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Fungal Genetics and Biology 26, 1–18 (1999)Article ID fgbi.1998.1103, available online at http://www.idealibrary.com on

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icroconidia of Neurospora crassa

amesh Maheshwari

epartment of Biochemistry, Indian Institute of Science, Bangalore 560 012, India

ccepted for publication October 27, 1998

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aheshwari, R. 1999. Microconidia of Neurosporarassa. Fungal Genetics and Biology 26, 1–18. Neuros-ora crassa produces two types of vegetative spores—elatively small numbers of uninucleate microconidiand very large numbers of multinucleate macroconidiablastoconidia and arthroconidia). The microconidiaan function either as spermatia (male gametes) or assexual reproductive structures or both. In nature theyrobably function exclusively in fertilization of proto-erithecia. The environmental conditions favoring theirormation and the pattern of their development areuite distinct from those of macroconidia. Mutants of. crassa have been isolated in which macroconidia-

ion is selectively blocked without affecting microco-idiation, showing that these two types of conidialifferentiation involve distinct developmental path-ays. Unlike microconidia of some related ascomyce-

es, those of Neurospora are capable of germination,roviding viable uninucleate haploid cells which areesired in several types of investigations. A techniquef selectively removing macroconidia from culture

nitiated on cellophane overlying agar medium allowsure microconidia to be obtained even from the wild-ype strains of Neurospora. The conditional microcy-lic strain, mcm, allows either macroconidia or micro-onidia to be obtained at will, depending on theonditions of culture. The new methods of obtainingure microconidia from normal laboratory strains willake it quick and easy to purify heterokaryotic transfor-ants following introduction of DNA into multinucleaterotoplasts. Moreover, these methods allow the detec-ion of genetic variability that remains hidden within an
ndividual fungus and the estimation of the frequency D
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087-1845/99 $30.00opyright r 1999 by Academic Pressll rights of reproduction in any form reserved.

f nuclear types in laboratory-constructed heterokary-ns. The discovery, function, and development oficroconidia are described and their research applica-

ions are discussed in this review. r 1999 Academic Press

ndex Descriptors: Neurospora; microconidia; sperma-ia; fertilization; spore; conidiation; microcycle conidia-ion; mutagenesis; germination; heterokaryosis; nuclearatio; transformation.

The most familiar aspect of Neurospora is its productionf a powdery mass of bright orange, vegetative spores, theacroconidia, which are easily disseminated by air cur-

ents and which can cause serious contamination in baker-es and in the laboratory unless precautions are takenPerkins, 1992). The fungus also produces relatively smallumbers of microconidia which are inconspicuous andhich do not become airborne. Since their discoveryearly 70 years ago and the demonstration that they areninucleate, microconidia have been used sporadically inutation research and for determining the nuclear types in

eterokaryons. Although microconidia are also known inome other species of fungi (Table 1), it is in the modelungus Neurospora that they offer the maximum promises an experimental tool. This review summarizes informa-ion on microconidia of Neurospora and illustrates theirse in some current and future objectives.

ISCOVERY OF MICROCONIDIA

In Neurospora, microconidia were first observed by
odge (1930) in ‘‘albinistic’’ cultures of N. sitophila which

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rose by spontaneous mutation in the laboratory. Inontrast to the parent strain which produced salmon-pinkacroconidia, the albinistic strains were characterized byhitish aerial hyphae that no longer produced macroco-idia but formed minute, hyaline, spherical to pear-shaped

ABLE 1

ome Examples of Fungi Which Produce Microconidia

Fungus Remarks Reference

ombardia lunata Macroconidia not pro-duced. Microco-nidia do not germi-nate; function asspermatia.

Zickler (1952)

ibberella fujikuroi Both macroconidiaand microconidiaproduced. Microco-nidia found usefulfor production ofauxotrophs andcolor mutants usingUV and nitrosogua-nidine mutagenesis.

Avalos et al. (1985)

loeotinia temulenta Macroconidia not pro-duced. Microco-nidia function asspermatia.

Griffiths (1959)

annizia gypsea, N.incurvata (Micros-porum gypseum)

Both macroconidiaand microconidiaproduced. Microco-nidia can germi-nate.

El-Ani (1968)

ectria haematococca Both macroconidiaand microconidiaproduced. Germi-nation of microco-nidia not reported.

Bistis and Georgop-oulos (1979)

eurospora crassa,N. sitophila, N. tet-rasperma

Both macroconidiaand microconidiaproduced. Microco-nidia germinatealbeit slowly.

Dodge (1932)

odsopora anserina Macroconidia not pro-duced. Microco-nidia do not germi-nate; functionpurely sexual.

Dodge (1936)

clerotinia fruticola Both macroconidiaand microconidiaproduced; microco-nidia do not germi-nate.

Woronin (1900); Wil-lets and Calonge(1969)

tromatinia narcissi Macroconidia not pro-duced; microco-nidia do not germi-nate.

Drayton and Groves(1952)

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opyright r 1999 by Academic Pressll rights of reproduction in any form reserved.

odies, about 2.5–3.5 µm in diameter, which were initiallyistaken for bacteria (Fig. 1). However, Dodge soon

earned that these structures could germinate like fungalpores and were morphologically comparable to the micro-onidia that had been figured by Woronin (1900) inotrytis cinerea and Sclerotinia fructigena. Dodge (1932)bserved that production of microconidia was not limitedo mutants; they were also produced by both mating typesven in the typical wild-type strains of N. sitophila, N.rassa, and N. tetrasperma.

In 1932 Drayton called attention to some ascomycetousungi in which microconidia behaved like the pycniospores

IG. 1. (a) Microconidia produced by albinistic strain of Neurosporaitophila, 3210. (b) Microconidiophore, 3210 . (c and d) Microconidia,240. (e) Swollen microconidia 40 h after sowing on agar, 3240.eprinted with permission from Mycologia, Vol. 22, No. 1, pp. 9–38,late 9, Copyright 1930, The New York Botanical Garden.

f rust fungi; i.e., they failed to germinate but functioned as

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Microconidia of N. crassa 3

ale gametes by donating their nuclei to trichogynes.herefore, microconidia were regarded as spermatia (maleells). However, Dodge (1932) emphasized that in N.itophila, in addition to their ability to spermatize, microco-idia can germinate if precaution is taken to ‘‘exclude allonilioid macroconidia and mycelial fragments, otherwise

he plate will be overgrown before the microspores have ahance to germinate.’’ By genetic tests, Dodge (1932)emonstrated that the mycelia produced by uninucleateicroconidia are exactly like those produced from multi-

ucleate macroconidia.

IOLOGICAL ROLEF MICROCONIDIA

Dodge was bewildered by the production of two types ofsexual spores in Neurospora which could perform theame function. To understand this, he studied Podospora5Pleurage) anserina, a related ascomycete which pro-uced microconidia exclusively (Dodge, 1936). Surpris-

arden.

ngly, Dodge made no mention of germinability of itsicroconidia. It is now known that microconidia of Podos-

ora do not germinate, or do so rarely (Esser, 1974). Tossess their role in fertilization, Dodge (1936) selectedtrains of opposite mating types, neither of which pro-uced microconidia, and confronted them on corn mealgar. Despite the lack of microconidia, the paired strainsormed perithecia in plate cultures. Dodge thereforeonsidered microconidia to be dispensable for fertilization.e remarked, ‘‘the . . . tendency . . . for those who discover

permatia of an ascomycete for the first time is to magnifyheir importance and to insist on their necessity, ignoringll the other ways these same fungi have of insuringross-fertilization.’’

The role of microconidia became shrouded in morencertainty when interaction between trichogynes andells of opposite mating types was studied in Neurospora.ackus (1939) reported that ‘‘macroconidia are even moreffective as spermatizing agents.’’ Bistis (1981, 1983)emonstrated that a microconidium produced a diffusibleheromone which attracted the tip of a trichogyne of thepposite mating type towards it, followed by its fusion withhe trichogyne’s tip (Fig. 2)—an observation supporting

IG. 2. Chemotropic interaction between microconidia and trichogyne of opposite mating type in N. crassa. (A) A 7-day old protoperithecium with arichogyne. Three microconidia (d) of opposite mating type were placed on agar surface approximately 80 µm from the trichogyne. (B and C) Directedrowth of trichogyne towards microconidia after 3.5 and 5.5 h, respectively. (D) Trichogyne has reached microconidium at 7.5 h. All micrographs are at theame magnification and the bar is 20 µm. Reprinted with permission from Mycologia, Vol. 73, No. 5, pp. 959–975, Copyright 1981, The New York Botanical

Copyright r 1999 by Academic PressAll rights of reproduction in any form reserved.

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he role of microconidia as fertilizing elements. However,his reaction was not a specific property of microconidialone; macroconidia acted just as efficiently. On the otherand, the situation in P. anserina in which macroconidiare not produced while the microconidia function only inertilization, came to be regarded as a paradigm of microco-idia. In Neurospora, the role of microconidia vis-a-visacroconidia remained unclear until recently.An understanding of the significance of production of

he two types of conidia by Neurospora has come frombservations of the fungus on burnt sugarcane in agricul-ural fields and from reconstruction experiments in theaboratory (Pandit and Maheshwari, 1996). In the sugar-ane ecosystem, the ‘‘infection’’ of the substrate originatesrom soilborne ascospores. The production of macroco-idia and of microconidia by N. intermedia on burntugarcane was separated both in time and in space. In thenitial phase of growth of the fungus in the sugar-rich

ABLE 2

ifferences between Microconidia and Macroconidia of Neurosporaa

Parameter Microco

ize 2.5 3 3.5 µm (Dodge, 1930(Barratt and Garnjobst, 1

hape Ovoid to pear-shaped (Lowuclear number Uninucleate (Dodge, 1930,

nucleate (Barratt and Gaand MacLeod, 1960; BayMaheshwari, 1991b)

olor Greenish-brownigments Melanin and carotenoids (Tongevity ,1 week (Ballou and Biancime of formation in culture Late, 6–9 days (Dodge, 193

and Yanofsky, 1989)evelopment Phialidic (Rossier et al., 197ndoplasmic reticulum Extensive (Lowry et al., 196itochondria Few, 1 µm long (Lowry et aydrophobicity Hydrophilicermination rate Slow and asynchronous, 6–

et al., 1998)lating efficiency Low, ,20–35% (see Table

nvironmental conditions for development(a) Humidity High, promoted by wetting(b) Temperature Cool (20–25°C)(c) Oxygen Micro-aerophilic or anaerobutritional conditions for development Low sugar and nitrogen (Pa

1996)roduction in nature In tissue pockets (Pandit anunction Fertilization (Pandit and M

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ubstrate, the formation of macroconidia and their con-tant discharge into the environment continued for severalays until sugar and nitrogen in the plant tissue werelmost depleted and the tissue had become desiccated. Byontrast, the production of microconidiophores occurredeveral weeks after macroconidiation had ceased and afterhe tissue was thoroughly soaked by the seasonal rains.evival of macroconidiation did not occur but microconid-

ophores along with protoperithecia were produced in tissueockets, strongly suggesting that in nature the microco-idia, and not the macroconidia, function in cross fertiliza-ion. Liberation of prodigious numbers of macroconidiauring the initial phase of growth of the fungus depleteshe plant tissue of its soluble sugars and thereby createsutritional conditions favoring differentiation of protoper-

thecia and microconidia and the onset of sexual reproduction.The microconidia differ from macroconidia morphologi-

ally, developmentally, cytologically, and in a number of

Macroconidia

3 3.98 µm 5–9 µm (Lowry et al., 1967)

, 1967) Oblong to spherical (Lowry et al., 1967)94–.99% uni-, 1949; Horowitzde Busk, 1967;

Multinucleate, 1–20 or more (Barratt and Garnjobst,1949; Huebschman, 1952; Pittenger, 1967)

Pink-oranget al., 1967) Carotenoids (Zalokar, 1957a, b)8) 2–3 years (Sussman, 1966)ger Early, 2–3 days (Dodge, 1932; Springer and Yanofsky,

1989)Blastic (Turian and Bianchi, 1972)Sparse to moderate (Lowry et al., 1967)

) Many, .2 µm (Lowry et al., 1967)Hydrophobic

alpana Rapid and fairly synchronous, 2–4 h

High, ,65–95% (Haard, 1967; Wellman, 1960;Fahey et al., 1978; and R. Maheshwari,unpublished observation)

Moderate, promoted by desiccation?Warm (30–37°C)Aerophilic (Turian and Bianchi, 1972)

d Maheshwari, High sugar and nitrogen (Pandit and Maheshwari,1996)

shwari, 1996) Liberated externally (Pandit and Maheshwari, 1996)ari, 1996) Dispersal; fertilization (Perkins and Turner, 1988)

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Observations largely on N. crassa, infrequently on N. sitophila, N. intermedia, and N. tetrasperma.

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hysiological parameters (Table 2). Although it is inferredhat microconidia facilitate cross fertilization in nature,heir germinability is apparently not required for thisunction, and germination may even be suppressed (seeistis, 1981). However, in Neurospora, where microco-idia can be induced to germinate, they are a usefulxperimental tool.

ICROCONIDIAL GENOTYPES

Microconidiation can vary considerably among strains ofeurospora (Moreau and Moreau, 1939; Wilson, 1985; A.andit, unpublished observations). Microconidia germi-ate at a slower rate than macroconidia (Table 2), enablinghe proportion of the two spore types in a mixture to bestimated by plating and counting as the early and the lateolonies. Using this method, Grigg (1960a, 1965b) deter-ined that in a culture of wild-type N. crassa only about

% of the spores are microconidia. Gillie (1967) used aoulter counter to estimate the relative numbers oficroconidia and macroconidia in a mixed population

aking advantage of the large difference in their sizes (Fig.). Using this technique he confirmed that wild-type N.rassa produced few or no microconidia when grown onogel’s minimal medium (see Davis and de Serres, 1970) at5°C but about 50% microconidia were present when theungus was grown on a complete medium without glycerol.

For experimentation, pure microconidia may be ob-ained from mutant strains of N. crassa in which macroco-idiation is selectively eliminated, for example, fluffy (fl)nd colonial-1 (col-1) (see Perkins et al., 1982). Someodifier genes increase microconidiation, for example,

each (pe) and dingy (dn). Double mutant combinations ofuch genes with a gene that blocks macroconidiation, forxample, pe fl, pe col-1, or fl dn produce microconidiaxclusively (see Perkins et al., 1982). The pe col-1 doubleutant offers an advantage in that the col-1 gene allows

utton-like colonies to be obtained for screening in muta-ion experiments (Barratt and Garnjobst, 1949).

In the microconidiating genotypes referred to above,utation simultaneously affects hyphal growth characteris-

ics and morphology. This limitation is overcome by the usef the microcycle-microconidiating mutant mcm (Ma-eshwari, 1991a,b). This mutant has the advantage in that

t controls microconidiation in a conditional manner. It is
ndistinguishable from the wild-type N. crassa on agar- m
rown culture but when its macroconidia are germinatedn liquid shake cultures, they undergo microcycle conidia-ion. Polarized growth of the conidial germ tube is arrestednd lateral protuberances are produced which separate,ncapsulating a single nucleus (Fig. 4). These structuresre morphologically and cytologically comparable to micro-onidia produced by surface-grown cultures and are effec-ive in fertilization. Millions of microconidia are producedn 24–36 h at 18 to 22°C. Thus either macroconidia or

icroconidia can be obtained at will depending on whetherhe strain is grown on agar or in liquid. (Above 25–30°C,ew or no microconidia are formed; the mcm strainroduces arthroconidia by septation of hypha and disjunc-ion of cells). A microcycle system was devised earlier thatnduced microconidiogenesis when microconidia werencubated in liquid medium (Rossier et al., 1977). How-ver, this method suffers from three limitations: (1) a pureicroconidial inoculum must be used to start the culture,

2) there is no synchrony, and (3) microconidiogenesis isery slow, requiring several days.

NVIRONMENTAL CONTROLF MICROCONIDIATION

umidity

Among the environmental variables affecting microco-idiation in Neurospora, moisture has the greatest influ-nce. This is inferred from the observations by Lindegrennd Lindegren (1941) in N. crassa with fluffy mutant, andy Grigg (1960a) with a peach colonial mutant, that mereetting of the mycelium for 2 days greatly stimulatedroduction of microconidia. In field conditions too, micro-onidiophores of N. intermedia developed profusely whenhe substrate (burnt sugarcane) had been soaked by rainfallPandit and Maheshwari, 1996). Moreover, microconidio-hores and protoperithecia were formed in tissue pockets.his apparently ensures humid conditions during theevelopment of reproductive structures and fertilization.

emperature

Grigg (1960b; 1965a) found that temperature affectsonidiation. When a fresh culture of a macroconidiateolonial-1 strain of N. crassa (derived from a culture of pe;ol-1 stock) was incubated initially for 2–7 days at 35°C,
acroconidiation was suppressed. When this aconidiate

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ulture was transferred to 25°C, the culture essentiallyroduced only microconidia. Even in the microconidiatingenotype fluffy, microconidiation was enhanced at 25°C ifhe culture was initially grown at 37°C for 2 days (Rossiert al., 1977, 1985). Observations on mcm mutant suggesthat cool environment favors microconidiation: nearly0 3 106 microconidia/ml of culture filtrate were producedn 24 h at 22°C while only one-fifth or one-tenth as manyere produced at 18 and at 26°C, respectively (Ma-eshwari, 1991b).

utrients

It is a common observation that development of proto-erithecia and microconidiophores occurs best in a me-ium with a low concentration of ammonium nitrogenWestergaard and Mitchell, 1947) and supplemented withellulose filter paper (a poor carbon source). By contrast,n the commonly used Vogel’s minimal medium supple-

ented with a sugar or on rich complete media, theseexual structures either are not produced or are producedn very low numbers (Sommer et al., 1989; Grigg, 1960a,965a). Bistis (1981) observed that in wild-type N. crassa,ormation of both microconidiophores and protoperithe-ia, but not of macroconidiophores, occurred under condi-

IG. 3. Size distribution of microconidia and macroconidia of N. crassas determined by a Coulter counter. After Gillie (1967).

ions of near starvation on 2% agar in distilled water and f


hat microconidia clustered about the protoperithecia.tarvation conditions also favor the development of repro-uctive structures in wild strains of N. intermedia inature. Their formation occurred after the scorched sugarane had been transformed from a nutrient-rich substratento a sugar- and nitrogen-depleted cellulosic substratePandit and Maheshwari, 1996).

The situation with respect to the mutant strains in whichacroconidial development is eliminated is different.ere, the development of microconidia is not affected by

utrient-rich conditions and, in fact, microconidiation mayven benefit from the supplementations of yeast extract,alt extract, and liver extract (Baylis and DeBusk, 1965a).icroconidial yield in submerged cultures of the mcm

train is optimal in Vogel’s N medium supplemented with.5% glucose or sucrose.

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Rossier et al. (1973) found that when wild-type N. crassaas grown in standing liquid cultures in a nitrate medium

o which 1 mM sodium iodoacetate was added, approxi-ately 95% of the conidia produced by mycelium wereicroconidia. It was hypothesized that microconidiogen-

sis is favored by the opening of the pentose shunt pathwaynder condition of diminished glycolysis. Ebbole andachs (1991) confirmed that iodoacetate increases theroduction of microconidia. They incorporated iodoac-tate in an agar medium and obtained 103–106 microco-idia per slant. The contaminating macroconidia andycelia were removed by a filtration procedure, although

ot without loss in recovery. This method of preparingicroconidia was used by Jarai and Marzluf (1991) for

electing nmr mutants of negative-acting nitrogen regula-ory gene of N. crassa from heterokaryotic primary transfor-

ants. Macino and co-workers also used Ebbole andachs’ method to obtain uninucleate microconidia fromild-type (al-11) N. crassa transformed with albino-1

al-1) gene and thereby analyze the phenotypes of nucleiecovered from ‘‘quelled’’ primary transformants witheduced pigmentation (Romano and Macino, 1992; Cogonit al., 1996). When the transgenic nuclei from ‘‘quelled’’lbino strains were combined with nontransgenic nuclei ineterokaryons, the dominance of quelled nuclei over thentransformed nuclei was observed. From this it was

nferred that quelling involves a transacting diffusible
actor that can enter the cytoplasm.

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PROCEDURE FOR OBTAININGURE MICROCONIDIA FROMILD-TYPE CULTURES

Based on the observations that in surface-grown culturesacroconidiation precedes microconidiation and that high

IG. 4. Microcycle microconidiation in mcm mutant of N. crassa. (A)rom which lateral protuberances have been formed into which single nucree microconidia are seen towards the left. Stained with Hoechst 33258.olume. Bar, 25 µm.

umidity and supplementation of medium with iodoac- i

tate stimulates microconidiation, Pandit and Maheshwari1993) improvised a method of obtaining almost pureicroconidia from surface-grown mycelium of wild-typeeurospora. A tiny macroconidial inoculum was placed at

he center of a cellophane circle layered over iodoacetate-upplemented medium in a Petri dish. The cellophane wasierced at the point of inoculation. The culture was

ned dark-field and fluorescence image of two macroconidial germ tubesentered. The macroconidia are towards the left and the right side. Three

µm. (B) Fluorescence image of isolated microconidia. Note large nuclear

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2–24°C. Under these conditions, production of macroco-idiophores by the mycelium spread over the cellophaneas reduced. After a determinative period of about 10ays, by which time macroconidiation had ceased, theellophane with conidiating mycelium over it was removednd the culture was incubated for 24 h. No new macroco-idiation was observed; the fine mycelial growth in agarecame programmed to produce aerial microconidio-hores. Successive crops of microconidia could be har-ested by swirling the culture with water. Approximately07–108 microconidia per Petri dish have been obtained 2o 5 days after removal of cellophane from wild-type N.rassa (Pandit and Maheshwari, 1993; K. K. Adhvaryu,npublished). Irelan and Selker (1997) have employed thisechnique to obtain microconidia from silenced transgenic. crassa strains harboring bacterial hph (hygromycinhosphotransferase) gene. The microconidia were used toxamine whether hph expression varies within a populationf clonally related cells. Expression of hph was variable andas correlated with epigenetically propagated differences

n methylation patterns. Shen et al. (1998) have used theellophane technique to examine whether the deletion ofhe rca-1 gene in N. crassa affects microconidial yield.ther applications of this technique are described later.

EVELOPMENT

Dodge (1930) described microconidophores of N. sito-hila as being ‘‘short or rather blunt branches arising at

ntervals along a hypha. The microconidia are borneaterally and terminally from cells of microconidiophoresnd are held together in drops of guttation fluid. Theyphae from which microconidiophores are formed areoticeably vacuolated.’’ This description applies to N.rassa as well (Pandit and Maheshwari, 1993). The hyphaerom which microconidiophores are produced are nar-ower than the vegetative hyphae. The hyphae whichroduce microconidiophores may originate as ‘‘intra-yphal hyphae’’ from older hyphae (R. Maheshwari, unpub-

ished observation). Ebbole (1996) noted that the microco-idiophore in wild-type N. crassa is produced from a ‘footell’ in the substrate mycelium and that the cells of theicroconidiophore are about 10 µm long. Lowry et al.

1967) made the first detailed study of ultrastructure andevelopment of microconidia in the peach-fluffy mutant of. crassa (Fig. 5). A microconidium originated as a
rotuberance on a short, somewhat swollen aerial branch

rom the surface mycelium. The outer layers of the hyphalall at this site degenerated and as the microconidiumrotruded, there was a deposition of more wall material athe base of the developing microconidium. At this stagehe cytoplasm of the developing microconidium was con-inuous with that of the parent cell. As the microconidium

IG. 5. Transmission electron micrograph of microconidiation in pe flutant of N. crassa. (A) Emergence of microconidium through aicroconidiophore hyphal cell. The outer layers of the hyphal wall at the

ite of emergence of the microconidium have been thrust aside andppear as collar. Note beginning of centripetal extension of the wall whichuts off microconidium at base (arrow) 316,000. (B) Centripetal exten-ion of the collar is complete (arrow) 315,600. (C) Mature microco-idium 322,500. The connection between the collar and the spore wall isisintegrating (arrow). Note single large nucleus. (D) Macroconidium13,800. Abbreviations: n, nucleus; m, mitochondrion; er, endoplasmic

eticulum; l, inclusion body. From Lowry et al. (1967) with permission ofhe publisher, American Society of Microbiology.

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rupted, the thickened layers of the parent cell were thrustside and appeared as a ‘‘collar’’ at the base of theeveloping microconidium. A nucleus entered the microco-idium and the microconidium was separated by a centripetalxtension of the collar. Microconidium development in Sclero-inia fruticola studied by electron microscopy by Willets andalonge (1969) resemble that of N. crassa. Lowry et al. (1967)

eported that the microconidia of N. crassa have a morextensive endoplasmic reticulum and fewer mitochondria thanhe macroconidia. Essentially similar ultrastructural features of

icroconidiogenesis were observed in iodoacetate-treated cul-ures of wild-type N. crassa (Rossier et al., 1973).

The number of microconidia produced from each cell ofhe microconidiophore remains uncertain. Dodge (1932)tated that ‘‘they are not formed internally as endospores,et they appear as though forced out through a collaredpening, one after another, occasionally adhering in a littlehain for a short time.’’ He felt that the collar structure mayepresent a very short sterigma. Later, Dodge (1936)bserved the same situation in P. anserina. In S. fruticolaicroconidia are produced in succession from bottle-

haped phialides and held together by mucilage. In iodoac-tate-stimulated microconidiogenesis in wild-type N. crassa,ossier et al. (1973) observed that often elongated microco-idia were successively budded at the same point on theollar of the mother cell, as is typical of phialoconidiaudded from a phialide. These authors therefore consid-red microconidia to be phialoconidia. Springer and Yanof-ky (1992) studied the highly microconidiating strain, fl;dn,y scanning electron microscopy and observed microco-idia originating ‘‘within the vegetative hyphae’’ beforemerging through the hole that had been left in the parentell by the first-formed microconidium (Fig. 6). In con-rast, the macroconidia are produced by repeated apicaludding of a hyphal tip (blastic development) (Springernd Yanofsky, 1989; Springer, 1993).

Light microscopy of wild-type N. crassa has shownicroconidia to be arranged in groups of three to eight

Fig. 7). Further study of the ontogeny of microconidia inifferent genotypes by a combination of scanning andransmission electron microscopy is desirable to clarifyheir mode of development and to elucidate the pattern ofrrangement of microconidia. Moreover, a study of theuclear events during microconidiogenesis is required tonderstand a critical aspect of microconidium formation,

.e., how uninucleate cells arise from multinucleate hyphalells.

The N. crassa double mutants, pe; fl; and fl;dn, in which s

acroconidiation is blocked, produce abundant microco-idia. This shows clearly that the microconidiation pathway

s distinct from the macroconidiation pathway. Ultrastruc-ural studies of Lowry et al. (1967) suggest the following

IG. 6. Scanning electron micrograph of microconidiation in fl;dnutant of N. crassa. (A) Microconidia borne on hyphae (bar, 10 µm). (B)microconidium is erupting through hyphal cell wall (bar, 1 µm). (C) A

econd microconidium is emerging through the hole in hyphal wall (bar,µm). From Springer and Yanofsky (1992) with permission of Cold

pring Harbor Laboratory Press.

teps in microconidium differentiation:


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Microconidiophore initial

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Separation of microconidium

on GENES

Berlin and Yanofsky (1985) isolated several genes, named

IG. 7. Microconidiophores of wild-type N. crassa. (A). Bright-fieldrocedure of Pandit and Maheshwari (1993) and stained with acid-fuchsinrising from very short protuberances from branches of microconidiophoar length, 50 µm in all micrographs.

onidiation (con) genes, by virtue of their being expressed m


referentially during macroconidiation over mycelialrowth. Springer and Yanofsky (1992) found that thexpression of five of these genes was not specific, beingxpressed not only in macroconidia but also in microco-idia, even though the two spore types are formed byompletely different processes. The observation of com-on con gene transcripts in macroconidia and microco-

idia (and also in ascospores) led Springer and Yanofsky1992) to suggest that these products might function inevelopment, dormancy, and germination. Protoperitheciand microconidiophores are formed simultaneously underonditions of carbon and nitrogen starvation and highumidity, and their development is favored when macroco-idiation has ceased (Pandit and Maheshwari, 1996) orven benefited when macroconidiation is blocked as in theluffy mutant. This suggests that some genes are commono sexual development and microconidiation but distinctrom the macroconidiation pathway. By subtractive hybrid-zation, Nelson and Metzenberg (1992) isolated severalexual development (sdv) genes that are expressed prefer-ntially under conditions favoring the differentiation ofrotoperithecia. Sommer et al. (1989) identified blue-light-egulated (bli) genes that are important for the formationf protoperithecia. It would be of interest to compare thexpression of sdv and bli genes during differentiation of

aph showing clusters of microconidiophores in culture initiated by thenlarged view of two microconidiophores. Groups of microconidia are seenFluorescence image of microconidiophores stained with Hoechst 33258.

microgr. (B). Ere. (C)

icroconidia, macroconidia, and protoperithecia.

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ERMINATION RATES

Although Dodge (1930) did not quantitate microconidialermination, his photograph of microconidia of N. sito-hila (Fig. 1) shows them as having swollen and elongatedfter 40 h of sowing on corn meal agar, indicating theirotential for near complete germination. The first measure-ent of microconidial germination in N. crassa was made

y Lindegren and Lindegren (1941). Their data show thaticroconidia of their fluffy mutant had high (up to 84%)

ermination. In their method microconidia were placednitially on beef extract agar in marked positions by meansf a micromanipulator. After 24 h, individual microconidiaere transferred to tubes containing potato dextrose agarnd the germination was scored. This laborious procedureas employed before Tatum et al. (1949) discovered thearamorphogenic effect of sorbose in inducing colonialrowth, making it possible to use plating on sorboseedium as a standard method of testing viability. The

iability of microconidia estimated by colony-formingbility on sorbose medium has been low or erratic (Table). Brockman and de Serres (1963) found that sorboseffects viability in macroconidial or ascospore platings ineurospora; the viability of mutant strains being affected

ABLE 3

iability or Plating Efficiency of Microconidia of N. crassa

GenotypeMethod of produc

microconidia

l Surface myceliumol; pem; fl; inl Surface myceliumem; fl; pan Surface myceliume; fl Surface myceliume; fl (FGSC No. 867) Surface myceliume; supe; col-1; acon-t Surface myceliumr; rg; pe; fl (FGSC No. 331) Surface myceliume; fl (FGSC No. 569) Surface myceliume; fl (F1 of FGSC No. 569 3 74-OR-23a) Surface myceliuml; dn Surface myceliume; fl Surface myceliumro-9; flL; inl; qa Surface myceliuml; dn (P9260) Surface myceliume; fl (P2178) Surface myceliumcm (FGSC 7091) Liquid shake cultucm (FGSC 7091) 3 Liquid shake cultu

4-ORS-6ae; fl (FGSC No. 3072) Surface myceliuml; dn (FGSC No. 3517) Surface mycelium

a Determined by comparison of number of colonies formed per plate tob
Germination of individual microconidia isolated on agar medium slants.
o varying extents by experimental variables (Brockmannd de Serres, 1963). Interestingly, when microconidia ofe;fl and mcm were put on the surface of a dialysisembrane overlying sorbose medium, their germinationas more than 80% (Kalpana et al., 1998). Munkres (1977)bserved that plating efficiency of microconidia of a peachluffy (pe; fl) mutant increased from 1–2% to 85–95% afterack crosses to wild-type N. crassa. Germination of mcmicroconidia was also reported to improve after a few

ackcrosses to wild-type N. crassa (Pandit, 1993). Theseesults indicate the existence of genes that influenceicroconidial germination.As noted earlier, microconidia of P. anserina do not

erminate in vitro; yet they effect fertilization. Dodge1932) reported that 25-day-old microconidia of N. sito-hila germinated well but functioned poorly in fertiliza-ion. He emphasized that in order to fertilize successfully,

icroconidia should be from fresh cultures. Whether theoss in the ability to fertilize is due to decline in theroduction of pheromone is not known. In Neurospora,he ability to fertilize and the ability to form vegetativeolonies may vary independently.

Grigg (1960c) reported an intriguing case of apparentow germination of microconidia. He synthesized a hetero-

%Germinationa Reference

84 Lindegren and Lindegren (1941)b

10–30 Giles (1951)0.1–6 Goodman (1958)14–30 Barratt (1964)

10 Baylis and DeBusk (1965a)10 Grigg (1965a)60 Chang and Tuveson (1967)

1–2 Munkres (1977)85–95 Munkres (1977)

6 Hedman and Vanderschmidt (1981)24–39 Hedman and Vanderschmidt (1981)30–60 Rossier et al., (1985)

5 Maheshwari (1991a)7 Maheshwari (1991a)

26 Maheshwari (1991a)60 Pandit (1993)

.80 Kalpana et al., (1998)40 Kalpana et al., (1998)

mber plated, as determined by haemocytometer count.

ing

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aryon between two strains, his-3; col-11; pe1; su-m1; al-2nd his-31; col-1; pe; su-m; al-21, and observed thathereas microconidia from the protrophic histidine (his-

1) homokaryon germinated on minimal medium, theis-31 microconidia from the (his 1 his-31) heterokaryonequired a specific supplement of histidine for germina-ion. The requirement for histidine, however, was a tran-ient phenomenon, limited to their initial germination.hus, even though a microconidium was genotypicallyis1, the cytoplasm it received from the heterokaryon waseficient in some factors required by his-31 nuclei to

nitiate histidine biosynthesis.The first visible sign of germination of microconidia is

heir swelling and they appear round, oval, or elongated (R.aheshwari and K. K. Adhvaryu, unpublished observa-

ions). The increase in size was associated with increase inuclear number prior to cellularization (Fig. 8).The low plating efficiency of microconidia has tended to

iscourage their use in genetic studies. Clearly, one of theuture objectives should be to improve plating media.erhaps, a comparative investigation of macroconidia andicroconidia of widely differing germinability (Table 3) in

erms of their ultrastructure, chemical composition, pro-ein synthesis machinery, and nuclear cycle may provideome clue to their generally low plating efficiency. Thenability of microconidia of G. temulenta to germinate wasttributed to the very low concentrations of RNA in theytoplasm resulting from cytochemical differentiation inhe microconidiophores (Griffiths, 1959).

IOCHEMISTRY

When harvested and pelleted, microconidia appearreenish- or blackish-brown. Turian et al. (1967) identifiedelanin in alkali extracts of microconidia. An acidic

arotenoid, neurosporoxanthin, was also present. Ballound Bianchi (1978) analyzed the lipid composition oficroconidia of peach fluffy; cot-1 mutant of N. crassa. In

-day-old microconidia maintained at 25°C, only freeterols and sterol esters were in measurable amountshereas triglycerides and phospholipids were present in

race amounts. Free fatty acids were not detected. Further,he authors reported that during microconidial maturationhe lipid components decreased rapidly. The loss in viabil-ty appeared related to the decrease in lipid content; 4-day-ld microconidia had no detectable lipid. Presence of lipidsas also suggested in microconidia of S. fruticola based on
ltrastructural observations (Willets and Calonge, 1969).

IG. 8. Nuclear division and septum formation in germinating micro-onidia of pe;fl mutant of N. crassa. Microconidia were germinated onorbose medium at 30°C for 18 h and stained with a combination ofalcofluor and Hoechst 33258. (A). Nucleus almost fills the cell volume.B) Swollen microconidia contain two (below) and four (above) nuclei.C) Septum formation. Each cell is binucleate. (D) Two-celled structureith divided nuclei. (E) Four-celled structure with multiple nuclei. (F)ompartments with multiple nuclei. (G) A germling showing multinucle-

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On the basis of chemical analysis, Horowitz and Ma-leod (1960) estimated the DNA content per nucleus inhe microconidium of rg;cr-1;al-2;pe;fl; A mutant to be.63 3 10214 g. From this value, the 1 C value of DNA in. crassa is 28 3 109 Da or ,42 Mbp. This value agrees

ery closely with ,40 Mbp based on microfluorometriceasurements of individual nuclei stained with Schiff

eagent (Duran and Gray, 1989) and with the value of 42.9bp, estimated by pulsed field gel electrophoresis (Or-

ach et al., 1992).

SES OF MICROCONIDIA

ransformation

In Neurospora, macroconidia are commonly used forNA-mediated transformation as they are readily ob-

ained, germinate rapidly, and are amenable for protoplast-ng. However, because they are mostly multinucleate, theransformed colonies are typically heterokaryotic, contain-ng only a few transformed nuclei. Purification of theransformed nuclei by passing it through the sexual cycle isheoretically possible but generally unsuccessful becausef the RIP phenomenon (Selker et al., 1987). Therefore,urification is now laboriously done by repeated platings ofacroconidia and isolations of the transformed phenotype

rom single macroconidial isolates. The use of uninucleateicroconidia for transformation would be a straight for-ard method of circumventing this difficulty and puteurospora on a par with Aspergillus in which new

ransformants are automatically recovered in uninucleateonidia. The feasibility of transformation of spheroplasts ofeurospora microconidia has been demonstrated usingis1 DNA (Ebbole and Sachs, 1990), hygromycin-resistanthph) plasmid (Royer and Yamashiro, 1992), and benomyl-esistant plasmid (Rossier et al., 1997).

Even when multinucleate protoplasts from macroco-idia or hyphal cells are used for transformation, primaryransformants can be purified quickly if they can be madeo yield microconidia, as demonstrated by the work of Jaraind Marzluf (1991) and of Romano and Macino (1992),eferred to earlier. Rossier et al. (1985) incorporated theluffy (flL) gene (which blocks macroconidiation) into theost strain, aro-9;inl;qa-2, and transformed mycelial proto-lasts with qa-21 DNA. The microconidia produced by therimary transformant were used to isolate individualransformation products. About half of the transformants
ere apparently homokaryotic. Microconidia from the n
eterokaryotic primary transformants were plated ontoelective and nonselective media to estimate the propor-ion of aro1 nuclei. Using microconidia to estimate nuclearatios, the authors found that catabolic dehydroquinasectivity was generally in agreement with the proportion ofransformed nuclei. Margolin et al. (1997) found that mostis1 transformants in N. crassa were heterokaryotic. How-ver, the desired homokaryotic transformants could bextracted from the primary transformants through microco-idia produced by the cellophane method (Pandit andaheshwari, 1993). Grigg (1965a) observed that a normalacroconidial strain could be induced to form microco-

idia if combined in a heterokaryon with a microconidialtrain with the nuclear ratio (1:20 to 40) in favor of theicroconidial strain. Preliminary tests with some

mcm1 1 mcm) heterokaryons have shown that heterokary-tic macroconidia will produce microconidia in shakeultures if the mcm nuclei are present in excess (K. Pitchaiani and R. Kalpana, unpublished). Thus, another option

or purification of transformed nuclei may be to put thempure transformant into a heterokaryon with an excess ofet-compatible mcm nuclei containing a forcing markernd growing the heterokaryon to conidiation. Macroco-idia from such a heterokaryon might then be shakenvernight to get microconidia and plated to obtain theransformed nucleus in pure homokaryotic form.

eterokaryon Analysis

The observation that uninucleate conidia of wild isolatesf some fungi give rise to morphologically distinguishableolonies led to the recognition of heterokaryosis as aidespread phenomenon in fungi (see Davis, 1966). Un-oubtedly different nuclear types present in the myceliumere segregating into uninucleate spores. Sansome (1947)

ynthesized heterokaryons of N. crassa using pairs oforphologically distinct mutations in a fluffy strain and

ecovered the mutant types by microconidial isolations.bbole and Sachs (1990) obtained microconidia from theeterokaryon (cyh-1 ad-3B aml 1 al-1 lys-4) grown inedium containing iodoacetate and resolved the hetero-

aryon into its individual adenine- and lysine-requiringomponents by microconidial plating. The cellophaneethod of Pandit and Maheshwari (1993) provides an easy,

fficient method for obtaining microconidia free of macro-onidia and this is being applied to detect genetic variabil-ty that remains hidden in strains collected from nature ashe following examples show.

A sample of microconidia produced by a phenotypically
ormal wild-collected N. intermedia strain from southern

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ndia was plated to derive single-colony homokaryoticultures. Among some 150 cultures, one homokaryon had atriking phenotype: it showed a senescence syndrome inubcultures (Pandit et al., 1994). Senescence of the mutantomokaryon was controlled by a single gene, senescencesen), which was introgressed into N. crassa and mapped ininkage group VR (A. Navaraj, A. Pandit and R. Ma-eshwari, unpublished). This is the first nuclear-geneenescence mutant identified in a strain of Neurosporasolated from nature. Microconidial platings of two other. intermedia wild isolates have also yielded senescent

trains. The senescence syndrome in these new strains hashown maternal inheritance (R. Maheshwari, A. Navarajnd P. Delhi, unpublished).

Among the homokaryotic isolates derived from a wildsolate (Vickramam) of N. crassa from southern India, fourtable types were plainly distinguishable by observation ofxternal morphology alone (A. Pandit, unpublished), reveal-ng that a wild isolate can be heterokaryotic for a number ofuclear types. Vickramam also yielded a para-aminoben-oic acid auxotroph which was originally leaky but becameather tight when backcrossed to wild-type N. crassa.omplementation test has indicated that the new mutant isifferent from pab-1 and pab-2 mutants of N. crassa.K. K. Adhvaryu, unpublished). Earlier, only two leakyuxotrophs (threonine and thiamine) were detected amonghe thousands of Neurospora strains collected from naturePerkins and Turner, 1988). Although only four wild strainsf Neurospora have been examined so far by microconidialnalysis, the ease with which morphological variants werebtained in all cases suggests that heterogeneity of nuclearypes (and possibly of mitochondrial types) within theycelium may be a common phenomenon. Perhaps more

ariability will be uncovered by molecular methods. Weave used microconidial plating to examine the purity of

aboratory stocks of wild-type N. crassa strains, 74-OR23-VA (FGSC No. 2489) and 74-ORS-6a (FGSC No. 4200).

orphologically abnormal variants were found in lowrequency (about 3%) among the colonies (K. K. Adhvaryu,npublished). These examples demonstrate that microco-idia are a valuable tool in detecting the spectrum ofutations arising in nature and in the laboratory stocks,

ncluding recessive lethal mutations.

uclear Ratios

It has been proposed that by altering the frequency ofuclear types, the fungal mycelium can make biochemicaldjustments to a changing environment (Jinks, 1952).
hether adaptive shifts in nuclear ratios occur remains m

ontroversial because of the technical problem of samplinguclei in mycelium. In the few studies on this subject, thestimation of nuclear ratios in Neurospora heterokaryonsas based on differential counts of the numbers ofomokaryotic and heterokaryotic macroconidia. A formulaased on the average number of nuclei per conidium waserived for estimating nuclear ratios (Prout et al., 1953;avis, 1959). Since the nuclear number per conidium can

e highly variable (Barratt and Garnjobst, 1949; Huebsch-an, 1952; Pittenger, 1967), a straight forward method of

ampling nuclear types and determining their frequency inhe mycelium would be through the sampling of microco-idia. If it is assumed that nuclei are randomly segregated

nto microconidia during their production, a sampling oficroconidial genotypes would be equivalent to a sampling

f nuclei in the heterokaryon. Grigg (1965b) found thatuclear ratio in a (his1 1 his) heterokaryon estimated byacroconidial plating was at least an order of magnitude

igher than by microconidial plating.A beginning has been made to study changes in nuclear

atios in heterokaryons in response to chemical composi-ion of the medium based on microconidial sampling (A.andit and R. Maheshwari, unpublished). As an example, aeterokaryotic association was forced between two glycosi-ase mutants into which the mcm gene and appropriateuxotrophic marker genes were introduced by crossing.he heterokaryon, (mcm; tre; pan; inv1; nic1 1 mcm; tre1;an1; inv; nic), was grown and allowed to conidiate inedia with either sucrose or trehalose as the carbon

ource. The macroconidia were collected and shakenvernight in liquid media containing individual sugars tobtain microconidia. These were plated on media supple-ented with pantothenic acid or nicotinic acid. The

roportion of the wild type inv1 and tre1 nuclei wasstimated based on the number of colonies on the twoedia, respectively. Simultaneous measurements wereade of invertase and trehalase activities in cultures to

etermine whether the nuclear frequency and enzymectivities are correlated. Different ratios of inv1 and tre1

uclei were found in mycelium grown in sucrose orrehalose. The activities of invertase and trehalase did notlways correlate with the frequencies of inv1 and tre1

uclei in the heterokaryotic mycelium.

utation

Because of their thin walls, uniform size, uninucleateondition, and high nuclear volume which reduces themount of radiation adsorbed by the cytoplasm, the
icroconidia of N. crassa were among the favored material

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or early investigations of the effect of radiation. Moreover,nlike other fungi in use at that time, the induced changesould be subjected to genetic analysis. The Lindegrensere the first to use microconidia in mutation research.hey obtained microconidia from a fluffy mutant—one of

he first morphological mutants that they had discoveredLindegren and Lindegren, 1941). X-ray treatment in-uced both gene mutations and chromosomal aberrations,

ncluding inversions. In microconidia exposed to ultravio-et radiation, 16–44% of survivors showed discerniblehanges in morphology but no chromosomal aberrationsere observed. In similar experiments, Sansome et al.

1945) showed that the mutant phenotypes were corre-ated with sterility and this was believed to be due tohromosomal aberration. A great diversity of morphologi-al mutants, from tiny colonial forms to vigorous forms,as obtained.In these early experiments the spreading growth habit of

eurospora made it very difficult to score germination onhe plate and determine the survival rate. The use oforbose in plating medium to restrict mycelial spread,ntroduced by Tatum et al. (1949), later overcame thisifficulty. These investigators demonstrated that X-ray-reated microconidia of peach fluffy could be plated tobtain mutants. Of the 1900 colonies recovered, 24% wereorphological variants and 3% were biochemical mutants.iles (1951) used the plating method to determine theose-response survival curves for microconidia and found a

inear log survival curve. In contrast, log survival curves foracroconidia had an initial shoulder which was attributed

o their multinucleate condition (Atwood and Norman,949). Later, however, Chang and Tuveson (1967) found ahoulder also for microconidia of a crisp ragged peachluffy strain (UVS-1) but not for a methionine-requiringuxotroph (cr rg; pe fl, me) (UVS-2). The initial shoulder ofVS-1 may be due to the UV repair capacity of the UVS-1

ells, which becomes saturated at a certain UV dose. Thetrictly exponential inactivation curve observed by Giles1951) and later by Norman (1954) may be due to UVepair-deficient strains used by them. Goodman (1958)solated a UV-resistant strain from microconidial strain of. crassa. From the comparison of the survival kinetics of

he resistant strain with the parental strain and witheterokaryon made between sensitive microconidial strainith macroconidial wild-type strain, he attributed inactiva-

ion of conidia to lethal mutation, nuclear inactivation, andytoplasmic inactivation.

Giles (1951) used microconidia of various nutritional
utants of N. crassa to investigate spontaneous and e
adiation-induced reversions. By genetic tests he deter-ined that most of the reversions were back mutation andfew were due to suppressor mutations. He subjectedicroconidia of eight inositol mutants to ultraviolet radia-

ion and observed marked differences in their reverseutation frequencies. The results indicated the existence

f multiple alleles at the inositol locus, distinguishable onlyy their relative abilities to undergo reverse mutation.aylis and DeBusk (1965b) subjected microconidia of a pe,

l mutant to X-ray to obtain cycloheximide-resistant mu-ants. Microconidia with their low amount of cytoplasm

ay prove very useful in the isolation of new mutants.

ONCLUSIONS AND FUTUREIRECTIONS

In 1936 Dodge was led to remark, ‘‘Few persons whoave studied species of Neurospora even intensively havever seen their spermatia.’’ This remark has not entirelyost relevance even today. For most biologists who useeurospora, the microconidia have remained unseen,

uperfluous structures. In nature microconidia function aspermatia. For this function their germinability is not ahysiological necessity. In Neurospora, however, whereicroconidia can be induced to germinate, they are a

aluable material for comparative investigations of sporeormancy and germination and for a molecular geneticpproach to the question of how differences arise in sporeypes within the same organism. Microconidia provide aowerful tool for uncovering the variability that arises inature and is stored hidden in the multinucleate mycelium.his knowledge is important for understanding changes in

he genetic composition of Neurospora in natural popula-ions. Furthermore, microconidial analyses of strains fromature can be a very effective method of obtaining geneticariants which may be difficult or impractical to obtain inhe laboratory. The ability to extract nuclei in this manners of great potential value in purifying new transformantsnd in estimating nuclear ratios in heterokaryons. Theethod should allow renewed investigations and an under-

tanding of the significance of heterokaryosis in fungi.

CKNOWLEDGMENTS

I am indebted to David Perkins, Stanford University, for his constant
ncouragement and enormous help in writing this review and for his

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ritical reading of the manuscript. I thank Matthew Springer, Stanfordniversity, for providing photographs (Fig. 6), George Bistis, Drewniversity, for comments on the manuscript, and Manjuli Maheshwari for

mprovements in style. I thank members of my laboratory, A. Pandit, A.avaraj, K. Pitchai Mani, K. K. Adhvaryu, Praveen Delhi, and R. Kalpana,

or their research contributions. Research support was provided by theepartment of Biotechnology and the Department of Science and

echnology, Government of India.

EFERENCES

twood, K. C., and Norman, A. 1949. On the interpretation of multi-hitsurvival curves. Proc. Natl. Acad. Sci. USA 35: 696–709.

valos, J., Casadesus, J., and Cerda-Olmedo, E. 1985. Gibberella fujikuroimutants obtained with UV radiation and N-methyl-N-nitrosoguanidine.Appl. Environ. Microbiol. 49: 187–191.

ackus, M. P. 1939. The mechanics of conidial fertilization in Neurosporasitophila. Bull. Torrey. Bot. Club. 66: 63–76.

allou, L. R., and Bianchi, D. E. 1978. Lipids associated with microco-nidial differentiation in a mutant of Neurospora crassa. Curr. Micro-biol. 1: 111–115.

arratt, R. W. 1964. Viability of microconidia. Neurospora Newslett. 6:6–7.

arratt, R. W., and Garnjobst, L. 1949. Genetics of a colonial microconidi-ating mutant strain of Neurospora crassa. Genetics 34: 351–369.

aylis, J. R., and DeBusk, A. G. 1965a. Notes on the use of microconidiat-ing strains in mutation experiments. Neurospora Newslett. 7: 7–8.

aylis, J. R., and DeBusk, A. G. 1965b. Actidione resistance: A forwardmutation technique and its application for mosaic analysis. NeurosporaNewslett. 7: 8–9.

aylis, J. R., and DeBusk, A. G. 1967. Estimation of the frequency ofmultinucleate macroconidia in microconidiating strains. NeurosporaNewslett. 11: 9.

erlin, V., and Yanofsky, C. 1985. Isolation and characterization of genesdifferentially expressed during conidiation of Neurospora crassa. Mol.Cell. Biol. 5: 849–855.

istis, G. N., and Georgopoulos, S. G. 1979. Some aspects of sexualreproduction in Nectria haematococca var. cucurbitae. Mycologia 71:127–143.

istis, G. N. 1981. Chemotropic interactions between trichogynes andmacroconidia of opposite mating-type in Neurospora crassa. Mycologia73: 959–975.

istis, G. N. 1983. Evidence for diffusible, mating-type-specific tricho-gyne attractants in Neurospora crassa. Exp. Mycol. 7: 292–295.

rockman, H. E., and de Serres, F. J. 1963. ‘‘Sorbose toxicity’’ inNeurospora. Am. J. Bot. 50: 709–714.

hang, L-T., and Tuveson, R. W. 1967. Ultraviolet-sensistive mutants inNeurospora crassa. Genetics 56: 801–810.

ogoni, C., Irelan, J. T., Schumacher, M., Schmidhauser, T. J., Selker,E. U., and Macino, G. 1996. Transgene silencing of the al-1 gene invegetative cells of Neurospora is mediated by a cytoplasmic effectorand does not depend on DNA–DNA interactions or DNA methylation.
EMBO J. 15: 3153–3163.

avis, R. H. 1959. Asexual selection in Neurospora crassa. Genetics 44:1291–1308.avis, R. H. 1966. Mechanisms of inheritance: 2. Heterokaryosis. In TheFungi, An Advanced Treatise (G. C. Ainsworth and A. S. Sussman,Eds.), Vol. 2, pp. 567–588. Academic Press, New York.avis, R. H., and de Serres, F. J. 1970. Genetic and microbiologicalresearch techniques for Neurospora crassa. Methods Enzymol. 27A:79–143.odge, B. O. 1930. Breeding albinistic strains of the Monilia bread mold.Mycologia 22: 9–38.odge, B. O. 1932. The non-sexual and the sexual functions of microco-nidia of Neurospora. Bull. Torrey Bot. Club 59: 347–360.odge, B. O. 1936. Spermatia and nuclear migrations in Pleurageanserina. Mycologia 28: 284–291.rayton, F. L. 1932. The sexual function of the microconidia in certaindiscomycetes. Mycologia 24: 345–348.rayton, F. L., and Groves, J. W. 1952. Stromatinia narcissi, a newsexually dimorphic discomycete. Mycologia 44: 119–140.uran, R., and Gray, P. M. 1989. Nuclear DNA, an adjunct to morphologyin fungal taxonomy. Mycotaxon 36: 205–219.

bbole, D. J. 1996. Morphogeneis and vegetative differentiation infilamentous fungi. J. Genet. 75: 361–374.

bbole, D. J., and Sachs, M. S. 1990. A rapid and simple method forisolation of Neurospora crassa homokaryons using microconidia. Fun-gal Genet. Newslett. 27: 17–18.

l-Ani, A. S. 1968. The cytogenetics of the conidium in Microsporumgypseum and of pleomorphism and the dual phenomenon in fungi.Mycologia 60: 999–1015.

sser, K. 1974. Podospora anserina. In Handbook of Genetics (R. C. King,Ed.), Vol. 1, pp. 531–551. Plenum, New York.

ahey, R. C., Mikolajczyk, S. D., and Brody, S. 1978. Correlation ofenzymatic activity and thermal resistance with hydration state inungerminated Neurospora conidia. J. Bacteriol. 135: 868–875.iles, N. H., Jr. 1951. Studies on the mechanism of reversion inbiochemical mutants of Neurospora crassa. Cold Spring Harbor Symp.Quant. Biol. 16: 283–313.illie, O. J. 1967. Use of the Coulter counter to measure the numbers andsize distribution of macroconidia and microconidia of Neurosporacrassa. Neurospora Newslett. 11: 16.oodman, F. 1958. A study of the mechanism of resistance to ultravioletlight in a strain of Neurospora crassa. Z. Vererbungslehre 89: 675–691.riffiths, E. S. 1959. Cytological and cytochemical aspects of thedifferentiation of the microconidia of Gloeotinia temulenta. Trans. Br.Mycol. Soc. 42: 123–124.rigg, G. W. 1960a. The control of conidial differentiation in Neurosporacrassa. J. Gen. Microbiol. 22: 662–666.rigg, G. W. 1960b. Temperature-sensitive genes affecting conidiation inNeurospora. J. Gen. Microbiol. 22: 667–670.rigg, G. W. 1960c. Phenotypic lag in Neurospora. Heredity 14: 207–210.rigg, G. W. 1965a. Induction of microconidiation or macroconidiationon the same Neurospora stock. Neurospora Newslett. 7: 12–13.rigg, G. W. 1965b. Determination of nuclear ratios of microconidialstrains. Neurospora Newslett. 7: 13–14.aard, K. 1967. Effect of age on relative plating efficiency of Neurospora
conidia. Neurospora Newslett. 11: 12–13.

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R

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edman, S. C., and Vanderschmidt, S. 1981. Germination of microco-nidia from selected Neurospora strains. Neurospora Newslett. 28: 14.orowitz, N. W., and Macleod, H. 1960. The DNA content of Neuros-pora nuclei. Microbial Genet. Bull. 17: 6–7.uebschman, C. 1952. A method of varying the average number of nucleiin the macroconidia of Neurospora crassa. Mycologia 44: 599–604.

relan, J. T., and Selker, E. U. 1997. Cytosine methylation associated withrepeat-induced point mutation causes epigenetic silencing in Neuros-pora crassa. Genetics 146: 509–523.

arai, G., and Marzluf, G. A. 1991. Generation of new mutants of nmr, thenegative-acting nitrogen regulatory gene of Neurospora crassa, byrepeat induced mutation. Curr. Genet. 20: 283–288.

inks, J. L. 1952. Heterokaryosis: A system of adaptation in wild fungi.Proc. R. Soc. B140: 83–99.

alpana, R., Adhvaryu, K. K., and Maheshwari, R. 1998. Germination andplating efficiency of Neurospora crassa microconidia. Fungal Genet.Newslett. 45: 19–20.

indegren, C. C., and Lindegren, G. 1941. X-ray and ultra-violet inducedmutations in Neurospora. I. X-ray mutations. J. Hered. 32: 404–412.

owry, R. J., Durkee, T. L., and Sussman, A. S. 1967. Ultrastructuralstudies of microconidium formation in Neurospora crassa. J. Bacteriol.94: 1757–1763.aheshwari, R. 1991a. Microcycle conidiation and its genetic basis inNeurospora crassa. J. Gen. Microbiol. 137: 2103–2116.aheshwari, R. 1991b. A new genotype of Neurospora crassa thatselectively produces abundant microconidia in submerged shake cul-tures. Exp. Mycol. 15: 346–350.argolin, B. S., Freitag, M., and Selker, E. U. 1997. Improved plasmidsfor gene targeting at the his-3 locus of Neurospora crassa by electropora-tion. Fungal Genet. Newslett. 44: 34–35.oreau, M., and Moreau, F. 1939. Sur les microconidies des Neurospora.Bull. Soc. Botan. France 86: 12–16.unkres, K. D. 1977. Selection of improved microconidial strains ofNeurospora crassa. Neurospora Newslett. 24: 9–10.elson, M. A., and Metzenberg, R. L. 1992. Sexual development genes ofNeurospora crassa. Genetics 132: 149–162.orman, A. 1954. The nuclear role in the ultraviolet inactivation ofNeurospora conidia. J. Cell. Comp. Physiol. 44: 1–10.rbach, M. J. 1992. No title. Fungal Genet. Newslett. 39: 92.andit, A. 1993. Germination of mcm microconidia of Neurospora crassa.Fungal Genet. Newslett. 40: 63.

andit, A., and Maheshwari, R. 1993. A simple method of obtaining puremicroconidia in Neurospora crassa. Fungal Genet. Newslett. 40: 64–65.

andit, A., Kannan, B., and Maheshwari, R. 1994. Presence of nucleicarrying a recessive lethal gene in a wild-isolate of Neurospora. FungalGenet. Newslett. 41: 66.

andit, A., and Maheshwari, R. 1996. Life history of Neurosporaintermedia in a sugar cane field. J. Biosci. 21: 57–79.

erkins, D. D. 1992. Neurospora: The organism behind the molecularrevolution. Genetics 130: 687–701.

erkins, D. D., Radford, A., Newmeyer, D., and Bjorkman, M. 1982.Chromosomal loci of Neurospora crassa. Microbiol. Rev. 46: 426–570.

erkins, D. D., and Turner, B. C. 1988. Neurospora from naturalpopulations: Toward the population biology of a haploid eukaryote.
Exp. Mycol. 12: 91–131. T
ittenger, T. H. 1967. Distribution of nuclei in conidia. NeurosporaNewsl. 11: 10–12.

rout, T., Huebschman, C., Levene, H., and Ryan, F. J. 1953. Theproportion of nuclear types in Neurospora heterocaryons as deter-mined by plating macroconidia. Genetics 38: 518–529.

omano, N., and Macino, G. 1992. Quelling: Transient inactivation ofgene expression in Neurospora crassa by transformation with homolo-gous sequences. Mol. Microbiol. 6: 3343–3353.

ossier, C., Oulevey, B., and Turian, G. 1973. Electron microscopy ofselectively stimulated microconidiogenesis in wild type Neurosporacrassa. Arch. Microbiol. 91: 345–353.

ossier, C., Ton-That, T-C., and Turian, G. 1977. Microcycle microconidia-tion in Neurospora crassa. Exp. Mycol. 1: 52–62.

ossier, C., Pugin, A., and Turian, G. 1985. Genetic analysis of transforma-tion in a microconidiating strain of Neurospora crassa. Curr. Genet. 10:313–320.

ossier, C., Brazil, G., and Utz-Pugin, A. 1992. Mitotic instability inbenomyl-resistant transformants of a fluffy strain of Neurospora crassa.Fungal Genet. Newslett. 39: 73–75.

oyer, J. C., and Yamashiro, C. T. 1992. Generation of transformablespheroplasts from mycelia, macroconidia, microconidia and germinat-ing ascospores of Neurospora crassa. Fungal Genet. Newslett. 39:76–77.

ansome, E. R. 1947. Somatic segregation by microconidial isolation insynthesized heterokaryons of Neurospora crassa. Proc. R. Soc. Edin-burgh Ser. B 62: 299–303.

ansome, E. R., Demerec, M., and Hollaender, A. 1945. Quantitativeexperiments with Neurospora crassa. I. Experiments with X-rays. Am.J. Bot. 32: 218–226.

elker, E. U., Cambareri, E. B., Jensen, B. C., and Haack, K. R. 1987.Rearrangement of duplicated DNA in specialized cells of Neurospora.Cell 51: 741–752.

hen, W.-C., Wieser, J., Adams, T. H., and Ebbole, D. J. 1998. TheNeurospora rca-1 gene complements an Aspergillus flbD sporulationmutant but has no identifiable role in Neurospora sporulation. Genetics148: 1031–1041.

ommer, T., Chambers, J. A. A., Eberle, J., Lauter, F.-R., and Russo,V. E. A. 1989. Fast-light regulated genes of Neurospora crassa. NucleicAcid Res. 17: 5713–5723.

pringer, M. L. 1993. Genetic control of fungal differentiation: The threesporulation pathways of Neurospora crassa. BioEssays 15: 365–374.

pringer, M. L., and Yanofsky, C. 1989. A morphological and geneticanalysis of conidiophore development in Neurospora crassa. GenesDev. 3: 559–571.

pringer, M. L., and Yanofsky, C. 1992. Expression of con genes along thethree sporulation pathways of Neurospora crassa. Genes Dev. 6:1052–1057.

ussman, A. S. 1966. Types of dormancy represented by macroconidiaand ascospores of Neurospora. Proc. Symp. Colston. Res. Soc. 18:235–257.

atum, E. L., Barratt, R. W., and Cutter, V. M., Jr. 1949. Chemicalinduction of colonial paramorphs in Neurospora and Syncephalastrum.Science 109: 509–511.

urian, G., and Bianchi, D. E. 1972. Conidiation in Neurospora. Bot. Rev.38: 119–154.

urian, G., Oulevey, N., and Tissot, F. 1967. Preliminary studies on


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Z

Z


CA

pigmentation and ultrastructure of microconidia of Neurospora crassa.Neurospora Newslett. 11: 17.estergaard, M., and Mitchell, H. K. 1947. Neurospora. V. A syntheticmedium favoring sexual reproduction. Am. J. Bot. 34: 573–577.illets, H. J., and Calonge, F. D. 1969. Spore development in the brownrot fungi (Sclerotinia spp.). New Phytol. 68: 123–131.ilson, C. H. 1985. Production of microconidia by several fluffy strains of
Neurospora. Neurospora Newslett. 32: 18.

oronin, M. 1900. Uber Sclerotinia cinerea und Sclerotinia fructigena.Mem. Acad. Sci. St. Petersburg VIII. e ser 10, Phys. Math. 1.

alokar, M. 1957a. Variations in the production of carotenoids inNeurospora. Arch. Biochem. Biophys. 70: 561–567.

alokar, M. 1957b. Isolation of an acidic pigment in Neurospora. Arch.Biochem. Biophys. 79: 568–571.

ickler, H. 1952. Zur Entwicklungsgeschichte des Ascomyzeten Bombar-
dia lunata. Arch. Protistenk. 98: 1–70.

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