'Chinese Spring' wheat addition lines containing ...

Hereditas 134: 53-57 (2001)

Expression of resistance to Blumeria graminis f.sp. tritici in ‘Chinese Spring’ wheat addition lines containing chromosomes from Hordeum vulgare and H. chilense D. RUBIALES’, T. W. L. CARVER’ and A. MARTIN’

’ Institute of Sustainable Agriculture, CSIC, Cordoba, Spain Institute of Grassland and Environmental Research, Aberystwyth, Ceredigion, UK Rubiales, D., Carver, T. W. L. and Martin, A. 2001. Expression of resistance to Blumeria graminis f.sp. tritici in ‘Chinese Spring’ wheat addition lines containing chromosomes from Hordeum uulgare and H. chilense. -Hereditas 134: 53-57. Lund, Sweden. ISSN 0018-0661. Received February 5, 2001. Accepted May 8, 2001 Blumeria graminis f.sp. tritici (syn. Erysiphe gruminis f.sp. rritici ) causes an important disease of wheat (powdery mildew) to which Hordeum uulgare and H . chilense are resistant. The study of chromosomal addition lines of H. uulgare and H. chilense in wheat showed that they possessed resistance to wheat powdery mildew. This was expressed as a reduction of disease severity but it was not associated with increased macroscopically visible necrosis. The resistance is of broad genetic basis, conferred by gene(s) present on different chromosomes of both H. uulgure and H. chilense. The feasibility of transferring this resistance to wheat is discussed. Diego Rubiales, Institute of Sustuinuble Agriculture, CSIC, Apdo. 4084. E- 14080 Cordoba, Spain. E-mail: [email protected]

Powdery mildew of wheat (Triticum aestivum L., T. durum L.), caused by B. graminis DC Speer fsp. tritici Marchal (syn. Erysiphe graminis DC fsp. tritici Marchal), here abbreviated as Bgt, is common, and of great economic importance, throughout temperate wheat-growing regions of the world. Breeding for resistance is considered to be the most effective and economically feasible means of powdery mildew control. Because mildew has the ability to overcome single gene resistance rapidly through evolution of virulence, there is a need for different forms of genetic resistance with greater durability (SZUNICS and SZUNICS1999). There is a considerable variation for resistance available in closely related species of Triticum, Aegilops, Agropyron, Secale, Haynaldia and Eremopyron (WAHL et al. 1978), and also in more distantly related genera including Hordeum (TOSA and SHISHIYAMA1984, RUBIALESet al. 1993a). Broadening the genetic base of cultivated wheat by the introgression of resistance genes from related species or genera may provide new sources of useful and durable resistance against this disease. Indeed, single resistance genes have been transferred successfully into bread wheat from several species of Triticum as well as from rye, but virulence to these genes evolved quickly in mildew populations (SzuNICS and SZUNICS1999, PEUSHA et al. 2000). Thus, the fact that a gene is introduced from an alien species gives no guarantee that it will confer durable resistance. Rather, it seems that durability may depend on the complexity of genetic control or the mechanistic basis of the resistance.

Several studies have described genetic resistance against Bgt in wheat, but none has addressed the genetics of wheat powdery mildew resistance in cultivated or wild barleys. Identifying the barley chromosome(s) carrying resistance, and demonstrating expression of this resistance in a wheat background, would aid efforts to transfer it to wheat. Hybridization of H. vulgare with wheat has often been attempted but fertile amphiploids have never been obtained (FEDAK1992). However, using the wild South American barley H . chilense (Roem. et Schult.) successful hybrids have been made with both durum and bread wheat, and fertile amphiploids have and CUBERO 1981; MART~N et been created (MART~N al. 1998, 2000). These have been named ‘tritordeum’. Also, chromosome addition lines from both H . chilense and H . vulgare into T. aestivum have been obtained (ISLAMet al. 1981, MILLER et al. 1982). These might provide bridges to transfer useful traits to cultivated wheat. The purpose of the present study was to study the expression of the H . chilense and H . vulgare resistance to wheat powdery mildew, to identify the chromosome(s) responsible for the resistance, and to assess the feasibility of transferring the resistance to wheat. MATERIALS AND METHODS Plant and pathogen materials

The chromosome nomenclature adopted in this paper is based on homoeology. We used six wheat-barley

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disomic addition lines obtained from Dr A. K. M. R. Islam, University of Adelaide, Australia, (ISLAMet al. 1981). The addition lines were based on the bread wheat line 'Chinese Spring' (CS) (2n = 6x = 42; AABBDD) as recipient, and H. vulgare 'Betzes' (2n = 2x = 14; H'H') as the chromosome donor. The addition lines are named CS/2H", CS/3HV, CS/4HV, CSjSH", CS/6H" and CS/7HV,and these are disomic (2n = 42 2) for H. vulgare chromosomes 2 H', 3 H', 4 H", 5 H", 6 H" and 7 H", respectively. The H. chilense addition used in this study were obtained from Dr S. M. Reader, John Innes Centre, Norwich, UK. Again, 'Chinese Spring' (CS) was the recipient, but here the chromosome donor was H. chilense accession H1 (4010001) (2n = 2x = 14; WhHCh).We used the 'CS'/ H. chilense ditelosomic addition lines lHchS,2HCha,5Hcha,6HGhS,7Hchaand 7HchP (2n = 42 2 t ) , a monosomic lHch monotelosomic lHchSaddition line (2n = 43 l ) , and disomic addition lines for chromosomes 4 Hch, 5 Hch, 6 HCh and 7 HCh(2n = 42 + 2). The progeny of self-fertilised plants of known karyotype (cytologically monitored) were used in the evaluations. Seedlings were grown in 1 litre pots of JI no. 3 potting compost in a spore proof compartment under natural light and temperature conditions for all experiments. Plants were grown to full expansion of the leaf required for experimentation i.e. the nextformed leaf was unrolling. According to experiment, test leaves were inoculated with B. graminis f.sp. tritici isolates H12 ( = Ty3 virulent on P m l , Pm3c, PmQ, Pm4b and Pm8 (HOVM0LLER 1989)), a population of unknown virulence collected at Cordoba, Spain, or with JIW2 (virulent on Pm3d but with no other known virulence (JKM Brown, pers. comm., John Innes Institute, U.K.)). The isolates were maintained on wheat cv. 'Cerco'. One day before inoculum was required for experimentation, plants heavily infected with the required fungus, were shaken to remove ageing conidia and ensure a supply of vigorous young spores for inoculation. We compared symptom development on, H. vulgare cv. Betzes, its various addition lines and susceptible Chinese Spring wheat, and similarly, on H. chilense H1, its various addition lines and Chinese Spring. For inoculation with isolate H12 and the field isolate, plants were grown until their second-formed leaf was expanded before five of each line were inoculated by shaking over them plants of 'Cerco' bearing freely sporulating Bgt colonies. In the case of isolate JIW2, separate sets of plants were grown by staggered planting so that one set reached secondformed leaf expansion, and the other set fifth-formed leaf expansion, at the same time. Second- and fifth-

+

+

+

formed leaves were then inoculated using a spore setting tower to give a spore density of ca 15 conidia mm - '. In all cases, following inoculation plants were maintained in a greenhouse compartment for 10 days before disease development was assessed. Two methods of assessment were used. For isolate H12 and the field isolate, the percentage of second-formed leaf area affected by powdery mildew was estimated with the aid of the key of LARGEand DOLING(1962). For was counted diisolate JIW2, colony number cm rectly on second- and fifth-formed leaves. In all cases, note was taken of whether leaves showed evidence of necrotic flecking. Data were submitted to an analysis of variance and the means were separated by the Duncan test using 'SPSS for Windows version 9.0'.

-'

RESULTS AND DISCUSSION H . vulgare cv. Betzes and H . chilense accession H1 were completely resistant to Bgt in all cases (Tables 1 and 2). Compared to Chinese Spring wheat, the addition lines generally showed reduced powdery mildew severity although there were many cases in which the difference was not significant. In no case was Bgt attack associated with visible necrosis of inoculated leaves. This was true even in H . vulgare, H . chilense and in addition lines showing good powdery mildew resistance. Factors conferring high levels of quantitative resistance to the Bgt isolate H12 and to the population from Cordoba were present in chromosomes 2H" and 6H" of H . vulgare (Table 1). However those factors did not convey resistance to isolate JIW2. Resistance to this isolate was conveyed by factors present on chromosomes 4H" and 5H", but these factors had no detectable effect on isolate H12 and only the factor on 4H" had a significant, though relatively small, effect on the population from Cordoba. Similar small but significant effects on the population from Cordoba were carried on chromosomes 3H" and 7H", but neither of these affected the other isolates significantly. When second- and fifth-formed leaves were attacked by JIW2, generally lower numbers of colonies developed on fifth- than second-formed leaves, indicating expression of adult plant resistance in all H. vulgare addition lines. The ranking of lines was generally maintained between leaf positions, indicating that no resistance factors were expressed differentially with respect to leaf position. However, the slightly reduced colony density conferred by factors present on chromosomes 6H' and 7H' was significantly different from Chinese Spring wheat in fifth but not in second formed leaves.

Exmession of mildew resistance in wheat

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Resistant factors were even more broadly distributed in the H. chilense genome (Table 2). The development of isolate H 12 was suppressed by factors present in chromosomes 1HChS,2Hcha, 4Hch, 5HCh, 6WhS, 7Wh and 7Hcha. Since addition line 5HCh showed suppression of isolate H12, while no effect was seen in the ditelosomic line 5HChL,it appears that the effective factor(s) is associated with the short arm of H . chilense chromosome 5. It was not possible to confirm this directly because no addition line incorporating only the short arm of the chromosome 5 is available. Development of isolate JIW2 was also suppressed by a factor(s) present on chromosomes 5Wh but unlike H12, JIW2 was also suppressed in the line 5HchL. Thus, the long arm of this chromosome carries a factor(s) that differentiates between H12 and JIW2. Similarly, 7Hch was associated with suppression of JIW2 as well as H12, but in this case the ditelosomic line 7Hchadid not affect JIW2, again indicating that different resistance factors affected the two fungal isolates. In addition, JIW2 was suppressed by a factor present on 6HCh,but this factor had no effect on isolate H12, and the remaining factors that affected H12 did not affect JIW2. Although generally lower numbers of colonies developed on fifth- than second-formed leaves attacked by JIW2, indicating expression of adult plant resistance in all H. chilense addition lines, the ranking of disease development between lines remained constant between leaf positions. Thus, there was no evidence that factors controlling adult plant resistance were carried by any individual H . chilense chromosome. An addition line of chromosome 1H' of H. vulgare has been obtained recently (ISLAMand SHEPHERD 2000) but it was not available when these experiments were performed, and thus could not be assessed in this study. Also, an addition line of chromosome

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3Hch of H. chilense has not yet been created. The possibility that additional resistance factors are present on those chromosomes cannot be excluded. As seen in Tables 1 and 2, the effects of factors suppressing powdery mildew development were quantitative, reducing although not eliminating disease development. However, there is good evidence that the resistance factors carried on chromosomes from both H. vulgare and H. chilense conditioned resistance that was specific to certain isolates but did not affect others. The cellular basis of this 'race specific', quantitative resistance requires further investigation, but the absence of visible necrosis due to Bgt attack may suggest that the resistance factors associated with particular chromosomes do not condition hypersensitive death of attacked cells. Detailed histological analysis will be necessary to confirm this conclusion, however, since single cell hypersensitivity cannot be detected macroscopically. Previous studies of H. chilense and H. vulgare have revealed that certain resistance mechanisms operate effectively against both appropriate and inappropriate formae speciales of the powdery mildew fungus (TOSA and SHISHIYAMA1984, RUBIALESand CARVER 2000). It may be that more detailed investigation of the addition lines used here will reveal the existence of such mechanisms that are race non-specific with respect to Bgt attack, and that are masked by factors governing the race-specific resistances detected in the current study. Interestingly, the resistance of tritordeum amphiploids was shown to be expressed via quantitative reduction in colony number and, as in the present study, this resistance was not associated with necrosis (RUBIALES et al. 1993a). Similarly, a H . chilense x rye amphiploid expressed quantitative resistance to the rye powdery mildew fungus without visible necrosis (RUBIALESet al.

Table 1. Reaction to Blumeria graminis Jsp. tritici in seedlings of Triticum aestivum cv. Chinese Spring (CS), Hordeum vulgare cu. Betzes and CSIH. vulgare single chromosome addition lines Line

T. aestivum cv. Chinese Spring (CS) H. vulgare cv. Betzes CS/2H' disomic CS/3H" disomic CS/4HVdisomic CS/5Hv disomic CS/6H" disomic CS/7H" disomic

% leaf area affected

Isolate H12'

Population from Cordoba'

Pdleaf 20 a3 Ob 3b 16 a 17 a 16 a 2b 20 a

zndleaf 33 a oc 9 bc 15 b 17 b 32 a l c 12 b

Isolate JIW22 2nd leaf 21 a Od 19 a 16 ab 6c 12 b 14 ab 15 ab

' Percentage of leaf area covered by mildew colonies assessed using the key of LARGEand DOLING(1962).

* number

of colonies/cm2. Letters in common indicate that differences are not statistically significant at P = 0.05, Duncan test.

sthleaf 12 a oc 13 a 8 ab l c 4 bc 7b 6b

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Hereditas 134 (2001)

Table 2. Reaction to Blumeria graminis Jsp. tritici in seedlings of T. aestivum cv. Chinese Spring (CS), H. chilense accession HI and CSI H. chilense single chromosome addition Line T. aestivum Chinese Spring (CS) H. chilense H1 CS/4Hchdisomic CS/5HChdisomic CS/6Hchdisomic CS/7Hchdisomic CS/l HchSmonosomic 1HCh+ monotelosomicl HChS CS/lHChSditelosomic CS/2Hchaditelosomic CS/5HchLditelosomic CS/6HChSditelosomic CS/7Hchaditelosomic CS/7HChpditelosomic I

Isolate H12' 2"* leaf

2"* leaf

Isolate JIW2* sthleaf

19 a3 0. f 13 abc 15 abc 16 ab 8 cde 11 bcd 6 def 14 abc 16 ab 12 bcd 4 ef 20 a

21 abc Of 23 ab 9 de 9 de 9 de 20 bc n.d. 28 a 4f 12 cde 17 bcd 14 bcde

13 bc Oe 16 ab 2 de 3 de 3 de 12 bc n.d. 22 a l e 5 cde 9 bcd I cde

Percentage of leaf area covered by mildew colonies assessed using the key of LARGEand DOLING (1962). of colonies/cm2. Letters in common indicate that differences are not statistically significant at P = 0.05, Duncan test.

* number

1993b), and quantitative resistance has also been reported in 'Chinese Spring'- A . longissima addition and substitution lines (ZELLERand HEUN 1985). These findings contrast to the situation seen in wheat x rye hybrids. In triticale and in addition, substitution and translocation lines incorporating rye chromosomal material into wheat, and where the genes Pm7 and Pm8 are transferred from rye into wheat, resistance to Bgt is clearly associated with reduced 'infection type' and visibly increased necrosis of attacked tissue (LINDE-LAURSEN 1977; HEUNand FRIEBE1990). The absence of necrosis associated with Bgt attack in wheat hybrids with either H . chilense or H. vulgare probably indicates that the resistance factors govern resistance mechanisms acting at an early stage of the infection process. Indeed, recent histological studies show that papilla-based resistance is a key feature of H. chilense resistance to B. graminis that prevents infection, and acts before initiation of cell death leading to necrosis (RUBIALES and CARVER2000). On the contrary, resistance contributed by rye in hybrids with wheat probably acts principally after penetration of the plant cell wall, and as for race-specific resistance conditioned by many Pm genes, incompatibility leads to plant cell death and the development of visible necrosis. The finding that factors conferring resistance to Bgt are present on several H. vulgare and H. chilense chromosomes, support conclusions drawn from histological investigations that resistance in H. chilense is under complex genetic control and due to several distinct mechanisms (TOSA and SHISHIYAMA 1984, RUBIALESand CARVER 2000). Exploiting such broadly-based resistance is clearly desirable in breed-

ing for resistance as the pathogen is unlikely to be able to overcome such genetic and physiological complexity by simple mutation to virulence, and the resistance is likely to prove durable. Obviously, it would be difficult to transfer multiple genetic factors present in different chromosomes to an agronomically valuable wheat cultivar, but transferring only a single resistance factor could prove valuable if that factor governs a novel resistance mechanism. Transference of traits from barley to wheat is hampered by the difficulties in hybridization of barley with wheat (FEDAK 1992). However, many hybrids and valuable cytogenetic stocks such as addition, translocation and substitution lines of barley in wheat have been produced (KOBAet al. 1997, ISLAMet al. 1981; SHEPERDand ISLAM 1987). Deletions and translocations have been induced using a gametocidal chromosome derived from Aegilops cylindrica (SHI and ENDO1999) and use of a p h l mutant (ISLAMand SHEPHERD 1992; MURAIet al. 1997), to allow homeologous chromosome pairing and recombination, has opened the possibility of using wheat-barley addition lines to transfer desirable traits from barley to wheat. In contrast with the difficulties in producing wheat x H . vulgare hybrids, fertile amphiploids were easily produced using H. chilense with both durum and bread wheat (MAR T ~and N CUBERO1981; MARTI"et al. 1998; MA R T ~ et N al. 2000). Such hybrids can be used as bridges to transfer genes to wheat. Addition, translocation and substitution lines of H. chilense in wheat have been obtained (MILLERet al. 1982; FERNANDEZ and JOUVE1988; MA R T ~ et N al. 1998; M A R T ~et N al. 2000). Also amphiploids of H. chilense with both durum and bread wheat carrying

Hereditas 134 (2001)

p h l mutants have been obtained ( M A R T ~ Net al. 2000) and spontaneous terminal, interstitial or centromeric translocations are frequently found in progeny of hybrid bread wheat x hexaploid tritordeum (AABBDHch) ( M A R T ~ N et al. 2000). These offer an alternative means for transferring traits from H. chilense to wheat. Specific markers for both H . chilense (HERNANDEZ et al. 1999) and H . vulgare (MURAIet al. 1997) chromosomes have been developed, making marker selected breeding a possibility in wheat.

ACKNOWLEDGEMENTS The authors acknowledge the Spanish C.I.C.Y.T. Proj. AGF99-1036 for the financial support and Ana Moral and Peter Roberts for technical assistance.

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