Trans. Br, mycol, Soc. 71 (2) 203-208 (1978)

[ 2°3 ]

Printed in Great Britain

THE MATING SYSTEM OF USTILAGO LONGISSIMA IN VITRO

By GILLIAN BUTLER, HILARIE BOUGHEY* AND HELEN CAUWOOD

Department of Plant Biology, University of Birmingham

In Ustilago longissima, colonies derived from individual basidiospores formed after brandspore germination were yeast-like whereas colonies derived from compatible pairs of strainsgrew as 'migrating' hyphae. The results of tests between strains derived from six infectedplants could be explained by a mating system in which there were three alleles at one locus(a) and five alleles at a second locus (b). The (a) locus controlled cell fusion and the (b) locusregulated the growth of the fusion hypha. Lesions from different parts of the same leaf orshoot contained the same four mating types.

During pioneer work on the mating systems ofthe Ustilaginales Bauch (1922) devised an agarplate method for the determination of mating types.This Bauch test depends on the formation ofhyphae by dikaryons (' Suchfaden ') in contrast tothe sporidia! growth of homokaryotic cultures.Using this method, together with studies ofsporidial fusions, Bauch (1923, 1930, 1931)claimed that in Ustilago longissima (Sow. exSchlecht.) Meyen, a parasite of Glyceria spp., themating system was tetrapolar with multiplealleles at one locus and three alleles at the other.More recently a tetrapolar system has been amplydemonstrated in Usti/ago maydis with two allelesat the cell fusion locus (a) and multiple alleles atthe locus (b) controlling dikaryotic growth (Rowell,19S5; Puhalla, 1968). However no comparablesituation with three alleles at one locus has beendescribed since that of Bauch. Whitehouse (1951)suggested that the appearance of a third allelecould be due to one of the tester stocks beinga heterokaryon carrying the other two alleles.The report of Bauch did not contain sufficientdetail to evaluate this possibility. This paperdescribes a re-investigation of the mating systemin U. longissimain vitro.

MATERIALS AND METHODS

Samples of infected leaves with brand sporelesions were obtained from two populations ofGlyceria maxima (Hartm.) Holmb., at Selly Oak,Birmingham (S) and Honington, Warwickshire(H). Sporidial cultures were maintained on com-plete medium (Day & Jones, 1968), kept at SOand subcultured every few weeks.

* Present address: Department of Genetics, Univer-sity of Birmingham,

Isolation of meiotic productsBrand spores were removed aseptically fromlesions which were mature but had not yet splitopen. They were suspended in sterile distilledwater, streaked onto water agar or completemedium and incubated at 25° for 30-36 h. For theisolation of the progeny of single brand sporesyoung isolated colonies were checked to see thateach was derived from a single brand spore.Each colony was removed and spread in c-osmlsterile distilled water on complete medium(Holliday, 1961). After incubation, lines derivedfrom single sporidia were obtained from theresultant colonies. In some instances colony mor-phology and mating reactions were used to recoverthe different products of one meiosis. For randomisolation of meiotic products a suspension wasmade from the combined products of five to tenbrand spores.

Mating tests on plates

'Puhalla method' (after Puhalla, 1968). Needlepoint inocula were placed 2 rom apart on com-plete medium and incubated at 2So. After 3 dayswhite aerial hyphae could be seen at the junctionbetween compatible colonies and sometimes a fanof hyphae was formed on the agar surface at thejunction.

'Bauch method' (after Bauch, 1932). A loopful ofsuspension of each of two sporidiallines, made upin sterile distilled water, were mixed on CzapekDox agar and incubated at 2So. Mter 3 days a dis-tinctive fringe of 'migrating' hyphae (Bauch's, Suchfaden ') could be seen on the agar surfacearound mixtures of compatible strains.

Sporidial fusion tests (after Bauch, 1930)

Sporidial suspensions were prepared from youngstreak colonies grown on complete medium for

204

A

I A: I B

lOA: I B

100A : I B

Mating in Ustilago longissima

B

I A : 10 B

I A: 100 B

Figs. 1-6

Fig. 1. Plate mating between compatible sporidiallines from inoculum containing different proportionsof the two sporidiallines.Figs. 2 and 3. Phase micrographs of migrating hyphae derived from fusion between two fully compatiblesporidial strains ( x 320).

Figs. 4, 5 and 6. Phase micrographs showing poor development of fusion products in hemi-compatible(common b) matings (x 320).

Gillian Butler, Hilarie Boughey and Helen Cauwood 2°51 day at 25°. The concentration of the suspensionswas approximately standardized by using similarnumbers of colony scrapings in 2 ml of steriledistilled water (c. 5 x 107 sporidia/ml), Suspen-sions were dispersed using a Whirlimix and anequal volume from each strain to be mated weremixed. Drops (0'05 ml) were incubated on sterileglass slides in Petri dish moist chambers for 2-3days at 15

0• Fusions were assessed under a phase

contrast microscope of samples diluted in water.In all experiments mixtures were tested at

least in duplicate, with single strain controls.

RESULTS

Morphology of monokaryotic and dikaryotic colonies

On complete medium monokaryotic colonieswere yeast-like and consisted of sporidia 10-

100 /lm long with no septa or just a few. Daughtersporidia were formed on fine necks either ter-minally or from intercalary segments. Aerialhyphae were formed only where sporidia of twocompatible mating types were in contact. Thesehyphae had the characteristic 'migrating' form(' Suchfaden ') of a growing apical cell full ofprotoplasm with a basal trail of septate more orless empty compartments. During subsequentgrowth the hyphae tended to be overwhelmed bysporidia.

On Czapek Dox medium monokaryotic cultureswere slimy, grew slowly and soon staled. Thesporidia tended to be longer (up to 150/lm) andmore segmented than on complete medium. Insome strains the hyphal growth form occurredaround older colonies. Such growth was readilydistinguishable from the compatible reaction in

Isolate

Table 1. Within isolate plate mating in U. longissima

Originof single No. of single sporidiallinessporidiallines compatible with testers

SI testers

Total no.sporidia

SI (1 lesion) A B C DIsolated bs* colony 6 0 2 1 9Isolatedbs colony 1 5 0 4 10

Total 7 5 2 5 19

SII testersA.-

SH (3 lesions on 1 leaf) E F G H

11 singlebs progenies 60 41 43 34 178

SIll (3 leaves on 1 shoot)

Total

5-10 bs leaf 15-10 bs leaf 25-10 bs leaf 3

SII I testers,..------A-------,

J K L M

351441 31 164

8 10 8 8

101212

34

* bs, Brand spore.

Table 2. Analysis of single brand spore progenies from different lesionson the same leaf (SII)

No. matingtypesNo. sporidia recovered/bs

No. sporidia with compatible ,Lesion No. bs* isolated/bs phenotype 1 2t 2* 3 4

1 4 17,6,16,16 3 1 0 1 2 02 3 20, 19, 18 0 0 0 1 1 13 4 16, 20, 18, 20 5 0 0 3 0 1

Total 1 0 5 3 2

* bs, Brand spore; t Incompatible; * Compatible.

Table 3. Between isolate plate mating in U. longissima

Isolates

No0\

SI SII Sill SIV HI HII*, ,

\ ,-----A----.-., IA , , , , , ,

\ ,-----"-----.Tester alleles SA SB SC SD SE SF SG SH SJ SK SL SM SN SP SQ SR HA HB HC HD HI HKSI A albl - + - - - + - - - + - - - + + - - + + - + +

B a2b2 + - - - + + + + + + + + - + - + - + - + - +Calb2 - - - + - + - + - + - + + + + + - + - - + +Da2bl - - + - - + + - - + + - - + - - + + + + - +

HI B a3b3 + + + + + - + - + - + - + - - - + - - - + - ~Oalb3 - + - + - + - + - + - + + - - + - - + - + + ...HII I a2b4 + - + - + - - + + + + + - + - + + + + + - - ~'

Ka3b4 + + + + + - - - + - + - + - + - + - - + - - ;s'SIll L aibs - + - + - + - + - - - + + + + + - + + - + + ~

Ka3 bs + + + + + - + - + - - - + - + - + - - + + - ..."....Isolate alleles albl a2b2 alb2 a2bt atbl a3b4 alb4 a3b1 al bt a3bs aibs a3bt a2bl a3 b3 a2b3 a3bt alb2 a3b3 a3b2 alb3 a2b4 a3b4 ~

0tI* Incomplete isolation. +, Compatible, forming migrating hyphae, -, Not compatible. 0-0

::10.9 .

Table 4- Occurrence of cell fusions (F) and migrating hyphae (S) in matings of U. longissima ..."..."

S·Isolates Il:I

I ---- -- ------ ,SI SII SlY Isolate SlY

-.... A ,---------" , , A

SA SB SC SO SE SF SG SH SN SP SQ SR SN SP SQ SRTester albl a2b2 atb2 a2bl albl a3b4 atb4 a3bl a2bt a3b3 a2b3 a3bl Tester a2bt a3b3 a2b3 a3b1

SI SA albl - FS - F - FS - F F FS FS F SIl SE atbt F FS FS FSB a2b2 - F - FS FS FS FS - FS - FS SF a3b4 FS - FSSC atb2 - FS - FS - FS FS FS FS FS SG a1b4 FS FS FS FSSO a2bt - F FS FS F - FS - F SH a3bl F - FS

Gillian Butler, Hilarie Boughey and Helen Cauwood 2°7

Sporidial fusions

In crosses which were compatible when mated onplates successful fusions between sporidia could berecognized by the formation of a 'migrating'

Table 5. Frequencies of individual mating typealleles in 6 infected plants of G. maxima

al az a3 b1 b, b. b4 b6

435 42221

DISCUSSION

This work supports Bauch's scheme for the matingsystem in U. longissima.

Mating on plates involves compatibility at allloci concerned in the mating process up to andincluding growth of the dikaryon (Puhalla, 1968).In U. longissima this reaction was clear cut andthere was good agreement between results obtainedby different observers. Although stability of thedikaryon varied with cultural conditions, on somemedia it was much more stable than that ofU. maydis (Day & Anagnostakis, 1971) and couldbe maintained indefinitely. Bauch (1931) foundthat this phase was infective. The basis of theformation of ' migrating' hyphae on older coloniesof some single sporidial lines was not established.

hypha (' Suchfaden '), The protoplasm from bothmated cells entered the 'migrating' hypha whichdeveloped either from the fusion bridge or fromthe distal end of one of the mated sporidia(Figs. 2, 3). In certain other crosses there werefusions within the same time but the fusion hyphaehad very limited growth, were often gnarled anddistorted and did not reach sufficient length toform a trail of empty cells (' Wirrfiiden' of Bauch,1930). There was no further development in5 days and multiple fusions between more thantwo sporidia were frequent (Figs. 4-6). In bothtypes of interaction fusions were sufficientlyfrequent for easy assessment and there was verygood agreement between replicates.

Hemicompatible reactions (fusions without'migrating' hyphae) occurred within each of theisolates SI, SII and SIV when the two strainsbeing tested differed at the three allele locus (a)but were identical at the five allele locus (b) (e.g.SI, Table 4). This finding was confirmed in testsbetween isolates containing all possible combina-tions of alleles at the (a) locus (Table 4). More-over, matings of 10 sporidiallines containing thebs. allele revealed three groups which consistentlyformed fusions in inter-group but not in intra-group crosses.

The two loci in U. longissima were designated aand b in conformity with the nomenclature usedin U. maydis (Day, 1974), and the number ofoccurrences of the individual alleles in six infectedplants is shown in Table 5.Between isolate plate mating

Crosses between the tester strains of differentisolates yielded greater numbers of compatiblereactions than would be expected on the basis ofa tetrapolar system with two alleles at each locus.The results (Table 3) can be explained mostsimply by assuming that there are more than twoalleles at both loci. The results are consistent fora mating system having three alleles at one locusand five alleles at the other. No reactions occurredwhich would not fit this scheme. Further crossesbetween strains having the same allele did notyield any compatible reactions even when thepartners had different alleles at the other locus.

which a fringe of 'migrating' hyphae was detect-able after 2 days. From an inoculum of 104-106

sporidia per loopful the hyphal fringe was regu-larly formed when a 10 : 1 mixture of two com-patible strains was used. With a 100: 1 mixturethe fringe was at first patchy but soon extendedaround the colony margin (Fig. 1). The fringewas stable, growing at c. 1500 11m per day andretaining its hyphal form on subculture from a massinoculum or from a single hyphal tip. Individualhyphal tips sometimes stopped extending andfragmented to form sporidia. Sporidial suspen-sions taken from parts of compatible coloniesdistant from the position of the original inoculumwere streaked and most yielded monokaryoticcolonies. Mating tests on samples of 30 singlesporidial lines from three different compatiblecombinations only yielded the two parental mono-karyons.

Within isolate plate mating

Isolates SI, SII and SIll were analysed separatelyand all were tetrapolar (Table 1). In each isolate,sporidia of four different mating types wereobtained and every sporidial line was compatiblewith only one of the four testers . The actualnumbers of sporidia isolated of each type depen-ded on the relative rates of multiplication of themeiotic products. Different lesions on the sameleaf and also lesions from different leaves on thesame shoot contained the same four mating types.

Where single brand spore progenies wereinvestigated (Table 2) four mating types wererecovered from only a few of the brand spores andthere was a relatively high frequency of recoveryof two compatible sporidial types. A few isolateswere recovered with the compatible phenotypebut the basis of this appearance was not investi-gated.

208 Mating in Ustilago longissima

Analysis of brand spores from different partsof the same shoot suggested that whole shootswere infected by the same dikaryon, In sectionsof infected hosts the mycelium was systemic,being abundant in the nodal tissue and presentin the shoot apical meristem (Butler, unpubl.).The behaviour of single sporidial lines in bothwithin and between isolate crosses combine tosupport the claim of Bauch that there were morethan two alleles at both of the mating type loci.The results would be difficult to explain in termsof concealed heterokaryotic testers, as suggestedby Whitehouse (1951) to explain the results ofBauch. However, the tests were not carried tothe next generation.

Cell fusion seems to occur more readily inU. longissima than in U. maydis (Rowell, 1955).Although there was no macroscopic evidence ofhemi-compatible interactions, the distinctionsbetween fully compatible and hemi-compatiblefusion products and absence of fusions were clearcut. The present observations are fully consistentwith the (a) locus controlling cell fusion. The threealleles at this locus behaved in a similar way.

The three a alleles and five b alleles wereobtained from only six infected plants so that it isposs ible that more alleles of both the a and b locicould be found in a larger sample. However, froma large number of isolates, Bauch (1931) detectedonly three alleles at the cell fusion locus (his Blocus) and eight alleles at the dikaryotic growthlocus (his A locus). The apparent occurrence ofthree alleles at the cell fusion locus (a) in U.longis-sima is a unique and intriguing situation. Sucha three allele system would permit a greater fre-quency of outbreeding than a two allele system.

The authors thank Mrs Chris Henry forinvaluable technical assistance and Miss J. COXfor the photograph reproduced in Fig . 1. The work

was in part carried out during final year under-graduate projects. We are indebted to Mr T.Burrow, Miss H . Loveitt, Miss M . Huss, Miss C.Smart, Mr P. Stott and Miss J. Ufton, whosework paved the way for these findings.

REFERENCES

BAUCH, R. (1922). Kopulationsbedingungen undsekundare Geschlechtsmerkmale bei Ustilago oio-lacea. Biologisches Zentralblatt 42, 9-38.

BAUCH, R. (1923). Uber Ustilago longissima und ihreVarietat macrospora. Zeitschrift fur Botanik 15,241-279.

BAUCH, R. (1930) . Uber Multipolare Sexual itat beiUstilago longissima. Archiv fur Protistenkunde 70,417-466.

BAUCH, R. (1931). Geographische Verteilung undfunktionelle Differenzierung der Faktoren bei dermultipolaren Sexualitat von Ustilago longissima.Archiv fur Prot istenkunde 75,101-132.

BAUCH, R. (1932). Die Sexualitat von Ustilago Scar-zonerae und Ustilago Zeae. PhytopathologischeZeitschrifte 5,315-321.

DAY, A. W. & JONES, J. K. (1968). The productionand characteristics of diploids in Ustilago violacea.Genetical Research, Cambridge 11, 63-81.

DAY, P. R. (1974). GeneticsofHost-Parasite Interactions.238 pp. San Francisco: Freeman.

DAY, P. R. & ANAGNOSTAKIS, S. L. (1971). Com smutdikaryonin culture, Nature, London 231,19-20.

HOLLIDAY, R. (1961). The genetics of Ustilago maydis.Genetical Research, Cambridge 2, 204-23°.

PuHALLA, J. E. (1968). Compatibility reactions onsolid medium and interstrain inhibition in Ustilagomaydis. Genetics 60, 461-474.

ROWELL, J. B. (1955). Functional role of compatibilityfactors and an in vitro test for sexual compatibilitywith haploid lines of Ustilago zeae. Phytopathology45,370-374·

WHITEHOUSE, H. L. K. (1951). A survey of hetero-thallism in the Ustilaginales, Transactions of theBritish Mycological Society 34, 340-355.

(Accepted for publication 15 January 1978)