à la lumière, et remarquable car possédant

la propriété d'agir après un temps de latence très long. Cette toxine présentait de

fortes similitudes en spectroscopie d'absorption (infra-rouge et ultra-violette) avec

l'orellanine d'ANTKOWIAK & GESSNER; elle formait aussi un dérivé jai

non toxique, cependant des propriétés telles que stabilité thermique et solubi

semblaient les différencier.

HOLMDAHL & al. (1987) ont récemment isolé l'orellanine à partir de C.

speciosissimus et confirmé sa structure et sa néphrotoxicité par une étude sur le

rat. KELLER-DILITZ & al. (1985) ont également détecté par chromatographie

sur couche mince la présence d’orellanine et d’autres composés fluorescents chez

C. speciosissimus, C. orellanoides Henry et C. rainierensis Smith et Stuntz.

ANDARY & al. (1986) n'ont de plus détecté l'orellanine que dans les espèces

suivantes appartenant à la section Orellani : C. orellanus et C. speciosissimus

(sous-genre : Leprocybe ); elle n'a pas été détectée chez C. bolaris (Persoon ex

Fries) Fries. La teneur est de l'ordre de 2g/100g de champignon sec.

L'orellanine semble donc étre un des produits directement impliques dans la

toxicité de ces champignons, quoique possédant une formule chimique structu-

rale et fonctionnelle peu stable et originale pour cette classe d'organismes

végétaux. .

Des composés fluorescents distincts des précédents ont été extraits de C.

speciosissimus ils sont retrouvés dans la plupart des espèces de Cortinaires.

L'analyse structurale des composés purifies a indiqué une structure

cyclopeptidique (CADDY & al., 1982). Par la suite, à partir de C. speciosissimus,

trois principaux composés de structure analogue ont été isolés et identifiés : ce

sont les cortinarines A (IV), B. (V) et C (VI), dont les deux premières sont

néphrotoxiques chez la souris (TEBBETT & al., 1983; TEBBETT & CADDY,

1983, 1984a; TEBBETT, 1984); la cortinarine C différe principalement des deux

précédentes par l'absence du pont thioëther joignant deux acides aminés du cy-

cle: cette différence structurale peut rendre compte d'une absence de toxicité

(vide infra). Les extraits obtenus à partir de 61 espèces différentes appartenant

au genre Cortinarius ont été examines en Chromatographie Liquide Haute Per-

formance (HPLC) pour détecter la présence et la quantité des diverses

cortinarines. La cortinarine A a été détectée parmi toutes, mais avec des concen-

trations diverses entre les différentes espèces. Seule C. violaceus, qui est une

Source : MNHN, Paris

350 D. MICHELOT et I. R. TEBBETT

Phe — HN - CH — CO — Val — Orn Phe — HN — CH —CO— Val — Om

8 | ow |

CH; CH,

c LT Leu ne Leu

N N

lys

» H Nemh H

les —Thr — OC CH —NH— isoLeu Thr — Ala — isoLeu

IV Corlinarine A, R-OCH; Vi Cortinarine C

toxique non toxique

V. Cortinarine B. R- OH

toxique

espèce de Cortinaires couramment considérée comme sans danger en est exemp-

te. Les concentrations maximales sont de 0,47%, 0,43%, 0,45% respectivement

pour C. speciosissimus, C. orellanus et C. orellanoides, espéces considérées com-

me les plus toxiques du genre. Cette toxine est aussi presente en quantités no-

tables dans certaines espèces non encore recensées comme toxiques; ce sont C.

turmalis Fries : 0,33% et C. betulorum (Moser) Moser : 0,28%; le seuil minimal

de détection est 0,004% ( C. croceifolius (Peck) Moser). La cortinarine B n'a été

trouvée que chez trois espèces : C. speciosissimus (0,60%), C. orellanus (0,52%)

et C. orellanoides (0,47%). L'ensemble de ces données suggèrent que la toxicité

des espèces de Cortinaires est une fonction croissante de la somme des concen-

trations relatives en cortinarines A et B (TEBBETT & CADDY, 1984b). La

cortinarine C, non toxique, est présente également dans les 61 extraits étudiés.

A la lumière des connaissances actuelles, il semble donc que la toxicité des

Cortinaires ne puisse pas être attribuée spécifiquement et exclusivement à une

espèce moléculaire, orellanine ou cortinarines, mais plutôt à une combinaison de

ces substances en s'étendant éventuellement à d'autres encore inconnues.

ETUDE IN VIVO ET IN VITRO DE LA TOXICITE DES CORTINAIRES

La toxicité des Cortinaires sur les organismes vivants a fait l'objet de nom-

breuses études expérimentales.

GRZYMALA a été l'un des premiers à vérifier expérimentalement la toxicité

de C. orellanus sur des animaux de laboratoire (GRZYMALA, 1964b). Chez le

chat, le cobaye ou la souris, il a observé une toxicité aiguë quel que soit le mode

d'administration (voie buccale, injections sous-cutanées ou péritonéales), et des

modifications histopathologiques analogues à celles observées chez l'homme lors

d'intoxications graves.

Source : MNHN, Paris

INTOXICATIONS PAR LES CORTINAIRES 351

Le rein est toujours la cible élective; les lésions quasi-irréversibles atteignant

cet organe sont tenues dans la majorité des cas pour responsables de la mortalité

observée. Elles se manifestent au niveau de l'épithélium tubulaire par une

nécrose aiguë conduisant à une insuffisance rénale et leur gravité est fonction de

la dose. GRZYMALA a remarqué que la toxicité est indépendante du lieu et de

la date de la récolte et qu'elle reste qualitativement la méme, que le champignon

soit absorbé frais, sec ou cuit (GRZYMALA, 1964b, 1965). La dose léthale de

principe toxique sur l'animal, quoique approximative compte tenu de la purifica-

lion trés sommaire et d'accumulations éventuelles du toxique, est de l'ordre de 5

mg par kilogramme. Cette valeur a été confirmée par VIALLIER & al. (1967);

ils ont de plus montré que, chez le rat et le cobaye, C. speciosissimus et C.

orellanoides produisent les mémes effets toxiques que C. orellanus. D'autres

espèces de Cortinaires : C. cinnamomeus Linnė ex Fries, C. phoeniceus (Bulliard

ex Maire) Moser et C. sanguineus Wulf ex Fries provoquent également, mais

après une période de latence plus longue, la mort de l'animal. RICHARD & al.

(1985) ont donné une valeur de la dose léthale moyenne (DL.) de l'orellanine

administrée par voie péritonéale : elle est de l'ordre de 12,5 mg kg; cette dose est

sans commune mesure avec celle de Sg kg calculée par modelisation Q.S.A.R.

(Quantitative Structure Activity Relationship) en comparaison avec des systèmes

moléculaires analogues; au vu de cette différence, les auteurs remettent en ques-

tion la structure exacte de l'espèce chimique toxique effectrice.

La sensibi individuelle des animaux testés est très variable. En effet, une

même dose de champignon induit des lésions rénales très importantes chez cer-

tains sujets tandis que, chez d'autres appartenant au méme groupe testé, l'état

reste normal (MOTTONEN & al., 1975). En suivant l'évolution de l'intoxication

chez le rat, ces auteurs ont aussi remarqué que les premiers signes se manifestent

sous la forme d'infiltrats au niveau interstitiel deux jours après l'ingestion du

champignon et l'inflammation quatre jours après; enfin apparaissent les nécroses

dans les tubules de la zone corticale (NIEMINEN & al., 1975). Au cours de la

même étude, NIEMINEN & PYY (1976a) ont observé des différences de

sensibilité individuelle qu'ils expliquent par une variabilité génétique; 20 à 30%

des animaux apparaissent résistants à la toxine tandis que, pour les sujets sensi-

bles, la gravité des lésions est fonction de la dose. Ils ont observé aussi que, selon

le sexe, des différences apparaissent au niveau des lésions rénales induites par C.

speciosissimus les femelles y seraient moins sensibles (NIEMINEN & PYY,

1976b). Cette observation a été confirmée par FINAZ DE VILLAINE (1981).

Pour essayer de comprendre le mécanisme de cette toxicité et éventuellement de

proposer un traitement, NIEMINEN (1976) en a étudié les modifications sous

l'influence de certains médicamènts. Il a observé les effets de synergie avec du

furosémide, du phénobarbital, de la phénylbutazone et du cyclophosphamide.

Ainsi, chez le rat, l'administration du furosemide, diurétique, avant l'ingestion du

champignon, potentialise les nécroses rénales mais n'a pas d'incidence sur lin-

flammation (NIEMINEN & al., 1976a). Il en conclut que la toxine doit atteindre

le rein à forte concentration quelques heures après l'absorption et que l'appari-

tion de nécroses décelables en histologie requiert un délai minimum de deux

jours. Le prétraitement par du phénobarbital, qui augmente le métabolisme

hépatique, intensifie aussi les lésions rénales dans la zone corticale mais n'agit

pas sur l'inflammation; il parait donc fortement probable que la toxine “efficace”

est un produit de métabolisme hépatique, et non la protoxine elle-même.

NIEMINEN suggère ainsi l'existence de deux toxines structuralement différentes

et à propriétés biologiques distinctes, l'une à action rapide, l'autre à action plus

lente. La phénylbutazone ne semble avoir aucun effet sur la néphrotoxicité de C.

speciosissimus. Quant au cyclophosphamide, immunodépresseur, administré en

méme temps que le champignon, il prévient l'inflammation rénale et les seules

Source : MNHN, Paris

352 D. MICHELOT et I. R. TEBBETT

lésions observées se situent au niveau des canaux collecteurs à l'extérieur de la

zone médullaire. Il en a déduit alors que ces derniers doivent être les premiers si-

tes d'action de la toxine (NIEMINEN & al., 1976b).

HOLMDAHL & al. (1980) ont également montré que C. speciosissimus, C.

gentilis et C. orellanus sont néphrotoxiques chez la souris, ainsi que C. limonius,

espèce jamais incriminée jusqu'alors. C. speciosissimus et C. orellanus parais-

sent, en outre, plus toxiques que les deux autres. La DLso approximative pour la

souris est de 2g de champignon sec par kilogramme (soit approximativement 20g

de champignon frais par kilogramme d'animal de laboratoire); naturellement, ces

valeurs doivent être considérées avec circonspection car il doit être tenu compte

de la variabilitė de la concentration en toxine tout comme de la sensibilité indivi-

duelle chez le sujet; elles ne doivent pas être extrapolées directement à l'espéce

humaine.

GERAULT (1981) a démontré la toxicité de C. splendens sur la rate et

retrouvé les observations faites par NIEMINEN avec C. speciosissimus: c'est-à-

dire une éventuelle résistance de certains animaux et une evolution de l'atteinte

rénale identique. Une étude comparative entre C. orellanus, C. splendens et C.

vitellinus (Moser), réalisée par GERAULT et interprétée par FINAZ DE

VILLAINE dans sa thèse, a mis en évidence les mêmes phénomènes et confirme

donc la néphrotoxicité de ces trois espèces de Cortinaires. Dans le même article,

la toxicité potentielle de tous les Cortinaires est fortement suggeree (GERAULT,

1981).

L'action des toxines de C. orellanus a été étudiée au niveau cellulaire par

GSTRAUNSTHALER & PRAST (1983). Les bipyridines-2,2’ et -4,4° portent

des architectures moléculaires comparables au Diquat et au Paraquat et par delà

à l'orellanine. L'effet de ces substances a été étudié sur des cultures de cellules

épithéliales de rein de porc ainsi que sur l'activité des enzymes de la membrane

apicale du méme organe. La morphologie des cellules testées se rapproche de

celle des cellules des rats intoxiques in vivo par C. orellanus. Les enzymes qui ont

été dosées jouent un rôle essentiel dans l'utilisation et la réabsorption de certai-

nes biomolécules telles que les peptides et les polysaccharides, car elles clivent les

substances non directement assimilables. Les activités de la

y-glutamyltranspeptidase, de la phosphatase alcaline et de, la leucine-

aminopeptidase sont diminuées par les deux bipyridines à des degrés divers dans

ces deux cultures de cellules. Ces travaux ont été poursuivis par des tests avec la

toxine fongique purifiée. L'orellanine de C. orellanus est douze fois plus

inhibitrice de la croissance cellulaire que la bipyridine-2,2' et vingt fois plus que

le dérivé -4,4. Elle diminue davantage le$ activités enzymatiques de la

phosphatase alcaline membranaire et de la lactate deshydrogénase

cytoplasmique. L'absence d'atteinte de la membrane cellulaire et la rupture de la

couche monocellulaire confirment un mode d'action intracellulaire de l'orellani

(HEUFLER & al., 1987). Quoique ne restituant pas dans leur intégralité les

mécanismes régulateurs inhérents à un modéle expérimental in vivo, ce modèle

utilisant les cultures de cellules rénales permet une bonne évaluation de la

néphrotoxicité de l'orellanine.

Des tests de cytotoxicité de l'orellanine ont été aussi réalisés sur des cultures

d'organismes monocellulaires. Elle inhibe la croissance de l'amibe Dictyostelium

discoideum et de la bactérie Escherichia coli (KLEIN & al., 1986). La toxine

purifiée ne cause aucune inhibition significative de la phagocytose et de la

pinocytose de l'amibe, alors que HOLMDAHL et ses collaborateurs avaient

décrit une forte inhibition de la pinocytose chez l'amibe Amoeba proteus en utili-

sant de l'extrait brut de C. speciosissimus (AHLMEN & al., 1983).

Source : MNHN, Paris

INTOXICATIONS PAR LES CORTINAIRES 353

MÉCANISME DE LA TOXICITÉ

De nombreuses expérimentations ont été réalisées en vue de discerner la

responsabilité des substances identifiées et différentes hypothèses ont été

proposées pour déterminer le mécanisme de la toxicité.

Une des premières hypothèses s'est appuyée sur les analogies de structure et

donc d'action biologique entre, d'une part, l'orellanine et ses dérivés, et d'autre

part, le Diquat et le Paraquat, produits herbicides largement utilisés.

ne dc

(^ (e

f ES A

ES ^7

(S sm (M

We OE

CH,

vi vi

Diquat Paraquat

Le Diquat (éthylidéne-1,1’ bipyridylium-2:2’, Reglone, VII) et le Paraquat

(diméthyl-1,1’ bipyridylium-4:4’, Gramoxone, Méthylviologen, VIII) sont des

herbicides de structure bipyridinique dont la toxicité sur les mammifères et donc

sur l'espèce humaine se manifeste spécifiquement par des lésions pulmonaires.

Quoique les mécanismes fins de l'action toxique restent encore à approfondi

il est communément admis qu'ils se situent au niveau du système redox

caractéristique de la structure de cette molécule de Paraquat. Jn vivo et in vitro,

la molécule de Paraquat réduite en l'espèce intermédiaire PQ*- par action du

NADPH cellulaire (Nicotine-amide Adénine Dinucleotide Phosphate

Hydrogénée, qui est un des cofacteurs des enzymes participant aux réactions

d'oxydo-réduction) retrouve instantanément sa forme oxydée PQ** en présence

d'oxygéne moléculaire; les anions superoxydes et hydroperoxydes ainsi générés

contribuent alors à la formation d'espèces radicalaires très toxiques conduisant à

des réactions de peroxydation des lipides membranaires. Une autre conséquence

de ce transfert d'électrons est la transformation totale du NADPH en NADP,

faisant tomber le taux de NADPH en deçà d'un seuil limite en dessous duquel

les fonctions chimiques et biochimiques ne peuvent avoir lieu. L'une ou l'autre

voie, ou bien la combinaison des deux, conduisent à la mort de la cellule

(SMITH, 1985, 1987).

Par analogie avec le mécanisme cytologique du Diquat et du Paraquat,

HOILAND (1983) a proposé un mécanisme impliquant des réactions en chaine

d'oxydo-réduction intracellulaires avec formation de radicaux libres: au cours de

Source : MNHN, Paris

354 D. MICHELOT et I. R. TEBBETT

ce cycle; il y aurait production de superoxyde cytotoxique et diminution de la

concentration de NADPH. Il a également décrit l’action destructrice d'extraits de

C. speciosissimus sur les pigments et les chloroplastes de la lentille d'eau Lemna

minor couramment utilisee pour tester les herbicides, tandis que des extraits de

C. gentilis (Fries) Fries, C. limonius (Fries ex Fries) Fries, C. cinnamomeus

(Linné ex Fries) et C. armillatus (Fries) Fries restent sans effet. On peut suppo-

ser que, si l'orellanine est directement impliquée dans cette action, elle serait ab-

sente ou en faible quantité dans les espèces précitées. Par ailleurs, il faut noter

que la ressemblance structurale et fonctionnelle entre les deux effecteurs que sont

le Paraquat et l'orellanine est limitée; aucune lesion pulmonaire n/a été observée

chez les personnes intoxiquées par les Cortinaires. En réalité, il a éte montré que

les comportements électrochimiques des deux molécules sont totalement

differents: dans une expérimentation examinant l'action de l'orellanine sur

Lemna minor, RICHARD & al. (1987) ont montré que l'orellanine ne pouvait ni

être facilement réduite in vivo ni former une espèce radicalaire réactive; en effet,

les électrons issus de l'eau ou du NADPH ne peuvent réduire l'orellanine dans

les cellules animales et végétales suivant la modèle de HØILAND.

Ainsi, l'hypothèse rapprochant l'action du Paraquat et de Vorellanine ne peut

être retenue.

L’orellanine purifiée à l'abri de la lumière et administrée per os à des doses

inférieures ou égales à 50mg kg est sans effet sur les souris, tandis que des doses

équivalentes de ce compose purifié à la lumière du jour provoquent la mort de

l'animal. Compte tenu de l'innocuité de l'orellanine native et du produit de

photodécomposition final, l'orelline, ANDARY & al. (1986) ont explique la

toxicité du produit initial par la formation, au cours de la phototransformation,

d'espèces chimiques intermédiaires génératrices de structures à noyau

isoxazolinium, pouvant se lier de façon covalente avec de nombreuses protéines

de l'organisme. Il apparaît ainsi que les molécules de photodégradation

immediate de l'orellanine jouent un rôle primordial. Ceci peut être comparé au

réarrangement photochimique observé assez fréquemment avec certaines

molécules à fonction N-oxyde. Ces résultats expérimentaux rejoignent d'ailleurs

ceux qui ont montré que la toxicité observée de l'orellanine ne correspond pas à

la toxicité "théorique" calculée en appliquant les équations de quantification de la

relation structure-activité (QSAR) et avec lesquelles on obtient une DL; très

élevée par rapport à la DL; expérimentale (vide supra).

Si l'orellanine a bien la structure bipyridinique proposée par ANTKOWIAK

& GESSNER (1979), alors son mécanisme d'action doit étre différent de celui

des molécules de la méme série et l'implication de ses métabolites est plus que

probable.

Les cortinarines A et B sont des cyclopeptides pontés par un groupement

tryptathionine et, par certaines analogies entre son action pharmacologique et les

symptómes de l'intoxication cortinarienne, le mécanisme toxique des cortinarines

peut être comparé au mode d'action de la vasopressine (IX)(I EBBETT, 1984):

- les cortinarines et la vasopressine ont une structure cyclique. L'ouverture du

pont disulfure rend la vasopressine inactive et celle du pont thioëther transforme

les cortinarines A et B en cortinarine C qui n'est pas toxique,

- la présence du groupement phénolique est nécessaire pour leur activité; la

phénylalanine, indispensable à l'action de la vasopressine, est aussi présente dans

la structure des cortinarines,

- l'acide aminé basique (l'arginine chez la vasopressine et la lysine chez les

cortinarines) potentialise leurs activités.

Source : MNHN, Paris

INTOXICATIONS PAR LES CORTINAIRES 355

_— NH — cH —co

Gly — Arg — Pro — Cys | dr

CH,

NH, Phe

Gin

ere

COOH — Cys

T — sn

Vasopressine

En établissant de tels parallèles entre la vasopressine et les cortinarines, il est

possible de faire l'hypothèse d'une action sur des récepteurs identiques au niveau

du rein.

La vasopressine a une action sur le tubule distal et les canaux collecteurs, ce

qui conduit å une rétention d'eau. Elle stimule le tractus gastro-intestinal produi-

sant nausées, crampes et diarrhées. Elle est vasoconstrictrice et induit donc une

hypertension avec paleur.

D'après les observations d'intoxications cliniques ou expérimentales, on re-

marque que les cortinarines agissent sur le tubule distal et les canaux collecteurs,

ce qui entraine une oligurie où une anurie. Elles produisent une gêne intestinale

avec nausées, vomissements, diarrhées et douleurs intestinales. Elles provoquent

de l'hypertension et une sensation de froid.

Cette hypothèse repose sur des similitudes structurales sensibles, une autre

s'appuie sur la potentialité de bioactivation des cortinarines.

Les cortinarines A et B provoquent des lésions rénales similaires chez la sou-

ris, alors que la cortinarine C, qui n'est pas un sulfure, ne semble pas être toxi-

que (TEBBETT, 1984) ll est possible que les cortinarines A et B soient

métabolisées en un méme composé plus toxique: un sulfoxyde de cortinarine B

biologiquement activable (FLYNN & ASH, 1983; ASH & al., 1984; TEBBETT,

1984, 1986; MICHELOT & aL, 1985; MICHELOT & LABIA, 1989).

L'intermédiaire hautement réactif subséquent serait susceptible d'atteindre le

récepteur cible suivant la représentation du schéma 2. L'intervention du systéme

enzymatique hépatique sur la fonction sulfure est prévisible et serait intense de

par la présence de monooxygénases à flavine (FAD monooxygénases) et du

Cytochrome P 450 (MADESCLAIRE, 1986; HOLLAND, 1988) Cette

hypothèse est en accord avec les observations de NIEMINEN qui pense que la

toxine est un métabolite. Elle pourrait expliquer deux éléments particuliers de

l'intoxication. Le temps de latence d'une part: le métabolite lentement libéré par

le système hépatique doit être en quantité suffisante pour agir; la différence de

sensibilité individuelle et sexuelle d'autre part: la capacité de métabolisation n'est

en effet pas la même suivant les individus et le sexe (NIEMINEN & PYY,

Source : MNHN, Paris

356 D. MICHELOT et I. R. TEBBETT

1976a, b). Nombre d'organismes vivants possèdent une gamme d'effecteurs

étrangers et bien souvent faciliter leur élimination hors de l'organisme. Il arrive

e que les produits de transformation, les métabolites, se révèlent être de

ants poisons. Le Cytochrome P 450, capable de O-déméthylation et de

Ron (TAKATA & al, 1983), présente des variations génétiques et

sexuelles (KALOW, 1987) et pourrait etre impliqué dans la transformation de la

toxine.

cH,

cH—cHOH—cH,OH

NEC ne NH Nas Hi

isoLeu

HAM — CO — CH —NH— co—Gly

x xes DLso 03 mo/Kg

XI X= (R)S—0 (a-Amanitin)

OL 59 = 0,3 mg/Kg

Xl X2 (S)S—0 Dlg =20mg/Ka

La réactivité et la toxicité potentielle de la fonction sulfure au sein du grou-

pement tryptathionine du système cyclopeptidique avait déjà été montrée par

WIELAND (1984) dans le cas de l'x-amanitine. Il a démontré que le sulfure (X)

issu de la réaction chimique du groupement sulfoxyde de cette molécule est aussi

toxique que le produit naturel (XI). On doit supposer que ce sulfure est oxyde in

situ dans le foie, spécifiquement et de facon irréversible, en sulfoxyde (Xl) de

configuration R très toxique (le dérivé S-sulfoxyde (XII) de synthèse est cent fois

moins toxique) (WIELAND, 1984; MICHELOT & al., 1985). Ainsi, de la même

façon, on peut supposer que les cortinarines À et B, initialement sous forme de

sulfures, génèrent dans le foie in situ les cortinarines sous forme de sulfoxydes

qui par la suite sont acheminées à leur cible, le rein, où elles ont déjà été

détectées sous cette forme (TEBBETT & CADDY, 1984a).

Source : MNHN, Paris

INTOXICATIONS PAR LES CORTINAIRES 357

Oxydation hépatique

— s+ 0

(monooxygénases à flavine

ou du Cytochrome P450)

Cortinarine A ou B

Sne Cortinarine A ou B

sulloxyde

Transfert vers le rein.

Transformation en un

intermédiaire réactif

Action toxique rénale

Schéma 2 - Métabolisme des cortinarines A et B.

Scheme 2 - Metabolism of cortinarins A and B.

CONCLUSION

Si les responsabilités des principes actifs de intoxication “cortinarienne” mis

principalement en évidence chez C. orellanus, C. speciosissimus et C. splendens

ne sont pas encore clairement établies, leurs analogies d'action au niveau rénal

laisse supposer qu'ils pourraient participer à part égale à l'intoxication mais avec

des voies légèrement différentes.

De nombreuses autres espèces du genre Cortinarius en contiennent et sont po-

tentiellement très dangereuses, même si des intoxications par ces dernières n'ont

pas été explicitement signalées. L’avancement des connaissances complemen-

taires à un niveau interdisciplinaire, dans des domaines tels que la chimie, la bio-

chimie et la médecine, devraient permettre dans un avenir très proche d'en

préciser le mode d'action. Il est évident que les intoxications par les Cortinaires

ne sont un problème de santé crucial, ni en France, ni dans quelque autre partie

du monde, car un petit nombre de cas sérieux sont répertoriés chaque année.

Toutefois, des intoxications collectives se produisent parfois, ainsi que l'actualité

Va récemment souligné. L'étude du mécanisme de cette intoxication est plei-

nement justifiée non seulement pour les problèmes de santé qu'elle pose, mais

aussi et surtout par le modèle toxicologique unique qu'elle constitue à l'heure ac-

tuelle pour les néphrologues.

Concrètement et dans l'immédiat, le devoir du mycologue, du pharmacien et

du médecin est de déconseiller globalement la consommation des Cortinaires, en

insistant plus particulièrement sur ceux qui présentent des colorations jaune,

orange ou rouge parmi lesquels se comptent les toxiques majeurs.

Source : MNHN, Paris

358 D. MICHELOT et I. R. TEBBE

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360 D. MICHELOT et I. R. TEBBETT

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Source : MNHN, Paris

INTOXICATIONS PAR LES CORTINAIRES 361

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Source : MNHN. Paris

Cryptogamie, Mycol. 1988, 9(4): 363-372 363

EFFECT OF SHORT TERM FUNGICIDAL PROGRAMME ON

NON-TARGET PHYLLOPLANE FUNGI OF SOYBEAN

by Renu PANDEY and Vijay KUMAR *

ABSTRACT - Five fungicides (captan, ziram, mancozeb, zineb and carbendazim) were sep-

arately applied on field grown soybean plants to find out their effects on phylloplane fungal

population and particularly Alrernaria alternata. Ziram caused appreciable decline in popu-

lation of the latter with litile recovery afterwards, Fungicides reduced both composite phyl-

loplane population and number of taxa immediately after spray compared with control; but

the long term effect was subsequently nullified in later samplings. Fungal species tolerant to

spray included non-pathogenic Alternaria alternata, Cladosporium herbarum and Mortierella

subtilissima. Populations of Aspergillus nidulans and Penicillium citrinum were notably pro-

moted following treatments. Diversity values under individual fungicidal stress lowered with

age. Evidences of leaf surface early recolonization after short term spray programme is an

indicator to design appropriate chemical application.

RÉSUMÉ - Cinq fongicides ont été appliqués sur soja afin d'étudier leurs effets sur la po-

pulation fongique du phylloplan, et particuliérement sur Alternaria alternara. Le ziram pro-

voque un déclin de ce dernier avec peu de reprise ultérieure. Comparés aux contróles, et

immédiatement aprés application, les fongicides réduisent la population du phylloplan et le

nombre de taxa; cependant, les effets décroissent avec le temps. Des espèces non

pathogènes sont tolérantes: Alternaria alternata, Cladosporium herbarum et Mortierella

subtilissima, et les populations d’ Aspergillus nidulans et de Penicillium citrinum augmentent

après traitements. Les études sur la recolonisatin des feuilles de soja après un programme à

court terme peuvent permettre de mettre au point une application chimique appropriée.

KEY WORDS : fungicides, phylloplane fungi.

INTRODUCTION

During the last two decades much information has accumulated on phyllo-

plane saprophytic population (FOKKEMA, 1981). Recent interest in environ-

ment has aroused concern on interaction of fungicides and non-target micro-or-

ganisms (HISLOP, 1976; ANDREWS, 1981).In most of the studies impact of

assessment have typically dealt with the immediate effect of pesticidal treatment

on non-target flora (BAINBRIDGE & DICKINSON, 1972; DICKINSON,

1973). DICKINSON & WALLACE (1976) assessed the spectrum of activity of

expensive fungicidal programme with the objective of finding out immediate and

a relatively long term (one year later) disturbances in microbial community. In

* Department of Botany, Christ Church College, Kanpur-208001, India.

Source : MNHN, Paris

364 R. PANDEY et V. KUMAR

our experiments interactions of fungicides with leaf surface fungi were examined

using relatively short spray programmes designed chiefly in relation to leaf spot

pathogen of soybean.

MATERIALS AND METHODS

Soybean cultivar T49 was grown in the plots of botanical garden of the Christ

Church College (Kanpur) in winter season (Nov-March) 1984, Replicate plots

were sprayed with captan (WP 50), ziram (WP 80), zineb (WP 75), mancozeb

(WB 75) and carbendazim each being applied on two occasions; at the preflow-

ering and the flowering stages of the plant. Meteorological regime prevalent in

duration between cach sample dates are averaged and presented in Fig. 1. Fun-

gicides used in these experiments were suspended in sterile distilled water and a

concentration of 2.0ug ml svas prepared based upon their active ingredient (a.i.)

in the commercial formulation.

Untreated control

Fungi cm?

S ò

3 10

e 0.

P g 70 30 ©

z Te

5 s

ri Er

-reg fier

Sı S2 S3 4 Ss S6

Fig. 1 - Distribution of leaf surface fungi and meteorological variables during samplings,

Fig. 1 - Distribution des champignons à la surface des feuilles, et variables météorologiques

durant l'expérimentation.

Collection of leaf samples for the determination of fungal population was

made on the seventh day after application of fungicides. Fully expanded leaves

generally the fourth and the fifth alternate leaf counting from the top bud were

plucked with sterilized forcep and were collected in new polyethylene bags. The

leves belonging to one treatment were pooled to make a composite whole.

Sample population were obtained by isolating fungi using dilution plate meth-

od. Fifty leaf disks (5 mm) punched with sterile cork borer at 4 points from

treated and control leaves were introduced separately into 250 ml flasks contain-

ing 50 ml sterile distilled water and shaken vigorously for 10 min. The homogen-

Source : MNHN, Paris

EFFECTS OF FUNGICIDES ON PHYLLOPLANE FUNGI 365

ate was diluted so that 1 ml would yield 30-50 colonies per plate. Petridishes

containing Czapek-dox-agar medium were gently rotated clockwise and anti-

clockwise to ensure uniform distribution of homogenates. Incubation was done at

24 + 2C under a bank of fluorescent light. Determinations of fungal colonies

were made after 5 days until 15 days.

Statistical analysis of the data

Non-normal data preventing criteria for analysis of variance were obtained on

phylloplane population. In order to stabilize variance the data on colony count

per replicat treatment was transformed for analysis by taking the Jx + 1/2 val-

ues which was found a suitable transformation before proceeding to ANOVA.

In order to characterize pesticidal impact on phylloplane community follow-

ing indices of species structure have been used:

(1) Shannon index of general diversity ( H ):

E ni n

HX C Igea

N N

where n, is the number of individuals of each species and N is the total number

of individuals.

(2) Evenness index (e): -

logs

where H = Shannon index;s — number of species

(3) Index of similarity (S) between different treatments at different stages of

crop growth:

A+B

where A = number of species in sample A, B = number of species in sample B,

C = number of species common to both samples.

(4) Index of dissimilarity = 1-S

RESULTS

Quantitative data

Table | shows a picture of phylloplane community with distribution of

composite phylloplane fungi, number of fungal taxa, diversity ( H ), evenness

index (e), index of similarity (S) and index of dissimilarity (1-S). The spectrum of

activity of different fungicides on non-target phylloplane fungi and on the

pathogen population is shown in Fig. 2. The population of the pathogen

generally declined following fungicidal application except at the preflowering

Stage with zineb, when there was a promotion in population. An appreciable

decline in population of Alternaria alternata immediately after application was

noticed with ziram and carbendazim which prolonged with the former but got

Source : MNHN, Paris

366 R. PANDEY et V. KUMAR

nullified with the latter in subsequent samplings. Prolonged depression in

pathogen population was also recorded with mancozeb and zineb in comparison

with captan.

Total phylloplane fungi

zinam MANCOZEB Zines CAPTAN CARBENDAZIM

60 m 4 n 1 3

40. 4

20 a

E [] [| a nn

OR n (eds S

2 S3 Sa S5 Se S3 $ S5 Se ET a Sa Ss Se 93 54 9s 96

im [1 % inhibition

$ ‘le promotion alternata

5 ZIRAM MANCOZER E CAPTAN CARBENDAZIM

= 100 4 - E e P

154 4

z n

504 1 1

25 di:

sis

nn ll

S3 Sa Ss Se 53 54 9596 Sa Sẹ Ss Se S3 54 5s Se Sa S 5g 96

Fig. 2 - Effect of fungicides on population of total phylloplane fungi and on dlternaria

alternata during and after the application.

Fig. 2 - Effets des fungicides sur la population des champignons du phylloplan et sur

“Alternaria alternata pendant et après application.

Decline and promotion in compositive phylloplane population was obtained

with different fungicides on different occasions of sampling (Fig. 3). Ziram appli-

cation produced nearly 48% depression in composite phylloplane population

even after 50 days of application. Such a long term effect of residual toxicity of

chemicals was nullified or reduced with all other fungicides utilized. The vari-

ations in the distribution of phylloplane fungi with respect to stages of samplings

were statistically significant (P > 0.005).

The number of fungal taxa declined soon after fongicidal application but the

Jong term effect was nullified when at subsequent isolation more species were iso-

lated from treated leaves. The trend of decline in population in individual phyllo-

plane fungi (Fig. 4) was in conformity with the composite population.

A total of 2260 individuals from 55 species were isolated from treated and un-

treated leaves at different stages of growth of soybean. Table 2 enlists relative

abundance based on the total number of individuals of fungal species and their

ranking.

Source : MNHN, Paris

EFFECTS OF FUNGICIDES ON PHYLLOPLANE FUNGI 367

O— contro!

— M

3 À 8 zie

t I &— A Carbendazim

1° 50 I a Captan

D 1 Rc a zum

$ 30

% 20

104

Fig. 3 - Trends in the distributin of phylloplane fungi before, during and after fungicidal ap-

plications. Sampling at broken lines show fungicide stress

Fig. 3 - Distribution des champignons du phylloplan avant, pendant et après les applica-

tions de fongicides.

Qualitative data

Fungi isolated from control and treated leaves separately and together are list-

ed in Table 3. The inclusion of species in this table was attempted afler a judi-

cious elimination from the data on several routine isolations. This has enabled to

provide a balanced perspective of a broad overview of species abundantly pres-

ent over leaf surface. The following observations in this respect are pertinent:

- 28 species of fungi including nearly all frequently occurring phylloplane fun-

gi were isolated from both treated and control leaves justifying their being called

true phylloplane inhabitants,

_~ 18 species of fungi were isolated following fungicide treatment which other-

wise were not isolated from untreated leaves,

- 9 species of fungi did not appear on treated leaves revealing their sensitivity

to fungicidal treatment.

A selective promotion in population of certain components of mycoflora such

as Aspergillus nidulans and Penicillium citrinum by fungicides is evident from

Fig. 4. The negligible effect of treatment with promotion in population of non-pa-

thogenic Alternaria alternata, Cladosporium herbarum and Mortierella

subtilissima reflects their insensitivity to fungicides. Whether such an effect is ac-

cidental or positive needs to be further ascertained before arriving at final con-

clusions about spectrum of activity of fungicides.

Source : MNHN. Paris

368 R. PANDEY et V. KUMAR

Analysis of community (Table 1) shows that diversity index ( H ), index of si-

milarity and dissimilarity and number of taxa colonizing the leaves progressively

declined with age on untreated leaves. A progressive lowering in the values of

Stages Treatments Distribution Number of General Evenness Index of Index of

(cont) fungal diversity Index similarity dissimilarity

taxa (H) (e) (5) (1-5)

c 8.72 15 247 0® - :

NE sai 16 23% om - E

c 45.5 B aa, 069. es z

™ 21.0 no £94 07 042 0.58

m 36.0 14 149 0,56 04 0.59

3 ZT 70.5 15 137 0.460 0.60 0.40

ca x4 17 R08 — 076 — 0.56 0.44

sa 19.8 12 2.9 — 088 0.4 0.56

c 37.0 10 22% 0% - -

™ 47.5 Ho 19% O8 0.6 0.34

m 24.2 10 202 0o88 0.40 0.60

1-78 11.6 5 123 06 — 040 0.60

cA 12.8 8 168 0.9 0.3 0.67

Ba 54.7 12 148 0.59 0.54 0.46

c 11.6 6 172 9€ - 2

™ 7.0 12 229 092 044 0,56

Mas 4.60 10 — 213 — 0.92 0,50 0.50

5 rm 4.57 3 1.99 0.90 0.2 0.76

ca 13.0 U 204 O8 040 0.60

sa 12.2 10 187 — 081 0.50 0.50

c 65 5 as 0% - -

™ 33 6 162 — 030 0.36 0.68

m 72 8 202 09) 015 0.85

5 1-78 4.8 4 L2 09 04 0.56

cA 5.5 8 14 03 030 0.70

sa 6.5 6 1.73 — 096 0.35 0.64

5,» seedling; 55, pre-flowering; S5, flowering; Sq, post-flowering; Sg, seed setting;

Sg» senescent. C, controls sprayed with distilled water only; ZM, ziram; M-45,

mancozeb; Z-78, zineb; CA, captan; BA, carbendazim.

Table 1 - Numerical distribution, taxa number and indices of phylloplane fungal community

of untreated and fungicide treated leaves of soybean.

Tableau 1 - Distribution, nombre de taxa et indices de la communauté fongique du

phylloplan de feuilles de soja non traitées (C) et traitées.

Source : MNHN. Paris

EFFECTS OF FUNGICIDES ON PHYLLOPLANE FUNGI 369

Individual Relative w — Individual Relative

Fungal species tota] abundance Fungal spectes tota] abundance

colontes (%) (Contd. ) colonies ^ (X)

Cladosportun herbarun 670 29.64 Rhizopus nigricans Y 0.53

Alternaria alternata 207 9.18. Aspergillus terreus n 0.48

Candida albioana 148 6.54 Meremontella echinata n 0.48

Mortierella subtilissima — 137 6.06 Phoma kiberinion n 0.48

Phoma humicola 129 5.70 Phoma glomerata w 0.44

Aerophtalephoma fusispora 113 5.0 Fusarium solani 9 0.39

Aspergillus flavus 70 3.09 Vertéoitléun albo-atrum 5 0.22

Black sterile mycelia 68 3.0 Aapergitiue Tushuensis 5 0.22

Pentoëtiéun cétrinun 61 2.69 Alternarta temutseima 5 0.22

Epicocoun purpurascens 56 24]. Dendrophona Sp. 5 0.22

Curvularia tunata 54 2.38 — Penteiliiwm chrysogenum 5 0.22

Fusariun semiteotun 52 2.30 Thielavia terricola 5 0.22

Aspergillus candidus a 1.80. Zorula herbarum 4 0.7

Yellow sterile mycelia 40 1.76 Aspergillus funigatus 3 0.13

Stachybotrys atra 38 168 Maorophomina phaseoti 3 0.13

Drechelera bicolor 31 1.37. Aspergillus clavatus 2 0.08

Aspergillus niger 27 119. Aspergillus sulphureus 2 0.08

Sporobolomyces salmonicolor 26 1.15 Fusariun roseo-griseun 2 0.08

Aspergillus nidulans 24 1.06 Melanospora mamie 2 0.08

Sterile sclerotia n 0,92 Mirothecium verrucarta 2 0.08

Drichoderma pseudokonéngit — 21 0.92 Altemaría longipes 1 0.04

White sterile mycelia 18 0.79 Curoularia verruculona 1 0.04

Alternaria citri 18 0.79 Diptodia sp. 1 0.04

Aureobasidiim pullulans 16 0.70 Orange sterile mycelium 1 0.04

Fusarium ozysporun 16 0.70 Pink sterile mycelium 1 0.04

Migrospora oryzae n 0.57 Rhizootonia batattoola 1 0.04

Cephalceporium acremonium — 1 0.53 Utoctadiun sp. 1 0.04

Chaetomium globosun 12 0.53

Relative abundance based on percentage of total colonies of all individuals isolated from

dilution plate; total colonies of all individuals = 2260

"Taxa arranged as per their relative abundance.

Table 2 - Total relative abundance of fungal species isolated from untreated and fungicide

treated soybean leaves.

Tableau 2 - Abondance relative des espèces isolées à partir de feuilles de soja non traitées et

traitées.

these parameters was also obtained in different samplings of fungicide treated

leaves. An increased value in diversity consequent upon application of carbenda-

zim and captan individually reflects the predominance of most tolerant species.

Zineb produced marked reduction in diversity associated with the increasing pre-

dominance of most abundant species.

Source : MNHN. Paris

370

From both treated and control

Altemaria alternata (Fr.) Kessler

Aspergillus candidus Link ex Fries

Acrophialophora fusispora (Saksena) Sanson

Aspergitive terreus Thom

Black sterile mycelium

Chaetomium globos Kuntze ex Steud

Cuwvularia lunata (Wakker) Boedijn

Epicoceun purpurascens Ehrenb. ex Schlecht.

Fusarium semitectun Berk. & Rav.

Mermmontella echinata (Riv.) Galloway

Phoma hiberinioa Grimes O'Connor & Cummins

Sporoboloryoea salmonicolom ,

Torula herbarun

White sterile mycelia

From control only

Alternaria longipes (Ellis & Everh.) Mason

Aspergillus Tuchuensie Inui

Dendrophoma

Melanospora

Utoctadiun sp.

sp.

ante Corda

From treated only

Aspergillus clavatua Desmazteres

Aspergillus nidulans (Eidam) Winter

Cephatosportun acrenoniun Corda

Fusarium roseo-grisewn

MNigrospora oryzae (Berk. & Br.) Petch

Penicillium chrysogenum Thom

Phoma glomerata (Corda) Mallenw. & Hochapf

Fhizopus nigricans Ehrenberg

Verticillium albo-atrum Reinke & Berthold

Table 3 -

an.

R. PANDEY et V. KUMAR

Alternaria citri Ellis & Pierce apud Pierce

Aspergillus flavus Link ex Fries

Aspergillus miger Van Tieohem

Aureobasidium pullulene (De Bary) Arnaud

candida albicans

Cladoaporium herbarum (Pers.) Link ex S.F.Gray

Drechslera bicolor (Mitra) Subram.A Jain

Fusarium poae (Peck) Wollemweber

Macrophomina phaseoti (Maublanc) Ashby

Mortierella oubtitissima Oudenans

Phoma humicola Gilman & Abbott

Stachybotrys atra Corda

Trichoderma peeudokoningtt Rifat

Yellow sterile mycelia

Alternaria tenuissima (Kurke ex Pers. White Shire

Curvularia verruculosa Tandon & Bilgrami ex

¥.B. Ellis

Diplodia sp.

Rhizoctonia bataticola

Aspergillus fumigatus Fresenius

Aspergillus sulphureus (Fresenius) Thom & Church

Fusarium oxysporum Synder

Myrotheciun verrusaria (Albertini & Schweinitz)

Ditmar

Orange sterile mycelia

Penicillium citrinum Thom

Pink sterile mycélium

Sterile sclerotia

Thielavia terricola (Gilman & Abbott) Emons

amentous fungi isolated from fungicide treated and or control leaves of soybe-

Tableau 3 - Champignons isolés à partir de feuilles de soja traitées et/ou non traitées.

DISCUSSION

The described spray programme for evaluation of pesticide effect on non-tar-

get mycoflora was designed primarily to compare a long term effect in succes-

sional pattern over leaf of a crop by a relatively inexpensive chemical applica-

tion. Studies on this type do not eliminate the necessity of using several

complementary cultural techniques which when followed gave best results with

the dilution plate in the present case. The data presented here suggest that the

spray programme timed at two occasions brought maximum decline in fungal

population immediately after application and that the prolonged impact was ob-

Source : MNHN. Paris

EFFECTS OF FUNGICIDES ON PHYLLOPLANE FUNGI 371

i EN

A. alternata

"ad

M, sublilissima P. citrinum

„nidulans

*'lh FREQUENCY

* b

flavus

[en]!

C. herbarum

tate

Control

* Nan-pathogenic

Fig. 4 - Occurrence (% frequency) of some frequently isolated non-target fungi on fungi-

‘cide treated leaves. Treatments given at sampling I & II.

Fig. 4 - Fréquence de quelques champignons isolés fréquemment sur feuilles traitées avec

les fongicides. Les traitements sont effectués aux échantillonnages 1 & 11

tained with only ziram. KUTHUBUTHEEN & PUGH (1979) obtained mark

reduction in soil fungal population by thiram which remained persistant and po-

pulation did not recover sufficiently. Breakdown products of thiram, carbon di-

sulphide and dimethylamine, in soil were implicated by them for such persist

Source : MNHN, Paris

372 R. PANDEY et V. KUMAR

ence. Comparable effect with zineb could have been expected in the present

study but an apparent stimulation in population of Alternaria alternata explains

discripency. Extremely low persistence of captan in nature have been shown in

earlier studies (BURCHFIELD, 1960; GRIFFITH & MATHEWS, 1969; AG-

NIHOTRI, 1971; KUTHUBUTHEEN & PUGH, 1979). Benzimidazole, due to

specific mode of action against some selective group of fungi, have been demon-

strated with poor effect and lesser persistence on fungal population (SWIN-

BURNE & al., 1975; DICKINSON & WALPOLE, 1975; DICKINSON &

WALLACE, 1976). Wide range spectrum of activity of organo-sulphur com-

pounds as obtained here is an addition to many reports (DICKINSON, 1973;

BAINBRIDGE & DICKINSON, 1972; DICKINSON & WALLACE, 1976).

The schedule of fungicidal spray in the present study was based on occasions

when the pathogen population attained peak. Evidences presented here show

early recolonization by non-target fungi and a simultaneous depression in patho-

gen population on foliage following fungicidal application. It is possible therefore

to design a modified chemical programme once positive role of non-target fungi

towards disease control is determined.

REFERENCES

AGNIHOTRI V.P., 1971 - Persistence of captan and its effects on microflora respiration

and nitrification of a forest nursery soil. Canad. J. Microbiol. 17: 377-383.

ANDREWS 1.H., 1981 - Effect of pesticides on non-target micro-organisms on leaves. In.

J.P. BLAKEMAN, Microbial Ecology of the Phylloplane. London, Academic Press:

283-304.

BAINBRIDGE A. and DICKINSON C.H., 1972 - Effect of fungicides on the microflora

of potato leaves. Trans. Brit. Mycol. Soc. 59: 31-41.

BURCHFIELD H.P., 1960 - Performance of fungicides on plants and in soil-physical,

chemical and biological considerations, Jn: J.H. HORSFALL & A.E. DIAMOND,

Plant Pathology: an Advanced Treatise. New York, Academic Press: 477-520.

DICKINSON C.H., 1973 - Interactions of fungicides and leaf saprophytes. Pest. Sci. 4:

563-574.

DICKINSON C.H. and WALLACE B., 1976 - Effect of late application of foliar fungi-

cides on activity of micro-organisms on winter wheat flag leaves. Trans. Brit. Mycol.

Soc. 76: 103-112.

DICKINSON C.H, and WALPOLE P.R., 1975 - The effect of late application of fungi-

cides on the yield of winter wheat. Exp. Husb. 29: 23-28.

FOKKEMA N.J., 1981 - Fungal leaf saprophytes, beneficial or detrimental ? In: J.P.

BLAKEMAN, Microbial Ecology of the Phylloplane: 433-454.

GRIFFITHS R.L. and MATTHEWS S., 1969 - The Persistence in soil of the fungicidal

seed dressings captan and thiram, Ann. Appl. Biol. 64: 113-118.

HISLOP E.C., 1976 - Some effects of fungicides and other organochemicals on the micro-

biology of the aerial surfaces of plants. Zn: C.H. DICKINSON & T.F. PREECE,

Microbiology of Aerial Plant Surfaces. London, Academic Press: 41-74.

KUTHUBUTHEEN A.J. and PUGH G.J.F., 1979 - The effects of fungicides on soil fun-

gal population. Soil Biol. Biochem. 11: 297-303.

SWINBURNE T.R., FLACK N.J. and BROWN A.E., 1975 -The effects on fungicides on

the microflora of apple leaf scars. Ann. Appl. Biol. 81: 87-90.

Source : MNHN, Paris

373

ANALYSES BIBLIOGRAPHIQUES

CORNER EJ.H., 1987 - Ad Polyporaceas IV. The genera Daedalea,

Flabellophora, Flavodon, Gloeophyllum, Heteroporus, Irpex, Lenzites,

Microporellus, — Nigrofomes, Nigroporus, Oxyporus, Paratrichaptum,

Rigidoporus, Scenidium, Trichaptum, Vanderbylia and Steccherimum. Berlin,

Stuttgart, J. Cramer, Gebrider Borntraeger, Beihefte zur Nova Hedwigia,

Heft 86, 265 p., 11 pl., 35 figs.

Dans ce quatrième volume, le Professeur Corner étudie 17 genres dont le

nouveau genre monospécifique Paratrichaptum. Les descriptions sont le fruit

d'études accomplies durant plusieurs années sur des spécimens frais en prove-

nance essentiellement d'Asie du Sud-Est, des Iles Salomon et du Brésil. Corner

crée 56 espèces et 21 variétés dont certaines sont peut-être déjà connues puisque

l'auteur écrit: “It has not been in my power to ransack herbaria”. Après une

étude approfondie de chaque genre, l'auteur en précise les affinites et la

phylogénie: ceci n'a été rendu possible que grâce à l'abondance des espèces tro-

picales, les représentants des Polypores étant beaucoup moins nombreux dans les

régions tempérées (par exemple Rigidoporus aujourd'hui réduit à une seule

espèce tempérée est enrichi de 11 espèces tropicales nouvelles). La conséquence

en est que les limites intergénériques deviennent plus difficiles à cerner. D'au-

cuns pourront regretter l'absence de données tirées de l'étude de cultures

mycéliennes... maisl'auteur a clairement défini le but de son ouvrage: donner aux

mycologues locaux toutes les précisions permettant une détermination de ces trés

nombreuses espèces et variétés. A cet égard, l'ouvrage du Professeur Corner

nous parait trés réussi, les nombreuses clés se révélant particulièrement utiles. Si-

gnalons que le volume est complété par huit pages de dessins en couleurs

d'espèces de Polypores, soulignant l'importance de l'aspect des spécimens Sur le

frais. Le Professeur Corner nous offre ainsi le premier ouvrage récent et impor-

tant sur les Polypores du continent asiatique.

A. David

GROULT B.W.W. and MORRIS G.J., 1987 - The effect of low temperatures

on biological systems. London, Arnold, 500 p.

Cet ouvrage est composé d'un ensemble d'articles faisant le bilan des connais-

sances actuelles sur l'effet des basses températures sur les cellules vivantes. Le

théme est abordé du niveau moléculaire à celui de l'organisme tout entier. Les

applications pratiques des techniques du froid en médecine, agriculture et indus-

tries agro-alimentaires sont également développées. L'ensemble des articles est

divisé en quatre parties. La premiere concerne les principes fondamentaux de la

cryobiologie: principes physico-chimiques, effets des basses températures sur les

cellules, principalement sur les membranes, les protéines et le cytosquelette, effets

des chocs osmotiques et cryogéniques en fonction de la vitesse de congélation et

enfin, influence de celle-ci sur l'or. anisation cellulaire. La deuxieme partie

concerne les techniques d'étude du déroulement de la congélation en microscopie

électronique (où les basses températures peuvent être utilisées comme moyen de

fixation), et photonique où l'on peut suivre la cristallisation en congélation à vi

tesse contrôlée. La troisième partie aborde les phénomènes naturels de

résistance ou d'adaptation au froid chez les animaux et les végétaux tandis que

Source : MNHN, Paris

374 ANALYSES BIBLIOGRAPHIQUES

la quatrième porte sur les applications. On y trouve des données sur la conser-

vation des cellules, tissus et semences de végétaux en vue de l'amélioration des

pratiques agricoles ou de la conservation de clones. L'application des techniques

cryogéniques aux disciplines médicales et vétérinaires (chirurgie, analgésie)

complète ce bilan des connaissances et des utilisations actuelles de la cryogénie.

On remarque cependant que le matériel fongique n'est pas traité, ni même

mentionné. Les recherches sur les champignons restent donc à développer et cet

ouvrage, dans ses parties théoriques au moins, sera une aide précieuse pour cette

entreprise.

M.F. Roquebert

WATLING R. and TAYLOR G.M., 1987 - Observations on the Bolbitiaceae:

27. Preliminary account of the Bolbitiaceae of New Zealand. Berlin,

Stuttgart, J. Cramer, Gebrüder Borntraeger, Bibliotheca Mycologica, Band

117, 60 p., 17 fig. et pl.

Il s'agit d'un premier essai concernant les Bolbitiaceae de Nouvelle-Zélande.

Vingt-cinq espèces sont étudiées (12 Agrocybe, 3 Bolbitius et 10 Conocybe) ; 4

sont nouvelles: Agrocybe olivacea, Conocybe gracilenta, C. horakii et C.

novaezelandiae, avec en plus 4 récoltes de Conocybe et | d' Agrocybe (spsp., ad

interim) vraisemblablement nouvelles mais innominées. Deux clés (Agrocybe et

Conocybe) concernent les principaux sous-genres et sections mondialement

connus et les espèces néo-zélandaises; enfin 5 pages de références viennent

compléter les informations avec 16 planches de dessins au trait et 1 planche pho-

tographique de spores au microscope électronique.

M. Bon

La quatrième Conférence Internationale sur l'Aérobiologie se tiendra à

Stockholm (Suéde) du 3 au 7 Septembre 1990 (Administration Secretary,

Konferensservice AB, Box 4037, S-171 04 SOLNA, SUEDE).

The 4th International Conference on Aerobiology will be held at Stockholm

(Sweden) on September 3-7, 1990 (Administrative Secretary, Konferensservice

AB, Box 4037, S-171 04 SOLNA, SWEDEN).

Source : MNHN, Paris

375

TABLE DU TOME 9 - 1988

ABDEL-HAFEZ A.LL - Mycoflora of broad bean, chick-pea and lentil seeds in

Egypt cues Cation 335

ABDELLAOUI-MAANE SENG J.M, SAI À al EIX G.

= FosetylAI is effective against mutants of Phyrophthora capsici resistant to

metalaxyl 47

ADISA V.À. - Some nutritional requirements of spoilage molds of pineapple fruit n

ADISA V.A. - Voir OKEY E.N.

ANDARY C., BOURRIER M.J. et HAUPERT R. - Rôle des polyols et des acides

‘amines dans la différenciation des Bolets … MES]

ARPIN N. et BOUILLANT M.L. - Métabolites secondaires fongiq

structures mais unité de fonction, la protection .... 267

BARRASA J.M. - Voir CHECA J.

BETTUCCI L, LUPO S. and SILVA S. - Control growth of wood-rotting fungi

by non-volatile metabolites from Trichoderma spp. and Gliocladium virens 157

BOIDIN J., GILLES G. et HUGUENEY R. - Rehabilitation du Corticium rickii

Bres. (Basidiomycotina) ..... ^ 43

BOMPEIX G. - Voir ABDELLAOUI-MAANE

BONNET Ph. - Voir RICCI P.

BOTTON B. and KHOURY M. - Coremium and rhizomorph differentiation in

Sphaerostilbe repens 1. - Interactions between aggregated organs during

morphogenesis of the thallus Mo Wee. diio o

BOUILLANT M.L. - Voir ARPIN N.

BOURRIER M.J. - Voir ANDARY C.

BRUNETEAU M. et RICCI P. - Elicitation et résistance induite 173

BRUNETEAU M. - Voir RICCI P.

CHECA J., BARRASA J.M., MORENO G., FORT F. and GUARRO J. - The

genus Coniochaeta (Sacc.) Cooke (Coniochaetaceae, Ascomycotina) in Spain . 1

COURTECUISSE R. - Champignons de la région Nord - Pas de Calais; 16-20 57

DAVID A. - Voir SALIBA J

DEXHEIM J. - Voir GIANINAZZI-PEARSON V

DIROL D. - Dégradation par des champignons lignivores de la paroi cellulaire de

pin traité à des doses sub-letales de produits de préservation chloré

DUMAS E. - Voir GIANINAZZI-PEARSON V

FORT F. - Voir CHECA J.

GAY G. - Rôle des hormones fongiques dans l'association ectomycorhizienne …… 211

GIANINAZZI S. - Voir GIANINAZZI-PEARSON V.

GIANINAZZI-PEARSON V., GIANINAZZI S., DEXHEIMER J., MORANDI

D, TROUVELOT À. et DUMAS E. - Recherche sur les mécanismes

intervenant dans les interactions symbiotiques plante-champignons endomyco-

rhizogénes VÀ ..... 201

GILLES G. - Voir BOIDIN J.

GUARRO J. - Voir CHECA J.

HADAR T. - The host specificity of Urophlyctis leproides (Trab.) Magn. - 2 WB

HARRISON G.L. and STEWART A. - Cultural variation in Mycosphaerella

fragariae (Tul.) Lindau. EE ST

HAUPERT R. - Voir ANDARY C.

HILBERT J.L. et MARTIN F. - Modifications des profils polypeptidiques lors de

étalissement de la symbiose ectomycorhizienne .......- eo

HUGUENEY R. - Voir BOIDIN J

JANEX-FAVRE M.C. - Étude ontogénique et structurale des périthéces du

Dictyotrichiella semümmersa Candoussau et Sulmont (Pyrénomyctes, — ,..

Herpotrichiellaceae)

KHOURY M. - Voir BOTTON B.

KUMAR V. - Voir PANDEY R.

LUPO S. - Voir BETTUCCI L.

Source : MNHN, Paris

376 TABLE

MANACHERE G. - Regulation of sporophore differentiation in some

macromycetes, particularly in Coprini an overview of some experimental

studies from fruiting initiation to sporogenesis ... 291

MARTIN F. - Voir HILBERT J.L.

MICHELOT D. et TEBBETT LR. - Les intoxications par les Cortinaires 345

MOLOT P.M. - Voir RICCI P.

MORANDI D. - Voir GIANINAZZI-PEARSON V.

MORENO G. - Voir CHECA J.

MUGNIER J. - Exemples d'études d'organismes parasites des racines dans des

cultures de racines transformées par Agrobacterium rhizogenes .. 233

OKEY E.N. and ADISA V.A. - Some nutritional requirements and the

four environmental factors on spore germination and growth of Lasiodiplodia

theobromae and Pseudocercospora HiMOTENSES nn. d 67

PANDEY R. and KUMAR V. - Effect of short term fungicidal programme on

non-target phylloplane fungi of soybean «......s..s......1... s

PAUL B. - Une nouvelle variété de Pythium capillosum dans les sols algériens

PAUL B. - Une nouvelle espéce de Pyrhium isolée d'une saline de l'ouest algérien .. — 325

PONCHET M. - Voir RICCI P.

RICCI P., BONNET Ph., PONCHET M., MOLOT P.M. et BRUNETEAU M. -

Recherche et intérêt d'èliciteurs fongiques dans les interactions de trois espèces

végétales avec des champignons phytopathogénes du genre Phytophthora ....... 191

RICCI P. - Voir BRUNETEAU M.

SAINDRENAN P. - Voir ABDELLAOUI-MAANE S.

SALIBA J. et DAVID A. - Apports des caractères culturaux et des confrontations

dans l'étude des représentants européens du genre Steccherinum

(Basidiomycétes, Aphyllophorales) ... NIE SS

SENG J.M. - Voir ABDELLAOUI-MAA

$ A S. - Voir BETTUCCI L.

STEWART A. - Voir HARRISON G.L.

TEBBETT I.R. - Voir MICHELOT D.

TROUVELOT A. - Voir GIANINAZZI-PEARSON V.

TURIAN G. - Polarité de croissance-diffėrenciation des hyphes fongiques (modèles) 239

Analyses bibliographiques (fascicule 1) 75

Analyses bibliographiques (fascicule 2) 167

Analyses bibliographiques (fascicule 3) 289

Analyses bibliographiques (fascicule 4) 373

Instructions aux Auteurs ... 85

Commission paritaire n° 58611

Dépôt légal n° 14201 - Imprimerie de Montligeon

Sortie des presses le 20 décembre 1988

» Imprimé en France

Éditeur : A.D.A.C. (Association des Amis des Cryptogames)

Président : A. Couté; Secrétaire : D. Lamy

Trésorier : R. Baudouin; Directeur de la publication : H. Causse

Source : MNHN, Paris

CRYPTOGAMIE — MYCOLOGIE

BUREAU DE RÉDACTION

MM. DURRIEU G. pour les articles traitant d'Écologie et de Phytopathologie

Laboratoire de Botanique, Faculté des Sciences,

Allées Jules Guesde, 31000 Toulouse (France)

JOLY P., pour les articles traitant de Systématique

Laboratoire de Cryptogamie, Muséum National d'Histoire Naturelle

12, rue de Buffon, 75005 Paris (France).

MANACHERE G., pour les articles traitant de Physiologie

Laboratoire de Mycologie, Université de Lyon I,

43, Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex (France).

Mmes ZICKLER D., pour les articles traitant de Cytologie

Laboratoire de Génétique, Université de Paris Sud,

Bât, 400, Centre d'Orsay, 91405 Orsay (France).

ROQUEBERT M.F., s'occupera des autres spécialités.

Laboratoire de Cry ptogamie, Muséum National d'Histoire Naturelle

12, rue Buffon, 75005 Paris (France).

COMITÉ DE LECTURE

BOIDIN J., Lyon (France) MONTANT Ch., Toulouse (France)

CHEVAUGEON J., Orsay (France) MOREAU Cl., Brest (France)

GAMS W., Baarn (Hollande) PEGLER D.N., Kew (Grande-Bretagne)

HENNEBERT G., Louvain-la-Neuve SUTTON B., Kew (Grande-Bretagne)

(Belgique) TURIAN G., Genève (Suisse)

LACOSTE L., Paris (France)

Les manuscrits doivent être adressés (en 3 exem laires) directement à un

membre du Bureau de Rédaction, choisi pour sa spécialité. Chaque membre du

Bureau se charge d'envoyer l'article à 2 membres du Comité de Lecture (ou

autres lecteurs compétants).

Bien qu'étant avant tout une revue de langue française, les articles rédigés en

Anglais, Allemand et Espagnol sont acceptés.

Les recommandations aux auteurs sont publiées dans le l* fascicule de

chaque tome.

Source : MNHN. Paris

27 JAN. 1989

ABONNEMENTS A CRYPTOGAMIE

Tome 10,1989

CRYPTOGAMIE comprend troi sections : ALGOLOGIE, BRYOLOGIE-

LICHÉNOLOGIE, MYCOLOGIE. Om peut souscrire indépendamment à

chacune des sections.

Abonnement à 1 section

France (HT 320 F) 326,72 F

Étranger HT 350,00 F

Abonnement aux 3 sections

France n (HT990F) 918,90 F

Étranger ; HT 980,00 F

Les anciens tomes et fascicules séparés de la REVUE DE MYCOLOGIE et

de CRYPTOGAMIE - MYCOLOGIE sont toujours disponibles.

MÉMOIRES HORS SÉRIE

N9 2 (1942). Les matières colorantes des champignons, par |.

Pastac. 88 pages:15F,

gnons, par R. Ulrich. 44 pages : 15F,

NO 6 (1958). Essai biotaxonomique sur les Hydnés résupinés et les

Corticiés, par J, Boidin. 390 pages, plvet fig. : 120 F.

N° 7 (1959). Les champignons et nous (Chroniques) (Ñ), par G.

Becker. 94 pages : 25 F. x

N9 8 (1966). Catalogue de la Mycothéque dela Chaire de Cryproga

mie du Muséum National d'Histoire Naturelle. (1) Micromy-

cétes. Macromycétes (premiére partie). 68 pages p B8.

NO 9 (1967). Table des-Matières (1936-1965). 85 pages : 20 T.

(1966-1975).30 pages : IDF,

FLORE MYCOLOGIQUE DE MADAGASCAR ET DÉPENDANCES

publite sous ls direetion de M- Ragee HPIM

Tome i. LesLactario Russulés, par Roger Heim (1938) (épuisé)

Tome il, v Le par Henri Romagnesi (1941), 164 pages,

46 fig. :

Tgme ill. Les b. par Georges Métrod (1949). 144 pages.

88 fig. » 90 F,

Tome IV. Les Discomycétes de Madagascar , Marcelle Le Gal

(1953). 465 pages, 172 fig, : 150 F. i

Tome V. Les Urédinées, par Gilbert Bouriquet et J.P. Bassino

(1965). 180-pages, 97 fig., 4 pl. hors-texte : 90 F.

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