Characterization of iprodione tolerance in Ophiostoma ulmi

Notes and brief articles of the variation. This relationship might have been improved if the field had been ungrazed. Grazing interfered with disease development by rem oving much of the disea sed material and thus preventing the development of a severe di sease epidemic, and after which disease levels were reduced from a period of rapid production of replacement leaves. Similarly, the relationship between the number of conidia captured per week and the total weekly rainfall or the number of wet day s per week also only accounted for about one third of the variation occurring. The significant negative correlation between the number of conidia captured per week and the mean weekl y maximum or minimum temperatures may primarily be a reflection of the lower temperatures recorded during the growing season , when conidia relea se was high, rather than an indication of a true and significant relationship between the two. The mean weekly maximum temperatures from May through to mid-October (13' 5-21 '5 "C) were generally suitable for infection, as it ha s been demonstrated under artificial conditions that mean maximum temperatures of 15-21 ° are suitable for disease development with optimal development at 18° (Barbett i, 1985). The technical assistance of Mr N . R. Peterson is greatly appreciated. I wish to thank the Rural Credits Development Fund of the Reserve Bank for financial support.

REFERENCES

BARBETTl, M .]. (1983). Cercospora zebrina on pasture legumes in We stern Australia. 4th Int ernational Plant Pathology Congress, M elbourne, August 1983 . A bstracts of Papers, p . zz z (abstract no. 886). BARBETTl, M . ] . (1985). Infe ction stu dies with Cercospora

zebrina on pasture legumes in Western Austral ia. A ustralian J ourna l of E xperimental Agri cultu re 25, 850-855. BARBETTl , M . j., SIVASITHAMPARAM, K . & WONG, D . H . (1986). Root rot of su bterranean clover . R eview of Plant Path ology 65, z87-295 · BERGER, R. D. & HANSON , E. W. (1963). Pathogenicity, host-parasite relationship s, and morphology of some forag e legume Cercosporae, and factors related to disea se development. Phytopathology 53, 500-508. BOKOR, A. (1983). Cercospora z ebrina found on T rif olium subterraneum L. in Western Australia. Australasian Plant Path ology 12, 24. COCKS, P . S., MATHISON, M .]. & CRAWFORD, E.]. (1978). Fr om wild plants to pasture cultivars: annual medics and subterranean clover in southern Australia. In A dvan ces in L egum e S cience (ed. R. ] . Summerfield & A. H . Bunting), pp . 569-596. Ke w, England : Royal Botanic Gardens. COLLINS, W.]. & GLADSTONES,]. S. (1984). Breeding to improve subterranean clover in Australia. In Forage Legum es f or Energy-Effi cient Animal Pr oduction (ed. R. F . Barnes, P. R . Ball, R. W. Brougham, G. C. Marten & D .]. Minson ). Pr oceedings of a Trilateral Workshop held in Palm erston North, New Zealand, April 30-May 4, 1984. Springfield, U.S .A. : United States Department of Agriculture, Agricultural Research Ser vice. GLADSTONES, ]. S. (1975). Legumes an d Au stralian agriculture. Farrer Memorial Oration, 1975. J ournal of the Australian Institu te of Agricultu ral S cience 41 , 227- 240. GLADSTONES, J. S. & COI.LINS, W .]. (1983). Subterrane an clover as a naturalized plant in Australia. Journ al of the Australian Institute of Agricultural Scien ce 49, 191-202. ]OHNSTONE, G . R. & BARBETTl , M. J. (1986). Impact of fungal and viral diseases on pastures. Sydn ey, Australian Wool Co rporation. (In the Press.) POWELL, S . C. (1970). Subterranean clover , our most imp ort ant pasture legume. J ournal of Agricult ure, V ictoria 68, 274-277.

CHARACTERIZATION OF IPRODIONE TOLERANCE IN OPHIOSTOMA ULMI BY A. G. MITCHELL

Forest Research Station, Alice H olt Lodge, Farnham, Surrey, GUlO 4LH Iprodione tolerance in Ophiostoma ulmi appears to be conferred by allelic or closely linked single-locus mutations. The characteri stics of three stable iprodion e-tolerant phenotypes are described . Stable singl e-l ocus nuclear mutations conferring tolerance to the fungicide carbendazim or MBC (Brasier & Gibbs, 1975) have been widely used as markers for in vitro and in vivo studies with the Dutch elm disease pathogen, Ophiostoma tCe ratocystis) ulmi (Buis.) Nannfeldt (Brasier, 1977, T rans. Br. my col. S oc. 88 ( 2), (1987)

1983 a, 1984 ; Webber , 1983; Webber & Brasier, 1984). A second marker was required for use in conjunction with carbendazim tolerance to facilitate stud ies on myc elial interactions . Since tolerance to dicarboximide fungicides has been shown to result from nuclear gene mutations

Pr int ed ill G rear Br it ain

Notes and brief articles Table 1. Effect of increasing iprodione concentration on growth rate of O. ulmi *Radial growth rate (mm day-' ) at increasingiprodione concentration Iprodione concentration(/Ig rnl" )

ED,. (pg I-I Isolate subgroup 0 2 5 50 500 iprodione) AL-DI8 NAN 3'20 2'23 1'20 0,80 0'78 0'58 1'5 HAY-51 NAN 2'85 1'93 0'98 0'83 0,84 0'50 1'5 EAN 2'96 2'11 0'92 0'78 0,66 0,60 1'5 CKT -11 H241 EAN 3'04 2'20 0'92 0'90 0'75 0'78 1'5 SSatO Non-aggressive 2'43 1'48 0'73 0'64 0'33 0'35 1'3 SSa21 Non-aggressive 2'75 1'73 0'91 0'73 0'58 0'40 1'3 * Mostly mean values of four replicates, but someare meansof only two or three replicates, since any colonies with faster-growing tolerant sectors were ignored. O. ulmi

(Beever & Byrde, 1982; Grindle, 1984), the possibility that tolerance to fungicides of this group might provide suitable markers for work with 0, ulmi was investigated. Preliminary work showed that O. ulmi was sensitive to the dicarboximide iprodione, added after autoclaving to 2 % Oxoid Malt Extract Agar (MEA) as Rovral (Rhone Poulenc), a wettable powder formulation with 50 % active ingredient. Since dicarboximide fungicides are fungistatic in their action sensitivity was expressed as the ED,. for radial growth, calculated from measurements of growth on 2 % MEA containing a range of iprodione concentrations, after incubation for 2 and 7 d (T able 1). These measurements were made for two isolates of the non-aggressive subgroup and for two isolates each of the North American (N AN) and Eurasian (EAN ) races of the aggressive subgroup of O. ulmi (Brasier, 1982). All six isolates had a similar ED,. of about t : 5 Jig ml" iprodione ; a concentration of about 5 pg ml" reduced growth rate by 70-75 %, but there was little further reduction between 5 and 500 JIg mr '. In contrast, the ED,. for conidial germination at 20 °C of the two NAN isolates was about 19 JIg ml '": Colony morphology also altered with increasing iprodione concentration (Fig. I), Both NAN and EAN isolates lost their usual fibrous-petaloid appearance, developing a powdery grey aerial mycelium with dark pigmentation visible from beneath, Non-aggressive isolates lost their typical 'waxy' appearance and became similarly powdery and pigmented. In the growth-rate experiments, slow-growing sensitive colonies on 2 % MEA + iprodione often produced faster-growing sectors. Further studies showed that these were tolerant variants, which could be readily selected by inoculating mycelial plugs on 10 2 % MEA + 5 JIg ml ? iprodione, a concentration which slowed sensitive growth Trans . Br . mycol. Soc . 88 (2), (1987)

sufficiently to allow any tolerant sectors to grow out. After incubation for 10 d originally sensitive colonies usually developed at least one tolerant sector. Using the above selection procedure three morphologically distinct classes of iprodionetolerant variant were recognized. Variants of the three classes were obtained for the NAN aggressive isolate AL-DI8, and characterized in more detail by comparison with their sensitive counterpart for differences in colony morphology on 2 % MEA in the absence and presence of iprodione ; radial growth rate on 2 % MEA; and ED,. for mycelial growth and conidial germination (T able 2). Since several workers have shown iprodione-tolerant isolates of other fungi 10 be abnormally osmotically sensitive (Beever & Byrde, 1982; Beever, 1983 ; Grindle, 1984; Grindle & Temple, 1985), sensitivity to a high sodium chloride concentration was also tested. Growth rates of representatives of each class were measured on a medium containing 40 g I -I sodium chloride, a standard test for osmotic sensitivity in mutants of Neurospora crassa Sheare & Dodge (Perkins, 1959), Each of the three classes of AL-D18-tolerant variant had a near wild-type morphology when grown on 2 'X, MEA without fungicide, a characteristically different morphology on MEA + 5/1g ml" iprodione (Fig, 1), and characteristic differences in growth rate and osmotic sensitivity (T able 2), Class 2 variants had a similar growth rate to the sensitive AL-D18 on 2 °/., MEA, with a high ED,. and moderate osmotic sensitivity. Class 1 and 3 variants grew slightly faster on 2 % MEA than the sensitive AL-D18, and had a high osmotic sensitivity, although class 1 variants had a higher ED"" than class 3 variants. Representatives of all three classes gave normal vegetative compatibility reactions, in terms of the production of mycelial barrages, synnemata and perithecia

Pr int ed in Great Britain

Notes and brief articles 5

50

F ig. 1. Growth of an ipro dio ne -s ens itive N AN-aggress ive isolate AL-DI 8 an d its de rived tolerant mutants on 2 % MEA an d 2 % MEA + iprod ione. 0, 2 % MEA. 5, 50 and 500 ; 2 % MEA + 5, 50 and 500 Itg I- I iprodi one respect ively. S, sensi tive isolate. 1 , 2 an d 3; class 1 , 2 an d 3 mutants respe ct ively.

(Brasier, 1984). Var iant s of all classes reta ined their tolerance over fifteen ma ss subculturings in the ab sen ce of th e fungicide, and all were sen sit ive to carbendazim . In pathogenicit y tests on four-year-old clon al Ulmus procera Salisb., scored as percentage defoliation, a class 1 tolerant vari ant of AL-DI8 was less pathogeni c th an sensitive AL -D1 8 (T able 2). T olerance was retain ed on re-isolation from th e inoc ulated tree s. Wh en inoculated into the bark of fre shl y cut logs of healthy U. procera class 1 tolerant Tr ans. Br . mycol. S oc. 88 (2), (1987)

variants of four isolates pr oduced smaller lesions than th eir sensi tive counterparts. These results agr ee br oadl y with th ose of other worker s. M ost dicarboximide-tol eran t isolates of fung i isolate d from tr eated crops or fungi cideamended medi a show reduced mycelial growth rate , sporu lation and pathogenicity, with some correlatio n bet ween high levels of tolerance and gr eat er reduction in vigour (Beever & Byrde, 1982 ; L erou x & Fritz, 1984). It has been suggested th at inc rease d osmo tic sensi tivity may have a sign ificant

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286

No tes and brief articles

T able 2. Characte ristics of ip rodione-sensitiv e and class 1,

2

and 3 tolerant mutants of NAN isolate A L -D18

Radial growth rate on 2 % MEA (mm day- I)

ED,o ( ji g ml: ' iprodione)

Radial growth Conidial Without Pathogenicity rate on 2 0 0 MEA* gerrnina tiorr] fun gic ide " + 40 g I - I NaCl* ('~~ defoliation)f Sensitive 1"5 19 3"55±0"12" 1"72± 0"05" 88± 3" Class 1 tolerant 500 4"05± 0"12 h > 5°0 0"30 ±0"12 11 78±9 b Class 2 tolerant 5°0 1'09 ±0"04l" 3"69 ± 0'1O" > 5°0 Class 3 tolerant 15 > 5°0 4' 26 ±0"1311 0'21± 0'05h * Means for six mutants of each class. t Means for one mutant of each class. t Means of three replicates, using only a single class 1 mutant. Means in each column followed by a different letter are significantly different. P < 0"001 for differences between means of growth-rate data. P < 0"01 for differences between means of pathogenicity data.

T able 3. Seg regation of tolera nt pheno types in crosses involving ipr odione-tolerant isolates Number of progeny A mating type x B mating type Tota l Class 1 Class 2 Class 3 Sensitive Wild-typ e x ipr-i MM2/ 1 J x AL-D I8 ipr-l :.' (NAN) 213 110 0 1°3 ° MM2/1 ;,> x AL-DI8 ipr-i 3 (NAN) 188 81 0 107 ° MM 2/1 x AL-D I8 ipr-i (NAN) 110 263 153 ° ° H-A2 x H-CI4 ipr -i (NAN) 60 0 0 105 45 211 160 P32 ipr-i ,; x H8t o <;> (non371 ° ° aggressive) ipr x ipr crosses 101 HAY-51 ipr-i x AL-DI8 ipr-z 59 42 ° ° HAY-51 ipr-l x AL-D I8 ipr -q 62 1°3 41 ° ° 102 1 HAY-51 ipr-2 xA L -D I8 itr-s 0 46 55 a ,\'2 values marked * and ** differ significantly from the expected I : 1 ratio at P < 0'05 and

effect on th e pathogenic capabilities and competitive fitn ess of di carboxirnide-tolerant isolates under field condition s (Beever, 1983). The gene tic contro l of iprod ione to lerance was inv estigated in cross es between sensitiv e and class 1 ipr odione -toleran t isolate s. Sing le ascospore isolates were obtaine d using the me thod of Bra sier & G ibbs (1976), and tested for tol eran ce by assessing gr owth on 2 % MEA + 5 jJ, g mr ' iprodion e aft er incubation for 2 d . The data show th at class 1 tolerance is probabl y conferred by mutation at a single locus, ipr-i , although the ratio of sen sitiv e to tol erant pr ogen y in some crosses di verged sign ificant ly from th e expected 1 : 1 ratio (T able 3). There was no evide nc e for matern al inheritance of iprod ione tolerance in reciprocal crosses involving known male and female par ent s. When selected progen y of th e cross between MM2 /1 and AL-DI8 ipr-i were tested for mating Trans. Br . mycol. Soc. 88 (2), (1987)

Ratio between classes

value"

1:0'9 1:0·8 1:1"4 1: 1"3 I : 1'3

0"23 3'60 7"03** 2"14 7'01**

1:1"4 1:1"5 1:0,8

2·86 4'28* 0,8

,\' 2

< 0'01 respectively.

type , the mating typ e (m l) and putative ip r-i locus were found to be linked (T able 4). It is therefore likely that ip r- t is also link ed to the locu s conferring carbendazim tolerance, sinc e thi s too is linked to tnt (Webber, M itchell & Smith, 1986). T o exami ne th e relationship of gen etic cont rol betw een th e tolerant classes 1-3 , cross es were set up bet ween appropriate mutants of th e NAN aggressive isolates AL-D I 8 and H AY- 51, suc h th at all possible combina tions of the th ree classes were tested . The phenot yp es of 100 progeny fro m each cross were scored after incu batio n for 19 d on 2 °0 MEA + 5 fig m l"? iprod ion e (T able 3). In each case th e progen y showed a ne ar 1 : 1 rat io of th e par ental classes. In add ition, one sens itive recombinant pr ogen y was obtained in th e cross bet ween class 2 and class 3 tolerant isolates . This pr ovides further evidence for control of tol erance by nuclear mutati on, and ind icate s that th e three classes

Primed in Great Britain

Notes and brief articles Table 4 . Evidence for linkage between iprodione tolerance and the mating type (mt) locus Number of pr ogen y

Cross, A mating type x B mating type

MM2/1 x AL-D1 8 it»

x' values

Wild-type Tolerant For ratio of For rati o of Total Ame Bme Amt Bmt Amt :Bmt Wild-type :ipr-1

1

213

105

5

3

100

0'04"

For rati o of Arnt wild-typ e :Bmt wildtyp e:Amt ipr-i : Bmt ipr-i

0'23 h

182'47 "

X' value indicates no significant deviation from a 1 : 1 ratio in segregation of mating type. h X2 value indic ates no significant deviation from a 1: 1 ratio in segregation of iprodione tolerance. r- X' value indicates a significant de viation (P < 0'001) from the 1: 1: 1: 1 ratio expected for independent segregation of lilt and ipr- 1, a

probably represent mutations at either two or three separate but closely linked loci (ipr- i , ipr -z and ipr- 3, respectively). There have been few studies of the genetic control of dicarboximide tolerance, although Grindle (1984) has shown that mutants of Neurosp ora crassa tolerant to the dicarboximide vinclozolin were of nine di stinct, probably allelic phenotypes. Grindle listed several other mutations, including four osmotic mutants, which also conferred dicarbox imide tolerance. The inheritance of tolerance in crosses between the nine vinclozolin-tolerant phenotypes and OS- 1 suggested that the locu s conferring vinclozolin tolerance was the same as or tightly linked to OS-I. Investigations of the control of tolerance of aromatic hydrocarbon fungicides, which typically shows cross-tolerance with dicarbox imide fungicides and is probably conferred by mutations of th e sam e loci, suggest that tolerance can ari se through allelic mutations at several loci, e.g. Threlfall (1968) and Ge orgopoulos (1977). These re sults indicate tha t iprodione-tolerant mutants can pr ovide at least one su itable nuclear gene marker phenotypically sim ilar to wild-type O. ulmi isolates and sensitive to carbendazim. The author is now using iprodione-tolerant mutants in conjunction with carbendazim tolerance in experiments to investigate mycelial interactions between O. ulmi isolates, such as the 'penetration effect' (Brasier, 1984), and to study the interaction between isolat es of the aggressive and nonaggressive subgroups. It is hoped that the latter may provide further insight into the processes leading to the replacement of the non-aggressive by the aggre ssive during the current epidemics of Dutch elm disease (Brasier, 1983b ; Houston, 1985). The ab ove experiments have so far involved onl y class 1 mutants, but since the three classes are readily distinguished by th eir morphology on 2 % MEA + iprodione they are probably all suitable as markers for in vitro studies. However, the fungistatic nature of iprodione and the speed at Trans . Br . my col. S oc. 88 (2), (1987)

which tolerant sectors ari se from sensitive growth in its presence could be a serious disadvantage to the use of these markers in some circumstances. The low fitness of class 1 mutants in elm bark may also be a problem for in vivo studies. I would like to thank Drs Clive Brasier and Joan Webber for help with the manuscript, and I acknowledge the rece ipt of an NERC stu dentship . REFERENCES

BEEVER, R. E. (1983). Osmotic sens itivit y of fungal variants resistant to dicarboximide fungicides. Tran sactions of the British My cological So ciety 80, 327-331. BEEVER, R. E. & BYRDE, R. J. W. (1982). Resistance to dicarboximide fungicide s. In Fungal Resistance in Crop Protection (cd. J. Dekker & S. G . Ge orgopoulos), pp. 101-11 7. Wageningen : Pudoc. BRASIER, C. M . (1977). Inheritance of pathogenicity and cultural characters in Ceratocy stis ulmi ; hybridization of pr otoperithecial and non-aggressive strains. Tran sactions of the British M ycological S ociety 68, 45-52. BRASIER, C. M . (1982). Occurrence of three sub-groups within Ceratocytis ulmi . In Proceedings of the 1981 Dut ch Elm Disease Symposium and Workshop (ed. E. S. Kondo, Y. Hiratsuka & W. B. G . Denyer), pp . 298--321. Manitoba, Canada: Department of N atural Resources. BRASIER, C. M. ( 1983 a). A cytoplasmically transmitted disease of Ceratocystis ulmi. Nature, London 305, 220-223. BRASIER, C . M. ( 1983 b). The future of Dutch elm disease in Europe. Forestry Commission Bulletin 60, 96-104. BRASIER, C. M . (1984). Intermycelial recognition systems in Ceratocystis ulmi : their ph ysiological properties and ecologica l importance. In The Ecology and Phys iology of the Fungal M ycelium (ed. D. H . Jennings & A. D . M . Rayner), pp . 451-497. Cambridge University Pre ss. BRASIER, C. M . & GIBBS, J. N . (1975). MB C to lerance in aggr essive and non-aggressive isolates of Ceratocystis ulmi. Annals of Applied B iology 80, 231-235 . BRASIER, C. M. & GIBBS, J. N . (1976). Inheritance of pathogenicity and cultural characters in Ceratocy stis ulmi : hybridization of aggressive and non -aggressive stra ins. Annals of Applied Biology 83, 31-37.

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Notes and brief articles

GEORGOPOULOS, S. G . (1977). De velopment of fung al resistance to fungicides. In Antifungol Compounds z (ed. M . R. Siegel & H. D. Sisler), pp . 439-495 . New York : Dekker. GRINDLE, M . (19 84). Isolation and characteriz ation of vinclozolin-resistant mutants of Ne urospora crassa. Tran sactions of the Br itish M y cological So ciety 82, 635-643· GRINDLE, M . & TEMPLE, W. (1985). Sporulation and osmotic sensitivity of dicarboximide-resistant mutants of Ne urospora crassa. Transactions of the Br itish M y cological S ociety 84, 369-3 72. HOUSTON, D . R. (1985). Spread and increase of Ceratocystis ulmi with cultural characteristics of the aggre ssive strain in northeastern North Ameri ca. Plant Disease 69, 677-680. LEROUX, P . & FRITZ, R. ( 1984). Antifungal activity of dicarboximides and aromatic hydrocarbons and resistance to these fungicides . In M odes of A ction of Antifungal Compounds (ed. A. P. J. Trinci & J. F. Ryley), pp . 207-237. Cambridge University Press.

Trans . Br . my col. S oc. 88 (2), (1987)

PERKINS, D . D . (1959). Ne w markers and mult iple point linkage dat a in Ne urospora. Genetics 44, 1185-1208 . THRELFALL, R. J. (19 68). The genetics and biochemi str y of mutants of A spergillus nidulans resistant to chlorinated nitroben zenes. J ournal of General Microbiology 52,35-44· WEBBER, J. F . ( 1983). Implications of fungicide tolerance. Forestry Commission R eport on Forest R esearch 1983, p. 33. HMSO, London. WEBBER, J. F. & BRASIER, C. M. (19 84) . The transmission of Dutch elm disease : a study ofthe processes involved. In ln uertebrate-Microbial Int eractions (ed. J. M. Anderson , A. D. M. Rayner & D. W . H . Walton ), pp. 271-306. Cambridge Universit y Press . WEBBER, J. F., MITCHELL, A. G. & SMITH, F. ( 1986). Linkage of the genes determining mating type and fungicide toler ance in Ophiostoma ulmi. Plant Path ology (In the Pr ess).

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