Resistance of wheat cultivars and breeding lines to septoria tritici ...

Plant Pathology (2001) 50, 325±338

Resistance of wheat cultivars and breeding lines to septoria tritici blotch caused by isolates of Mycosphaerella graminicola in field trials J. K. M. Browna*², G. H. J. Kemab, H.-R. Forrerc, E. C. P. Verstappenb, L. S. Arraianoa, P. A. Bradinga, E. M. Fostera, P. M. Friedc and E. Jennyc a

Cereals Research Department, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK; Plant Research International, PO Box 16, 6700 AA Wageningen, the Netherlands; and cSwiss Federal Research Station for Agroecology and Agriculture, Reckenholzstrasse 191, CH-8046 ZuÈrich, Switzerland

b

Isolate-specific resistance of 71 cultivars and breeding lines of wheat (Triticum aestivum) to septoria tritici blotch was evaluated in six field trials in the Netherlands, Switzerland and the UK between 1995 and 1997. Each plot was inoculated with one of six single-pycnidium isolates of the pathogen Mycosphaerella graminicola. There were strong interactions between wheat lines and M. graminicola and the line-by-isolate interactions were stable over the six trials. Lines with specific resistance or specific susceptibility to each of the isolates were identified. Specific resistance to isolate IPO323 was especially common, being carried by 22 lines from 10 countries. The results confirm that lineby-isolate interactions in septoria tritici blotch of wheat are effective in adult plants in field conditions, and are not simply confined to seedlings. Wheat lines with good, quantitative resistance to all or most isolates were identified, including lines from Brazil, the USA and seven European countries. These may be useful as sources of resistance in wheat breeding. Keywords: cultivar-by-isolate interactions, median tetrad analysis, Mycosphaerella graminicola, septoria tritici blotch, wheat

Introduction Septoria tritici blotch is currently the major foliar disease of wheat in most European countries and is important in many other regions worldwide (Eyal et al., 1987; Polley & Thomas, 1991; van Ginkel & Rajaram, 1993). The disease is a major target for fungicide applications on wheat (Cook, 1999; Jùrgensen et al., 1999), but owing to the cost of fungicides, the use of resistant cultivars is increasingly seen as an attractive method of control. Since the early 1980s, therefore, European wheat breeders have increased their efforts to select cultivars resistant to septoria tritici blotch (Johnson, 1992). Septoria tritici blotch is caused by the ascomycete fungus Mycosphaerella graminicola, which has a heterothallic, bipolar mating system (Kema et al., 1996c) and reproduces both sexually and asexually throughout the growing season of wheat (Kema et al., *To whom correspondence should be addressed. ²E-mail: [email protected] Accepted 3 November 2000. Q 2001 BSPP

1996c; Hunter et al., 1999). Populations of M. graminicola have high levels of genetic variability, as shown by molecular markers (Owen et al., 1998; Schneider et al., 1998; McDonald et al., 1999). Extensive variation has also been detected in virulence of the fungus towards different cultivars in tests of seedlings (Eyal et al., 1973, Eyal et al., 1985; Perello et al., 1991; Ahmed et al., 1995; Ballantyne & Thomson, 1995; Kema et al., 1996a, Kema et al., 1996b) and of adult plants (Kema & van Silfhout, 1997). Some wheat cultivars have strong interactions with particular M. graminicola isolates, being specifically resistant or susceptible to them. However, very few trials of the progeny of crosses between wheat lines, intended to analyse the genetics of resistance, have been conducted with single isolates of M. graminicola. Instead, trials are generally exposed to natural infection or inoculated with mixtures of isolates (Wilson, 1979; van Beuningen & Kohli, 1990; Jlibene et al., 1994). Similar methods are used to infect wheat breeders' trials for selection of resistant cultivars. Partly because so few experiments have been done with singlegenotype isolates, research on the genetics of the resistance of wheat to septoria tritici blotch is in its 325

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J. K. M. Brown et al.

infancy, as is the application of this research to wheat breeding. This compares poorly with research on the genetics of other important diseases, such as powdery mildew and the three rust diseases of wheat (McIntosh et al., 1998). The highly specific interactions of some cultivars with M. graminicola isolates mean that the durability of some resistances is at risk from the evolution of specific virulence in pathogen populations (Cowger et al., 2000). It is important to know if interactions between adult plants of wheat cultivars and isolates of M. graminicola are stable over different locations and different years, so that breeders may know whether specific resistances, identified in one location, will be effective in another. Also, stability of interactions allows geneticists to have confidence in the replication of experiments over different locations and years. This paper describes interactions between M. graminicola isolates and wheat lines in a series of field trials done in three European countries over 3 years. Several cultivars and breeding lines are also identified which had good resistance to all or most of the isolates used in these trials, and which therefore may be useful sources of resistance to septoria tritici blotch.

Materials and methods Wheat lines Seventy-one wheat lines of diverse origins were tested in field trials (Table 1). Most are winter types and have been released as commercial cultivars, or are breeding lines developed by European breeders. The majority of the lines were grown in two or more trials but 27 lines, including parents of several precise genetic stocks maintained at the John Innes Centre, underwent trials only in the UK in 1997.

Pathogen isolates and inoculation methods The M. graminicola isolates used (Table 2) were obtained from the collection of monospore isolates maintained at Plant Research International, Wageningen, The Netherlands, and have been reported previously (Kema et al., 1996c; Kema & van Silfhout, 1997). Inoculum was applied using hand-pumped, motor-driven or propane-driven knapsack sprayers. Inoculation was done in the evening so that moisture was retained on the leaf surface during the following night, thus promoting infection. Trials were inoculated on one to three occasions in late May or early June (Table 2). In Switzerland, trials were sprayed with water every 2 h during the day after inoculation. In The Netherlands and the UK, trials were misted or sprinkled as required to maintain humidity within the plots until they were scored.

Experimental design Trials were conducted over 3 years in The Netherlands, 1 year in Switzerland and 2 years in the UK (Tables 1 and 2). Each trial was sown in a split-plot design with two replicate blocks. Within each block there were four or five main plots, each inoculated with one isolate. One additional main plot in each block was mock-inoculated with water but no spores. Within each main plot, there was one subplot of each wheat line. Isolates were randomized within blocks and wheat lines were randomized within main plots. Work on five cultivars included in the NL95 trial (see Table 1 for abbreviations) has been reported previously (Kema & van Silfhout, 1997). These five, Clement, Hereward, Kavkaz, Okapi and Vivant, were sown in square 3 £ 3 m subplots, isolated from each other by guard rows of winter barley. The other seven cultivars that underwent trials in NL95 were sown as hill plots. All the large subplots were separated from one another by rows of barley. For the hill plots, main plots inoculated with different isolates were separated from one another. In the 1996 and 1997 trials in The Netherlands and the UK, wheat lines were sown in sets of three 1 m rows, while six-row plots were used in Switzerland in 1996. Main plots, inoculated with different M. graminicola isolates, were separated from each other by guard rows of barley or oats.

Observations The percentage leaf area that was necrotic or covered by lesions bearing pycnidia was estimated visually. Trials were scored on one to three occasions (Table 2). In the NL95, NL97 and UK97 trials, scores were made on flag leaves. In CH96 and NL96, both flag leaves and second leaves were scored, while in UK96 only second leaves were scored because of low levels of flag leaf infection. Necrosis and pycnidia were scored in all trials except for UK97, where only pycnidia were scored. Ten leaves were scored per subplot at each time of scoring, except for CH96 and NL97, where a single score was given to each subplot.

Analysis of field trial data As pycnidia and necrosis were scored on a percentage scale, analysis of the data was done by generalized linear modelling (GLM) of binomial proportions (genstat 5, Genstat 5 Committee, 1993), using genstat for Windows, 4th edition (Numerical Algorithms Group Ltd, Oxford, UK). Where both random and fixed effects were involved, analysis was done by generalized linear mixed modelling (GLMM; Welham, 1993). In each of the six trials, mean pycnidium scores were estimated for each subplot (i.e. one wheat line, inoculated with one isolate, in one block). This was done separately for each Q 2001 BSPP


Resistance to Mycosphaerella graminicola isolates

trial by an appropriate GLM (not shown). Means were estimated from both uninoculated and inoculated plots. Mean pycnidium scores for subplots were analysed by GLM and GLMM to examine the performance of lines and isolates throughout the series of six trials. In order to estimate the effects of the inoculated isolates, disregarding symptoms caused by natural inoculum, mean pycnidium scores from the control, uninoculated plots in each block were used as covariates in the analysis of data from the inoculated plots. This was done by fitting the effect of the control plots before that of other factors in the GLM or GLMM. In the GLM, the significance of the various factors was tested by F-tests of the deviance for each factor divided by the residual deviance. In the GLMM, the significance of the fixed effects was tested by x2 tests of Wald statistics.

Identification of interactions between lines and isolates Median tetrad analysis (MTA) (Bradu & Hawkins, 1982; Brown, 2001) was used to identify specific resistance or susceptibility of wheat lines to particular isolates. MTA is a robust method of identifying several statistical interactions simultaneously in a two-way table of data, such as disease scores for a set of wheat lines and isolates (Kroes et al., 1999). MTA was used in preference to analysis of residuals as a means of detecting interactions because, unlike a large residual, one large median tetrad has little influence on the size of other median tetrads for the same line or isolate. The analysis was done on mean pycnidium scores (pyc), transformed to logits {log[pyc(100±pyc)]}. Means were calculated for combinations of lines and isolates. The median tetrad estimates the amount by which an observed score deviates from that expected if there were no interaction, that is, if no line had isolate-specific resistance or susceptibility. In the absence of any lineby-isolate interaction, the median tetrads have a normal distribution. If there is a specific interaction, the median tetrad for the relevant cell of the table of disease scores is low if a line is specifically resistant to an isolate, or high if there is a specifically susceptible interaction. These median tetrads are outliers from the normal distribution of the median tetrads of cells that are not affected by line-by-isolate interactions. The statistical significance of the deviation of the most extreme median tetrads from a normal distribution was tested by Grubbs' method (Barnett & Lewis, 1978; Sokal & Rohlf, 1981). MTA was done for groups of lines and isolates in several stages, for two reasons. Firstly, many more data were obtained for some lines than for others, so the random error in the data was least (and the median tetrads were therefore most accurate as estimates of line-by-isolate interactions) for lines tested in the most trials. Secondly, the large number of lines that were specifically resistant to isolate IPO323 meant that interactions involving other isolates were difficult to Q 2001 BSPP


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detect in the presence of data on IPO323. MTA was therefore done first on data for the other five isolates. The nine lines grown in all six trials were tested first; then the 27 lines, including the first nine, grown in four or five trials; then all 33 lines grown in the 1996 trials; then all 40 lines grown in two or more trials except for four spring wheats grown only in the two 1997 trials. (The establishment of spring wheat was poor in the NL97 trial and data are not likely to be as reliable as those for winter wheat.) Lastly, all 71 lines were analysed. Then the data for IPO323 were included and the five groups of lines tested in the same order, except that the set of nine lines tested in all six trials was not analysed separately (the reason for this is explained below in the Results section). Following each stage of the MTA, the score for any combination of a wheat line and an isolate identified as having a statistically significant interaction (P 0´05) was omitted before proceeding to the next stage. At some stages of the MTA, some interactions only became evident once data for more extreme observations were removed (for example, see Fig. 1). Finally, all median tetrads that were larger (positive or negative) than the smallest median tetrad that was statistically significant, that for Kavkaz with IPO323, were identified. All interactions identified in this final step involved the 27 lines that underwent trials only in UK97 or the four spring wheats tested only in 1997.

Results Disease symptoms Levels of necrosis and pycnidia were highly correlated in all five trials where both were scored (Table 2). The major exception was for Veranopolis in the CH96 trial, which had relatively high necrosis but few pycnidia. However, Veranopolis had similar levels of necrosis in uninoculated plots in CH96, indicating that the necrosis was not caused by the M. graminicola isolates with which the trial was inoculated. It is most likely that necrosis was induced partly by early senescence and partly by other diseases, including powdery mildew, brown rust and yellow rust. As necrosis and pycnidium scores were highly correlated, detailed analysis is presented of the pycnidium data only.

Comparison of trial sites Variation between trials was investigated by GLM, in which the effects of line, isolate and trial and their two and three-way interactions were analysed. There was a large, significant interaction between line and trial (not shown), implying that the relative levels of disease on different wheat lines varied between trials. Much the greatest contribution to this interaction came from Veranopolis in the UK96 trial, where it had high pycnidium scores compared with those in other trials; as in CH96, the performance of this cultivar was

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Table 1 Wheat lines, their flowering habit and country of origin, grown in field trials of resistance to isolates of Mycosphaerella graminicola in three European countries over 3 years Trialsa Wheat line Amarok Amigo Andante Apollo Arina Arminda Atlas 66 Axona Baldus BerseÂe Bezostaya 1 Cappelle Desprez Ceb 957 Centauro CH75-892 CH76-000 CH76-106 CH76-263 CH76-264 CH76-337 CH76-351 CH95-413 Champlein Cheyenne Chinese Spring Clement CS(Syn3D)e CS(Syn5D)e Dynamo ENMP-LSA1 ENMP-LSA2 Eufrates Favorits Flame Frontana Galaxie Golia Hereward Hobbit sib Hope Kavkaz Kavkaz-K4500 l.6.a.4 Longbow Lutescens 62 Mara Mironovskaya 808 NSL92-5719 Obelisk Okapi Pastiche Poros ReÂcital Riband Ritmo RU-51A RU-586-96 RU-5-96

Habitb W W W W W W W S S W W W W S W W W W W W W W W W S W S S W S S S W W S W S W W S W S W S S W W W W W W W W W W W W

Typec cv cv cv cv cv cv cv cv cv cv cv cv BL cv BL BL BL BL BL BL BL BL cv cv LR cv cyto cyto cv BL BL cv cv cv cv cv cv cv BL cv cv BL cv cv cv cv BL cv cv cv cv cv cv cv BL BL BL

Origind France USA UK Germany Switzerland Netherlands USA Netherlands Netherlands France Russua France Netherlands Italy Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland France USA China Netherlands UK UK UK Portugal Portugal Portugal Romania UK Brazil France Italy UK UK USA Russia Kenya UK Hungary Italy Russia UK Netherlands Netherlands UK Germany France UK Netherlands Czech Rep. Czech Rep. Czech Rep.

NL95

x

CH, NL, UK 96

NL97

x

x

x x x x

x x x x x x

x x x x x x x x x

x

x

x

UK97 x x x x x x x x x x x x x

x x x x x

x x x x x

x

x x x x x x x

x

x

x

x x x x x x x x x x x x x x x x x x

d

Total no. 5 1 6 5 5 4 1 2 2 1 1 1 5 1 3 5 5 5 5 5 3 5 1 1 1 6 1 1 3 1 1 1 1 6 1 5 1 6 1 1 6 1 6 1 1 1 6 3 6 5 1 1 5 5 2 2 2

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x x x x

x

x x

x x x x x

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x x

x

x x x x x x x x x


329

Table 1 continued Trialsa Wheat line

Habitb

Typec

Origind

Runal Ï aÂrka S Sava SG-RU-5007 Shafir Shango Slejpner Spark TE 9111 Tonic Vdh 1141-92 Veranopolis Vivant Vlasta Total

W W W W S W W W S S W S W W

cv BL cv BL cv cv cv cv BL cv BL cv cv cv

Switzerland Czech Rep. Serbia Czech Rep. Israel UK Sweden UK Portugal UK Netherlands Brazil UK Czech Rep.

NL95

CH, NL, UK 96 x

NL97

UK97

Total no.

x x

x x x x x

5 2 2 2 2 3 1 5 1 2 4 5 6 2

x x x x x

x

x

x x x

12

33

x x x x x x x x 65

x x x x 35

a Trial sites: CH, ZuÈrich-Reckenholz, Switzerland; NL, Wageningen, The Netherlands; UK, Norwich, England; 95, 1994±95; 96, 1995±96; 97, 1996±97. b S, spring wheat; W, winter wheat. c BL, breeder's line; cv, cultivar; cyto, cytogenetic stock; LR, landrace. d x, line undergoing trials at that site. e CS(Syn3D), chromosome 3D of Synthetic 6£ substituted into Chinese Spring; likewise CS(Syn5D) (Nicholson et al., 1993).

affected by rust and by early senescence. Since Veranopolis in UK96 was such a significant outlier, it was removed from the remainder of the analysis. This reduced the deviance for the line±trial interaction substantially, by 4´2%. After excluding the data on Veranopolis in UK96, the remaining, significant line± trial interaction effect (Table 3) was caused by relatively minor variation in the performance of numerous lines in different trials. The analysis presented in Tables 3 and 4 and Figs 2 and 3 omits data for Veranopolis in UK96. There was no significant effect of trial on the interaction between line and isolate (L £ I £ T term in the GLM columns of Table 3). This implies that interactions between wheat lines and M. graminicola

isolates were stable over the six environments investigated in this series of trials. (Inclusion of data on Veranopolis in the UK96 trial had little effect on the L £ I £ T term.)

Interactions of lines and isolates Since the line±isolate interaction was stable over trials, the data were re-analysed in such a way that a single set of mean scores for lines and line-by-isolate interactions could be estimated from data pooled across trials. This was done by GLMM, with trial and isolate within trial treated as random effects, and line and the interaction between line and isolate as fixed effects. The main effect

Table 2 Mycosphaerella graminicola isolates used in field trials and correlation coefficients between necrosis and pycnidium scores Isolates Triala

IPO 001

IPO 290

IPO 323

IPO 89-011

NL95 CH96 NL96 UK96 NL97 UK97

xb x x x x x

x x x x

x x x x x x

x

x

x x

IPO 94-265

IPO 94-269

No. inoculations

No. times scored

Units of observation

Correlation of necrosis and pycnidia

x x x x

x x x x x

3 2 2 1 1 2

2 3 2 1 1 2

Leaves Plots Leaves Leaves Plots Leaves

0´83 0´82 0´93 0´85 0´89 ±c

a Trial sites: CH, ZuÈrich-Reckenholz, Switzerland; NL, Wageningen, The Netherlands; UK, Norwich, England; 95, 1994±95; 96, 1995±96; 97, 1996±97. b Isolate tested in that trial. c Necrosis not scored in UK97.

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Figure 1 Identification of significant interactions between wheat lines and isolates of Mycosphaerella graminicola in successive stages. The data are for the 27 lines grown in four or more trials and for all isolates except IPO323 (stage ii in the series of MT analyses). The set of points is a halfnormal plot of MT; if the MT were perfectly normally distributed, the points would fall on a straight line. The vertical dashed line is the 5% significance level for Grubbs' test of outliers from a normal distribution. At each of stages a, b and c, one line-by-isolate interaction was identified as significant. No further interaction was detected at stage d; Veranopolis and IPO89011 had the strongest interaction that was not statistically significant.

of isolate was not analysed separately because of variation between trials, particularly in the concentration of inoculum; the isolate main effect was therefore confounded with the trial effect. The line±isolate interaction term was large and highly significant, as was the main effect of line (Table 3). Mean pycnidium scores for lines and isolates are given in Table 4. Median tetrad analysis was used to identify instances where wheat lines were specifically resistant or susceptible to particular isolates, with especially low or high pycnidium scores, respectively. The following interactions were identified when data for IPO323 were omitted from the analysis. 1 Among the nine lines grown in all six trials, NSL92-5719 was specifically resistant to IPO001 (Fig. 2a). 2 Three interactions were successively identified for the 27 lines grown in four or five trials (Fig. 1).

Veranopolis was specifically susceptible to IPO001 (Fig. 2a), and Amarok and CH-95413 were specifically resistant to IPO89011 (Fig. 2d). 3 No further interactions were identified for the 33 lines tested in the 1996 trials, when data on the six lines that underwent trials only in 1996 were included. 4 Among the 40 lines tested in two or more trials, excluding four spring wheats that underwent trials only in NL97 and UK97, RU-51A was specifically resistant to IPO94269 (Fig. 2f) and SG-RU-5007 was specifically resistant to IPO001 (Fig. 2a). 5 Among the complete set of 71 lines, three interactions were identified successively, by the same procedure as that illustrated in Fig. 1. Tonic was specifically resistant to IPO89011 (Fig. 2d), ENMP-LSA2 was specifically susceptible to IPO290 (Fig. 2b), and Frontana was specifically susceptible to IPO001 (Fig. 2a). Data for IPO323 were then included in the analysis. It was obvious, by inspection, that many lines were Q 2001 BSPP



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Table 3 Summary of generalized linear modelling (GLM) and generalized linear mixed modelling (GLMM) of percentages of leaf area with lesions bearing pycnidia of Mycosphaerella graminicola in six trials in the Netherlands, Switzerland and the UKa

Term

GLM d.f.c

Deviance

Deviance ratio

P1

Controlf Trial Isolate T£I Line L£T L£I L£I£T Residual

1 5 5 18 70 133 314 471 909

1351 12083 19598 5288 21535 4735 13067 2819 9301

132´0 236´2 383´0 28´7 30´1 3´5 4´1 0´6

*** *** *** *** *** *** *** ns

d

GLMM (fixed effects)b d.f.c Wald statistic

P2e

1

104´6

***

70

1057´5

***

319

846´2

***

a

Excluding Veranopolis in UK96 trial. In the GLMM, random effects were trial and isolate within trial (trial £ isolate). c d.f., degrees of freedom. d P1, F-test probability of deviance ratio. ns, P . 0´05; ***, P , 1023. e P2, x2 test probability of Wald statistic. ***, P , 1023. f Control: score in uninoculated plot of appropriate block. b

resistant to that isolate (Fig. 2c; Table 4). Among the nine lines grown in all six trials, five, namely Flame, Hereward, Kavkaz, NSL92-5719 and Vivant, appeared to be relatively resistant to IPO323. When only those nine lines were analysed, three of the remaining four lines, Andante, Longbow and Okapi, were paradoxically identified as specifically susceptible to IPO323 because they contrasted with the majority of lines, which were specifically resistant to IPO323. Analysis of these nine lines is therefore not presented separately. 6 Among the 27 lines tested in four or more trials, four groups were successively identified as being specifically resistant to IPO323. First, Arina, CH76000, Flame and Hereward had the highest median tetrads (MT), indicating the strongest specific resistance to IPO323. The next highest MT were those for Vivant, CH-76106 and Apollo, then those for Vdh1141-92 and NSL92-5719, and finally that for Kavkaz (Fig. 3). The resistance of Kavkaz to IPO323 had the lowest MT of all the specific interactions that were statistically significant. 7 Again, no further interactions were detected among the 33 lines tested in 1996, as in step 3. 8 Among the group of 40 lines tested twice or more, RU-5-96 and SG-RU-5007 were specifically resistant to IPO323 (Fig. 3). Then Veranopolis was identified as susceptible to IPO89011 (Fig. 2d). This interaction consistently had the largest MT from stage 2 to stage 7 above (Fig. 1c), other than those identified as statistically significant. 9 Finally, the two ENMP-LSA lines, followed by Shafir, were identified as specifically resistant to IPO323 (Fig. 3). Among the 31 lines that underwent least extensive trials, 27 grown only in the UK in 1997 and four spring wheats tested in both 1997 trials, there appeared to be many more isolate-specific interactions than those listed above. Interactions stronger than that of Kavkaz with Q 2001 BSPP


IPO323, as indicated by a larger absolute value of the MT, were as follows. 1 Kavkaz-K4500 (KK) was specifically susceptible to IPO001 (Fig. 2a). 2 Centauro was specifically susceptible to IPO290 (Fig. 2b). 3 Poros, Atlas 66, TE9111, Axona, Chinese Spring, Bezostaya 1 and Amigo were specifically resistant to IPO323 (Fig. 3). 4 KK was susceptible to IPO89011, while Axona and Chinese Spring were specifically resistant to this isolate (Fig. 2d). 5 Tonic was specifically resistant to IPO94265 (Fig. 2e).

Resistance of wheat lines There was a wide range of resistance among the wheat lines grown in the trials. The existence of strong line± isolate interactions and the small number of isolates used in these trials mean that one should be cautious about identifying particular lines as good sources of resistance for breeding wheat in Europe. However, it was possible to identify lines that are likely to be worth submitting for further trials, perhaps with more isolates from a wider geographic area. The overall mean scores for the wheat lines, given in Table 4, were calculated by omitting data for isolates with which they had a specific interaction. These means therefore reflect the general level of resistance to the isolates tested. A wide range of germplasm had good resistance. Among the 40 lines that underwent trials at least twice, the Brazilian cultivar Veranopolis was most resistant; this is a well-known source of resistance (Rosielle, 1972). Three of the next four most resistant lines were from Switzerland, including the cultivar Arina and two CH breeding lines. Other lines with a mean disease level of 20% or less were two RU breeding

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Figure 3 Mean level of infection of wheat lines by Mycosphaerella graminicola isolate IPO323, plotted against the mean level of infection across all isolates (data from Table 4). X, Resistant responses of lines to IPO323 that were statistically significant (P 0´05); O, resistances that were not significant (P . 0´05) but had larger MT than that for Kavkaz with IPO323 (the smallest significant MT); S, lines that did not show an interaction with IPO323 stronger than that of Kavkaz. Axes are logit-scaled so that specific line-by-isolate interactions of similar size are at similar distances from the trend line of S throughout the graph.

lines from the Czech Republic; two cultivars, Okapi and Apollo, from Germany; the breeding line NSL92-5719 and three cultivars, Andante, Spark and Pastiche, from the UK; the breeding line Vdh1141-92 and the cultivar Arminda from the Netherlands; and three more CH breeding lines from Switzerland. Of the remaining 31 lines, the Portuguese line TE9111 was most resistant, being second only to Veranopolis overall. The French cultivars BerseÂe and Cappelle Desprez; the CIMMYT line KK; a Brazilian cultivar, Frontana, which is one of the parents of Veranopolis; and Atlas 66 from the USA also had good resistance (mean score ,20%).

Discussion Methodology The trials were inoculated around the time of heading,

when flag leaves were fully expanded. This was intended to minimize the effect of plant height and developmental rate on the level of disease, as early flowering and short stature tend to increase susceptibility to septoria tritici blotch (Danon et al., 1982; van Beuningen & Kohli, 1990; Camacho-Casas et al., 1995). It is not possible to ensure that either factor has no effect at all on disease development. However, while flowering time and height may affect the mean performance of a wheat genotype, they are not expected to affect specific interactions between wheat lines and M. graminicola isolates, which are the focus of this paper. It is notable that two of the most susceptible lines (Table 4), Chinese Spring and Hope, are very tall, while the most resistant line, Veranopolis, is one of the earliest flowering of all those tested. The analysis presented here is based on scores of leaf area affected by lesions bearing pycnidia of

Figure 2 Mean level of infection of wheat lines by six isolates of Mycosphaerella graminicola plotted against the mean level of infection across all isolates (data from Table 4). X, Line-by-isolate interactions that were statistically significant (P 0´05); O, line-by-isolate interactions that were not significant (P . 0´05) but had larger MT than that for Kavkaz with IPO323 (the smallest MT that was statistically significant); S, combinations of lines and isolates that did not show an interaction effect greater than that of Kavkaz with IPO323. Points above the trend line of S indicate specific susceptibility; points below indicate specific resistance. Axes are logit-scaled so that specific line-by-isolate interactions of similar size are at similar distances from the trend line of S throughout the graph.

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Table 4 Percentage leaf area of wheat lines covered by lesions bearing pycnidia of Mycosphaerella graminicola isolates in six trials in the Netherlands, Switzerland and the UK in 1995±97 Percentage leaf area affecteda Wheat line

Number of trials

Overall meanb

IPO 001

IPO 290

IPO 323

IPO 89011

(i) 40 lines, excluding those undergoing trials only in UK97 and spring wheats trialled only in NL97 and UK97 1 0 3 (0) Veranopolis 5 0 7 (0)c CH76-337 5 8 0 16 17 1 CH76-106 5 9 2 10 0 (10) 7 SG-RU-5007 2 9 0 (1) 17 0 (10) 7 Arina 5 10 2 12 0 (12) 16 Okapi 6 12 4 16 20 14 NSL92-5719 6 12 0 (1) 16 1 (12) 4 Vdh1141-92 4 13 0 24 0 (9) 3 CH76-264 5 14 1 11 39 8 RU-5-96 2 14 5 15 0 (17) 13 Apollo 5 16 1 18 1 (15) 13 CH95-413 5 17 1 20 21 1 (10) CH76-000 5 18 1 27 0 (18) 30 Arminda 4 18 1 24 31 *d Andante 6 18 3 23 45 4 Spark 5 18 2 27 27 20 Pastiche 5 20 0 22 38 9 Vlasta 2 22 4 26 38 17 Ceb 957 5 23 2 27 38 23 Clement 6 25 4 34 8 39 Flame 6 25 9 38 1 (31) 24 CH76-263 5 26 8 31 46 27 CH75-892 3 27 16 31 39 * CH76-351 3 29 19 39 37 * Kavkaz 6 29 5 35 5 (32) 35 Obelisk 3 30 2 45 41 * Runal 5 30 1 40 13 47 Dynamo 3 31 4 41 44 * Vivant 6 32 2 27 1 (31) 29 RU-51A 2 32 5 70 24 33 Shango 3 34 6 36 45 * Hereward 6 36 14 44 1 (43) 38 RU-586-96 2 38 11 76 54 24 Sava 2 40 5 56 37 25 Ritmo 5 42 6 52 57 49 Galaxie 5 45 2 53 46 58 Longbow 6 47 10 58 50 60 Riband 5 47 10 63 55 46 SÏaÂrka 2 48 3 79 67 43 Amarok 5 59 34 73 68 11 (55) (ii) 27 lines undergoing trials only in UK97 and four spring wheats trialled only in NL97 and UK97 TE 9111 1 2 1 2 0 {3}e Kavkaz-K4500 (KK) 1 3 3 {0} 10 0 BerseÂe 1 3 1 3 5 Frontana 1 5 15 (1) 12 5 Atlas 66 1 11 3 24 0 {14} Capelle Desprez 1 16 1 33 21 Champlein 1 21 10 37 41 Mironovskaya 808 1 26 1 61 15 Favorits 1 27 6 49 8 Cheyenne 1 30 3 79 10 Poros 1 30 8 53 2 {36} Mara 1 31 3 55 10 Tonic 2 33 9 53 37 Golia 1 36 7 45 43 Lutescens S 1 37 19 42 23

Q 2001 BSPP

6 38 {1} 2 11 6 13 4 27 16 9 26 29 0 (30) 20 53

IPO 94265

IPO 94269

0 15 13 11 12 12 22 23 18 11 26 9 29 24 20 23 28 23 30 35 32 27 30 33 30 37 41 37 52 26 49 49 19 * 41 54 59 54 40 45

0 8 21 21 18 18 27 26 22 38 35 53 15 29 30 27 42 42 39 49 37 36 38 36 56 46 57 50 62 6 (52) 54 47 60 84 58 69 56 64 66 76

* * * * * * * * * * * * 3 {42} * *

2 3 8 2 22 26 26 42 68 61 42 70 48 74 58



335

Table 4 continued Percentage leaf area affecteda Wheat line

Number of trials

Overall meanb

IPO 001

IPO 290

IPO 323

IPO 89011

IPO 94265

IPO 94269

Hobbit sib CS (Syn 3D) ENMP-LSA1 Axona CS (Syn 5D) Bezostaya 1 Slejpner ReÂcital Shafir Centauro Eufrates Amigo Baldus Chinese Spring ENMP-LSA2 Hope

1 1 1 2 1 1 1 1 2 1 1 1 2 1 1 1

38 43 45 47 48 50 51 51 52 54 60 66 72 80 90 95

10 16 16 6 13 4 18 14 7 19 42 12 65 32 59 76

58 77 73 81 81 72 66 72 67 99 {74} 72 85 96 95 100 (96) 100

56 23 1 3 22 6 53 69 3 35 26 21 83 24 1 99

14 38 29 3 {40} 47 48 41 49 39 60 63 71 45 18 {82} 90 89

* * * * * * * * * * * * 72 * * *

59 68 63 57 78 73 77 53 91 83 88 81 59 93 95 99

(52) {49} {51}

(59)

{70} {87} (93)

a

Means from generalized linear mixed modelling (GLMM; Table 3). Overall means calculated by GLMM of mean disease levels, with wheat line as a fixed effect and isolate as a random effect (scores for line-isolate combinations for which P 0´05, or, for part (ii) of the table, showed interactions more extreme than that of Kavkaz with IPO323 (Figs 1 and 2), were excluded from the data set analysed). c Round brackets indicate expected scores in the absence of line-by-isolate interaction, where the MT was significant (P 0´05, Grubbs' test). d *, not tested. e Curly brackets indicate expected scores in the absence of line-by-isolate interaction, where the MT was not statistically significant (P . 0´05) but was larger than that for Kavkaz with IPO323 (see Fig. 1). b

M. graminicola. The pycnidium score is a direct estimate of the proportion of a leaf colonized by the pathogen and is probably more reliable than necrosis as a guide to a plant's susceptibility (Kema et al., 1996a). However, necrosis is easier to score, and the high correlation between necrosis and pycnidium scores in five trials (Table 3) indicates that necrosis may be used to score septoria tritici blotch provided that no other factors, such as senescence or other diseases, cause extensive necrosis. The unusual results for Veranopolis in the CH96 and UK96 trials, however, emphasize the need for caution about using necrosis as the only measure of disease.

Line-by-isolate interactions At least one line was specifically resistant or susceptible to each of the isolates tested (Figs 2 and 3). Specific interactions between wheat lines and isolates of M. graminicola are now well established as features of septoria tritici blotch (Eyal et al., 1973, Eyal et al., 1985; Perello et al., 1991; Ahmed et al., 1995; Ballantyne & Thomson, 1995; Kema et al., 1996a, Kema et al., 1996b; Kema & van Silfhout, 1997). However, almost all the work cited was done on seedling leaves. In the one exception, interactions involving five cultivars and three isolates were broadly consistent between 2 years of field trials of adult plants (Kema & van Silfhout, 1997). Q 2001 BSPP


In most previous work, the existence of cultivar-byisolate interactions has been inferred from a significant interaction term in anova or generalized linear modelling. The methods used here, notably the MT analysis which appears to have been first used in plant pathology by Kroes et al. (1999), allow individual interactions to be identified clearly. This paper reports a large number of line-by-isolate interactions that are effective in adult plants. As in the work of Kema & van Silfhout (1997), the interactions were stable over the six trials (Table 3), implying that geneticists and breeders may indeed be confident that the expression of specific resistance to avirulent genotypes of M. graminicola will be consistent over sites and years. Most of the specific interactions marked with solid circles in Figs 2 and 3 were studied in two or more trials. The exceptions are the susceptibility of Frontana to IPO001 (Fig. 2a) and of ENMP-LSA2 to IPO290 (Fig. 2b), as well as the resistance of both ENMP-LSA lines to IPO323 (Fig. 3). Given the absence of a significant line £ isolate £ trial (L £ I £ T) term (Table 3), these interactions are likely to be an accurate indication of the true responses of the lines to the isolates concerned. The results of these trials therefore strongly support the view that specific interactions of wheat cultivars with M. graminicola isolates are important in field conditions, and are not merely confined to seedlings (Kema & van Silfhout, 1997). Specific interactions identified on the basis of data

336


from only one trial should be regarded as somewhat tentative. This is especially so where a line's mean score over all isolates, and its mean for an isolate with which it appears to have a specific interaction, are close to the same end of the percentage range. For example, TE9111 was identified as specifically resistant to IPO323, but it also had good resistance to the other four isolates with which it was tested. On the other hand, ENMP-LSA2 was specifically susceptible to IPO290, but was also very susceptible to other isolates (Figs 2 and 3; Table 4). In these cases there were large differences between the logits of the observed pycnidium scores and those expected in the absence of a specific line-by-isolate interaction, but only small differences between the observed and expected scores on the original, percentage scale. Nevertheless, some specific interactions identified only in the UK97 trial have been confirmed in tests of detached seedling leaves, including the resistance of Bezostaya 1 and Chinese Spring to IPO323 (Fig. 3). KK was susceptible to IPO89011 (Fig. 2d) in detached leaf tests, but not to IPO001 (Fig. 2a) (Arraiano et al., 2001). Tonic and Amarok were resistant to IPO89011 in detached leaf tests, but Tonic was not resistant to IPO94265 (Fig. 2e) (J.C. Makepeace, C.M. Raitt & J.K.M. Brown, unpublished results). Specific resistance to IPO323 (Fig. 3) was detected in diverse lines, including cultivars and breeding lines from China, Israel, the USA and seven European countries. The source of this resistance is not known. Indeed, as is the case in powdery mildews and rusts, IPO323 may have more than one avirulence gene, recognized by different resistance genes in different wheat lines. The strength of expression of resistance to IPO323 varied between lines, from ENMP-LSA2 at one extreme to Kavkaz at the other (Fig. 3). The relatively weak resistance of Kavkaz to this isolate is consistent with previous results, in which it was specifically resistant to IPO323 but not as strongly as Hereward or Vivant (Kema & van Silfhout, 1997). The analysis presented here may be somewhat conservative in that there may have been some line-by-isolate interactions which were not identified. In the work of Kema & van Silfhout (1997), Clement showed some specific resistance to IPO323, although, like Kavkaz, more weakly than Hereward or Vivant. The specific resistances to IPO323 of Eufrates, KK, Favorits and CS(Syn5D) were stronger than that of Clement, but weaker than that of Kavkaz, as indicated by their MT. In detached leaf tests, KK was specifically resistant to IPO323 (Arraiano et al., 2001). A further complication is that specific resistance may be masked by a high level of nonspecific, background resistance. For instance, Veranopolis had the highest resistance among the lines tested, once isolate-specific effects were removed; in any case, it was only lightly diseased by the two isolates, IPO001 and IPO89011, to which it showed specific susceptibility (Table 4; Fig. 2a). Veranopolis has consistently shown specific resistance to IPO323 in tests of whole seedlings

(Kema et al., 1999) and detached seedling leaves (L.S. Arraiano, unpublished results), but this resistance was not detected in the trials reported here. This is presumably because Veranopolis' nonspecific, `background' resistance was so strong that its specific resistance to IPO323 had little additional effect in reducing disease. The two lines with significant specific susceptibility to IPO001 (Fig. 2a) are closely related, as Frontana is a parent of Veranopolis (Fox et al., 1997). The susceptibility of Veranopolis to IPO001 in field trials reflects the results of seedling tests reported previously (Kema & van Silfhout, 1997). Two breeding lines, SG-RU5007 and NSL92-5719, were identified as specifically resistant to IPO001 even though they also had good resistance to the other isolates. NSL92-5719 was tested with IPO001 in all six trials, and was also resistant in detached leaf tests (J.C. Makepeace, C.M. Raitt & J.K.M. Brown, unpublished results), so even though there was only a small difference between the actual level of infection of NSL92-5719 by IPO001 and that expected in the absence of line-by-isolate interaction (Table 4), the identification of this specific resistance appears to be sound. The specific susceptibility of Veranopolis to IPO89011 is consistent with previous work, in which this was one of several isolates which were virulent on seedlings of Veranopolis (Fig. 2d; Kema et al., 1996a). IPO89011 was not specifically virulent on seedlings of KK in previous experiments (Kema et al., 1996a), but was virulent on detached seedling leaves (Arraiano et al., 2001). The response of KK to IPO89011 therefore needs further clarification. The specific interactions described here are characteristic of gene-for-gene relationships, but it is not possible to conclude that they are indeed controlled by corresponding single genes in the host and pathogen without formal genetic analysis of both partners in the pathosystem. Furthermore, where a wheat line was specifically susceptible to an isolate, it is not possible to determine whether that line has a gene that actually confers susceptibility, or lacks a gene, carried by the majority of lines, that confers resistance to that isolate.

Breeding for resistance Many wheat lines had high levels of quantitative resistance to the isolates tested. They include diverse cultivars and breeding lines from Brazil, Portugal, France, Switzerland, the Czech Republic, Germany, the USA, the UK and The Netherlands (Table 4). An important source of quantitative resistance in northern European cultivars may have been Cappelle Desprez, which is the most important single progenitor of modern cultivars in northern Europe (Martynov et al., 1992, Martynov et al., 1996; Fox et al., 1997) and which has moderately good resistance to septoria tritici blotch (Table 4). However, several European lines had Q 2001 BSPP



better resistance than Cappelle Desprez, which suggests that resistance genes must have been accumulated from several sources. Quantitative resistance to septoria tritici blotch in European wheat cultivars is therefore probably polygenic, controlled by many genes, each with a small effect, as is similar resistance in American material (Jlibene et al., 1994; Camacho-Casas et al., 1995). Progress in improving resistance to septoria tritici blotch may be made by intercrossing lines from different European breeding programmes. Even better resistance could be developed from crosses between resistant lines from Europe and America, although agronomic problems would have to be overcome to develop commercially successful cultivars. It is possible that some of the quantitative resistance identified may in fact be adult plant resistance because Veranopolis, which had good resistance to all isolates (Table 4), was susceptible to IPO94269, but not IPO323, at the seedling stage (Kema et al., 1999). The breeding programme at FAP (Forschungsanstalt fuÈr Landwirtschaftlichen Pflanzenbau), FAL (Forschungsanstalt fuÈr AgraroÈkologie und Landbau) since 1996, Switzerland, has been notably successful in developing wheat with good resistance to septoria tritici blotch, as indicated by the low scores for Arina and five CH breeding lines (Table 4). This is the result of a longterm programme aimed at selecting lines with broadspectrum disease resistance and increased tolerance, in the sense that yield losses caused by disease are minimized (BroÈnnimann, 1968). Until 1996, breeding trials were conducted in 13 locations with widely varying climatic conditions at a wide range of altitudes. No fungicides were used, in order to intensify selection for resistance. Since 1990, trials have been artificially inoculated with M. graminicola (Anon., 1992). Several other breeding programmes have been successful in selecting lines with good quantitative resistance to septoria tritici blotch. However, the existence of line-by-isolate interactions in adult plants implies that some lines, selected as having good resistance, may actually have resistance of a race-specific, nondurable kind. In Oregon, USA, the resistance of the cultivar Gene, which was popular in the early 1990s because of its resistance to septoria tritici blotch, has been broken down by virulent genotypes of M. graminicola (Cowger et al., 2000). One option for breeders is to produce a `pyramid' of known, race-specific resistance genes, by developing lines with several resistance genes which are jointly effective against the pathogen population. Another is to avoid the use of race-specific resistance altogether, and instead to select lines with good quantitative resistance in the absence of specific resistance genes. More knowledge of the genetics of resistance to septoria tritici blotch is needed for either strategy to be implemented.

Acknowledgements We thank Mr W.J. Angus (Nickerson UK Ltd), Dr P. Q 2001 BSPP


337

BartosÏ (Research Institute for Crop Protection, Czech Republic), Mr T.W. Hollins (Plant Breeding International, UK), Eng B. MacËaÄs (National Plant Breeding Station, Portugal), Dr R.K. Rai (Cebeco Zaden BV, The Netherlands), Dr L.T.M.M. van Beuningen (Koninklijke Van der Have NV, The Netherlands; now part of Advanta Seeds Ltd), Dr H. Winzeler (Delley Seed and Plants, Switzerland) and Mr A.J. Worland (John Innes Centre) for providing many of the wheat lines studied. We thank Maria Todorova, Sonia Hamza, Suzanne Verhaegh and the experimental service of Plant Research International for assistance with the trials carried out in The Netherlands. Support for the research was provided by the Ministry of Agriculture, Fisheries and Food (J.K.M.B., E.M.F.), the EU Framework IV Biotech programme (G.H.J.K., E.C.P.V., P.A.B.) and Praxis XXI ± FundacËaÄo para a CieÃncia e a Tecnologia, Portugal (L.S.A.), and by travel grants from COST 817.

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