EXPERIMENTAL MYCOLOGY 15, 346-350 (19%)

BRIEF NOTE

A New Genotype of Neurospora crassa That Selectively Produces Abundant Microconidia in Submerged Shake Culture

RAMESH MAHESHWARI

Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India; and Department of Biological Sciences, Stanford University, Stanford, California 94305-5020

Accepted for publication June 17, 1991

MAHESHWAFU, R. 1991. A new genotype of Neurospora crassa that selectively produces abun- dant microconidia in submerged shake culture. Experimental Mycology 15, 346-350. A new mi- crocycle microconidiating strain (mcm) of Neurospora crassa was derived by successive back- crosses of a soil isolate to a laboratory wild-type strain. Surface grown cultures show normal wild-type vegetative morphology. However, under continuous agitation in liquid cultures at 22°C macroconidial germlings bypass the usual mycelial phase and produce abundant numbers of uni- nucleate microconidia within 24 h. Temperature and culture media affect the production of micro- conidia. 0 1991 Academic Press, Inc.

INDEX DESCRIPTORS: Microconidia; Neurospora crassa; microcycle microconidiogenesis.

The majority of the vegetative spores produced by Neurospora crassa are round or oval macroconidia measuring 4-7 pm in diameter and contain a variable number of nuclei, from 1 to 20 or more (Barratt and Garnjobst, 1949). Approximately 1% of the spores, which are produced late in culture, are uninucleate microconidia, measuring 2.5-3.5 Frn in diameter (Dodge, 1932; Bar- ratt and Garnjobst, 1949; Grigg, 1960; Lowry et al., 1967). Microconidia were thought to be homologous to spermatia but Dodge (1932) demonstrated that they are capable not only of fertilizing protoperithe- cium but also of germinating and producing vegetative cultures.

Microconidia are desired for many pur- poses-for example, for DNA-mediated transformation, for recovering new reces- sive mutants, for investigating the muta- genic and lethal effects of various chemical and physical agents, for determining nu- clear ratio in heterokaryons, or for estab- lishing homokaryotic cultures.

The few studies on microconidia have been of those produced by the double mu- tants pe fl or fl;dn, in which macroconidia-

tion is suppressed and the cultures produce microconidia almost exclusively (Perkins et al., 1982). However, microconidiation is asynchronous, and most microconidia re- main viable for less than 4 days after their formation (Ballou and Bianchi, 1978). Fur- thermore, these microconidiating strains are less vigorous and show decreased fer- tility.

Rossier et al. (1973) reported that micro- conidiogenesis in wild-type N. crassa is se- lectively induced in standing liquid cultures in the presence of iodoacetate. Ebbole and Sachs (1990) modified this method to obtain microconidia from agar medium supple- mented with iodoacetate. The yield varied from lo3 to lo6 microconidia per slant. However, the filtration procedure used to purify microconidia free from macroconidia and mycelia resulted in severe loss in re- covery. Moreover, the viability of micro- conidia was low, from 1 to 20%.

Rossier et al. (1977) also devised a micro- cycle system of inducing microconidiogen- esis in heat-treated microconidia of fluffy mutant or wild-type N. crassa (obtained by iodoacetate treatment) on phosphate buffer

346 0147-5975/91 $3.00 Copyright B 1991 by Academic Press, Inc. AU rights of reproduction in any form reserved.

MICROCONIDIA IN N. C~USSU

FIGS. 1-3. N. craaa strain mcm (RM 39-8A) grown at 22°C in liquid shake culture. Fro. f . Bright-KeId image of cotton blue-stained 18-b culture showing microcycle ~cro~ni~i~~~~-

esis. Note lateral protuberances and free microconidia. (x300). FIG. 2. Combined dark-field and fluorescence image of microcycle structures showing microconid-

iogenesis. Sample was stained with Hoechst 33258 (Raju, 1982) and mounted in 90% glycerine con- taining ~phenyen~~ne (1 rn~rnl~ to reduce fading of ~uorescen~e. Note entry of a~~~e~~ in protuberance (x600).

FIG. 3. Fluorescence image of isolated microconidia (48 b) stained with Hoechst 33258 (X

348 RAMESH MAHESHWARI

agar. Because the germination of micro- conidia was asynchronous and the total time required for microcycle microconidio- genesis was long (120-144 h), this system is not suitable for experimental studies. A re- liable and rapid method of obtaining micro- conidia should open up new possibilities in genetic studies.

Recently I derived a microcycle strain by backcrosses of a soil isolate of N. crassa. This strain allows selective and rapid pro- duction of microconidia in large numbers without affecting any of the features of the wild type. The microcycle strain differs from the wild type by a single gene, mcm, which has been mapped in linkage group IIL, very close to w-7 (Maheshwari, 1991). This paper describes the methodology of using this strain and the optimal conditions for the production of microconidia and for their viability.

Microcycle microconidiating (mcm) strains were derived, respectively, by five (RM 5-1~ and RM 5-21A) and six (RM 39-6a and RM 39-8A) backcrosses of soil isolate Vickramam A (Maheshwari, 1989) to Oak Ridge wild-type N. crassa. The method of producing microconidia by strains of mcm genotype involves two steps.

i. Preparation of macroconidial inocu- lum. The two mcm strains, RM 5-21A and RM 39-8A, were grown on a glycerol com- plete agar medium (Tatum et al., 1950) at 25°C for 7-10 days with 12 h/day illumina- tion to maximize the production of macro- conidia. The macroconidia were harvested by gentle shaking with sterile saline (0.9%, w/v) solution and the suspension was cen- trifuged to obtain a bright-orange conidial pellet, The conidia were suspended in ster- ile water by vortexing and stored in a capped tube at 4°C. Conidia remained fully viable for at least 1 month.

ii. Submerged liquid culture. Erlenmeyer flasks, one-fifth filled with liquid Vogel’s medium N (Davis and de Serres, 1970) + 1.5% glucose, were inoculated with a sus- pension of macroconidia to give a final con-

centration of 5 x 106-lo7 conidia/ml. The flasks were continuously shaken on a gyra- tory shaker at 240 rpm at 22°C.

MICROCYCLE MICROCONIDIOGENESIS

The polarized growth of the macroconid- ial germ tube became arrested after approx- imately 12 h and it was transformed into a microcycling structure by 14 h. The hypha differentiated lateral and apical protuber- ances and presented a vacuolated appear- ance (Fig. 1). Generally a single nucleus en- tered into each of the protuberances (Fig. 2) following which the protuberance appeared to be abstricted, giving rise to a micro- conidium. The present observations by light microscopy could not establish whether the microconidia are produced as phialospores (Rossier et al., 1977) or as po- rospores budded as protuberances through a hole in the hyphal wall and then ab- stricted. The formation of microconidia im- parted a greenish-brown color to the micro- cycling cultures. At 18 or 22°C mcm pro- duced microconidia exclusively. Based on a count of 614 microconidia stained with Hoechst 33258, 92% were uninucleate and the remainder were binucleate (Fig. 3). The nucleus filled the microconidium with little free cytoplasm.

OPTIMAL CONDITIONS FOR MICROCONIDIOGENESIS

Microconidial production was strongly influenced by the temperature of incubation (Table 1). The optimal temperature in Vo- gel’s medium was close to 22°C. Micro- conidiogenesis was inhibited at higher tem- peratures and was totally suppressed above 30°C. The microconidial yield was some- what improved in Westergaard and Mitch- ell’s synthetic cross medium at 26 or 30°C (Davis and de Serres, 1970) but Vogel’s me- dium N was better at the lower tempera- tures.

Whereas in microconidia, microcycle mi- croconidiogenesis by heat treatment was

MICROCONIDIA IN N. crassa 349

TABLE 1 Effect of Temperature and Medium on Microcycle Microconidiogenesis in mcm Strain RM 39-8A in

Different Mediaaab

Number of microconidia (X lo-“) produced/ml in 24 h’

Temperature Vogel’s Synthetic (“Cl medium N cross medium

18 2.30 0.78 22 21.50 4.30 26 4.15 6.50 30 0.08 1.45

’ Liquid media were supplemented with 1.5% glu- cose.

b Inoculum was 10’ macroconidia/ml. ’ Yield determined by hemacytometer counting of

suspensions after removing microcycling structures by filtration, as described by Newmeyer (1990).

reported to occur in nonnutritive medium (Rossier et al., 1977), by contrast the pro- cess in macroconidial germ tubes of mcm strains occurred in liquid shake cultures only in the presence of a utilizable carbon source. Glucose at 1.0 or 1.5% was optimal for microcycle microconidiogenesis. The number of microconidia liberated increased with the time of incubation. In 72 h, their numbers exceeded 107/ml.

VIABILITY

The viability of microconidia from 24- to 36-h cultures of mcm (RM 39-SA) ranged from 17 to 35% in eight determinations with an average value of 26% (Table 2). Colonies from microconidia developed slowly com- pared to those from macroconidia. Few col- onies were visible at 48 h. Approximately 90% of the colonies were conspicuous in 72 h. The number of colonies was comparable at 25 and 34°C but colonies. at 34°C were slightly more compact. On appropriately supplemented sorbose media, germination of microconidia of auxotrophic strains mcm;his-3;A and mcm;arg-5;A was compa- rable to that of the mcm control. The via- bility of microconidia of mcm was signifi-

TABLE 2 Viability of Microconidia in Diierent Genotypes of

Neurospora crassaa

Method of producing Number of % Average

Genotype microconidia determinations -Vi&iity

PUXl;A Submerged 8 26 microcycle

mcm;his-3;Ab Submerged 1 30 microcycle

mcm;arg-5;AC Submerged 1 39 microcycle

jl;dn;Ad Surface 4 5 mycehum

pe .tUe Surface 2 7 mycelium

--

4 Viability was determined as colony-forming units on Vo- gel’s sorbose agar medium (Davis and de Serres, 1970). Serial dilutions were made in 0.9% NaCI, and 1.0 ml suspension containing IO0 microconidia was plated in each of three to six replicate plates. Colonies were counted on the fifth or the sixth day.

’ Constructed by crossing strain mcm (RM 5-k) to &-.?A (Y 175M614).

’ Constructed by crossing strain mcm (RM 5-k) to al-l;org- 5A (P9125).

d Strain P9260 provided by D. D. Perkins. ’ Strain P2178 provided by D. D. Perkins.

cantly higher than those produce face mycelium of fl;dn;A or pe JE

Previously r the only reliable taining exclusively microconid macroconidia, was to use mo mutant strains,.jIfZ, peg, orfl;dn (see Perkins et al., 1982). Strains of mcm genoty the great advantage that the strain is w type in morphology and all other respects under ordinary conditions. With the deriva- tion of a microcycle microco~diati~g strain in the Oak Ridge wild-type N. craSsa back- ground, the difficulty of obtaining viable uninucleate cells for genetic and rn~lec~~ar studies has been overcome. It is this work will encourage the use conidia in several types of invest

ACKNOWLEDGMENTS

This study was completed during the tenure of a Fulbright fellowship at Stanford University in the lab- oratory of David D. Perkins. I thank members ofthat laboratory for valuable discussions, encouragement, and comments on the manuscript. Travel and fellow- ship were provided by the University Grants Commis- sion, New Delhi, and the Council for International Ex-

350 RAMESH MAHESHWAJU

change of Scholars, Washington, D.C. Research work LOWRY, R. J., DURKEE, T. L., AND SUSSMAN, A. S. at Bangalore was supported by the University Grants 1967. Ultrastructural studies of microconidium for- Commission and at Stanford by Public Health Service mation in Neurospora crassa. J. Bacterial. 94: 1759- Grant AI-01462. 1763.

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