Partial resistance to Wheat dwarf virus in winter wheat cultivars

Authors


E-mail: silhavy@abc.hu

Abstract

Four Hungarian winter wheat cultivars were investigated for their susceptibility to the geminivirus Wheat dwarf virus (WDV). Previously, two cultivars (Mv Regiment and Mv Emese) were assessed by breeders to exhibit virus symptoms in the field, whereas Mv Dalma and Mv Vekni showed few symptoms. Two inoculation techniques for WDV, vector transmission with the leafhopper Psammotettix alienus and agroinoculation, were used. Leafhopper transmission was more efficient than agroinoculation. However, irrespective of the technique used, no Mv Dalma or Mv Vekni plants showed clear WDV symptoms. In contrast, 3/30 Mv Emese and 4/36 Mv Regiment plants showed dwarfing and chlorosis after agroinoculation and 13/17 and 14/15 plants, respectively, had clear WDV symptoms after vector transmission. WDV-specific PCR showed that Mv Vekni and Mv Dalma plants could be infected, especially following vector transmission (approximately 50% infection), but at significantly lower frequency than Mv Emese or Mv Regiment plants (100% infection). Furthermore, real-time PCR showed that WDV DNA accumulated to much lower levels in infected Mv Vekni and Mv Dalma plants than in infected Mv Regiment and Mv Emese plants. The data strongly suggest that Mv Vekni and Mv Dalma are partially resistant to WDV infection. As WDV resistance has not previously been identified in wheat, and because WDV can cause significant yield losses, the resistance of Mv Vekni or Mv Dalma will provide a valuable breeding resource.

Introduction

Wheat dwarf virus (WDV, family Geminiviridae; genus Mastrevirus) is an important pathogen of wheat and barley. WDV is a single stranded (ss) DNA virus which encodes four proteins: the coat protein (CP), the movement protein (MP) and two replication associated proteins (Rep and RepA) (Schalk et al., 1989). Two WDV strains have been described, barley and wheat strains. These two strains can infect the other host only at very low efficiency although they share approximately 84% nucleotide identity (Koklu et al., 2007; Ramsell et al., 2009). The wheat strain isolates share >98% nucleotide sequence identity (Ramsell et al., 2008).

WDV is transmitted by the leafhopper Psammotettix alienus in a circulative, non-propagative manner (Vacke, 1961). WDV infections have been detected in many European countries including Hungary (Bisztray et al., 1989), the Czech Republic (Vacke, 1961), Sweden (Lindsten & Lindsten, 1999), Finland (Lemmetty & Huusela-Veistola, 2005), Spain (Achon & Serrano, 2006), France (Bendahmane et al., 1995) and Germany (Huth, 2000). In addition, WDV has recently been reported from Turkey (Koklu et al., 2007), Bulgaria (Tóbiás et al., 2009), Africa (Najar et al., 2000; Kapooria & Ndunguru, 2004) and China (Wu et al., 2008). WDV has a wide host range infecting most of the economically important cereals including barley, wheat, oats and rye as well as many wild grasses (Vacke, 1972). Symptoms of WDV infection on wheat include chlorosis, streaking and dwarfing.

Although WDV is not regarded as a major wheat pathogen, local WDV epidemics can cause severe yield losses (Lindblad & Waern, 2002; Sirlováet al., 2005). During the last two decades WDV has become the most important viral pathogen of winter wheat in Hungary (Pribék et al., 2006) and predictions are that climate change will increase the incidence of vector-transmitted viruses worldwide (Habekußet al., 2009). WDV protection currently relies on chemical control of the vector or on agrotechnical measures but it is likely that the former methods will become less effective as the use of pesticides is limited by European Union directives. Cultivation of WDV resistant wheat cultivars would be a cheaper and environmentally friendly alternative but only minor quantitative differences in the degree of WDV infection have been reported (Habekußet al., 2009). Indeed, in a recent study in which 25 registered winter wheat cultivars were tested for WDV resistance in small plot trials, even the least susceptible cultivars showed strong symptoms and severe (87–93%) grain yield loss (Sirlováet al., 2005).

Recently, wheat breeders observed that two Hungarian cultivars (Mv Dalma and Mv Vekni) developed only weak or no viral symptoms even in years when other cultivars showed severe WDV-like symptoms (G. Vida, Martonvásár, Hungary, unpublished results). In order to determine whether Mv Dalma and Mv Vekni could serve as a source of WDV resistance in wheat breeding programs, their susceptibility to the Czech isolate of WDV (WDV-[CZ]) was tested using two inoculation procedures: agroinoculation (Hayes et al., 1988; Boulton, 1995, 2008) and vector transmission. The susceptibility of Mv Dalma and Mv Vekni was compared with that of two Hungarian cultivars (Mv Emese and Mv Regiment) that usually show strong WDV-like symptoms in breeding plots.

Materials and methods

Plant materials

Seeds of Triticum aestivum (hexaploid wheat) lines were sown individually in 500 mL pots containing Levington M2 compost and grown in a containment B glasshouse at 25°C (day) and 20°C (night) with an 18 h photoperiod until used for inoculation. Cultivars Mv Dalma, Mv Emese, Mv Regiment and Mv Vekni (all derived from the Martonvásár, Hungary, Wheat Breeding Program) were used for agroinoculation and insect transmission tests; additionally cvs Chinese Spring, Piko and Trintella were agroinoculated.

Mv Dalma is an extra-early ripening high yielding cultivar with good bread making characteristics. It carries a chromosome segment from rye (1AL:1RS translocation) and is present in the pedigree of several Hungarian breeding lines. Mv Vekni is a high-yielding, mid-late cultivar containing several resistance genes from Aegilops ventricosa (VPM-1) (Sr38, Lr37 and Yr17 stem, leaf and yellow rust resistance genes, respectively). Mv Emese is an extra-early cultivar which is resistant to abiotic stresses and carries the Barley yellow dwarf virus resistance gene Bdv1 (Vida et al., 2009). Mv Regiment is a wheat cultivar with outstandingly high yield potential, but poor bread making quality. Cultivar maintenance is impeded by its susceptibility to WDV.

WDV agroinoculation assays

Plants were agroinoculated with a tandem dimeric copy of the genome of the Czech isolate of WDV (WDV-[CZ], Woolston et al., 1988) present in Agrobacterium tumefaciens strain C58C1 (pGV3850, Zambryski et al., 1983) as described by Boulton et al. (1989) for Maize streak virus (MSV) but with the modifications described for wheat in Boulton (2008). Plants (approximately 40 for each cultivar, Table 1) were inoculated at the three leaf stage (Zadoks stage 13, Zadoks et al., 1974) and examined for symptoms visually each week for 4 weeks after inoculation. All plants with symptoms, and some symptomless plants, were sampled 25–30 days after inoculation and the samples stored at −80°C until used for PCR analysis.

Table 1.   Number of plants showing symptoms and infection rates (detected by PCR analysis) following agroinoculation of wheat (Triticum aestivum) cultivars with the Czech isolate of Wheat dwarf virus (WDV-[CZ])
Plant cultivarNumber of plants with symptomsaPhenotype distribution (H/M/S)bPhenotypes of PCR tested plantsc (H/M/S)PCR positives at 20 cyclesd (H/M/S)
  1. aNumber of plants with strong or mild symptoms/number of inoculated plants. Symptoms were evaluated 4 weeks after agroinoculation. Phenotypic differences between Mv Dalma-Mv Emese, Mv Dalma-Mv Regiment, and Mv Vekni-Mv Regiment are significant (Fisher’s exact test, < 0·05).

  2. bH: healthy symptomless plants, M: plants with mild symptoms, S: plants with strong symptoms.

  3. cDNA from 16 plants of each of the four Hungarian cultivars were used for PCR analysis.

  4. dPlants were regarded as WDV infected if products were produced following 20 cycles of PCR. Numbers in parenthesis denote the phenotypes assigned to these plants (see footnote b) The agroinoculated non-Hungarian cultivars were not subjected to PCR (NT: not tested).

Mv Dalma2/43, ∼5%41/2/014/2/00/16
Mv Vekni2/36, ∼5·5%34/2/014/2/01/16 (0/1/0)
Mv Emese7/30, ∼23%23/4/39/4/35/16 (2/0/3)
Mv Regiment9/36, ∼25%27/5/47/5/44/16 (0/2/2)
Piko6/40, ∼15%34/3/3 NT
Trintella14/35,∼40%21/4/10 NT
Chinese Spring4/44, ∼31%30/4/10 NT

WDV vector transmission tests

Psammotettix alienus vectors were provided by Professor T Lundsgaard, Dept. of Plant Biology, Agricultural University, Denmark in 2003 and have since been maintained on Avena sativa cv. Forward at 26°C (day) and 20°C (night) with a 16 h photoperiod. Prior to the current work, the leafhoppers were transferred to wheat cv. Trintella and established over two generations. The WDV transmission tests were performed within Perspex cages (25 × 45 × 55 cm), vented at both ends with 50 μm mesh. Approximately six young plants of each Hungarian wheat cultivar were used, with cultivars caged separately, and the experiment was repeated twice such that 15–19 plants were tested for each cultivar. Twenty four hours before adding the test plants to cages, a plant showing symptoms (cv. Trintella) produced by agroinoculation with the WDV construct, was placed into each cage with 40 P. alienus adults to enable the leafhoppers to acquire WDV. For the first experiment, seedlings were at Zadoks stage 13, whilst plants with tillers emerging (stage 21–22) were used for experiments 2 and 3. After 14 days of transmission access, all P. alienus were removed from the experimental cages and the plants were sprayed with a synthetic pyrethroid insecticide. Plants were then moved to a containment glasshouse and observed for the appearance of WDV symptoms over the following 4 weeks, after which all were sampled for PCR analysis.

Virus acquisition and transmission tests were all undertaken within the high containment area of the John Innes Centre’s Entomology facility, under DEFRA FERA licences PHL 185E/5884 and 185E/6067. WDV clones were held and agroinoculation carried out according to DEFRA licence PHL185E/6182(10/2009).

Statistical analysis

Two-tailed Fisher’s exact test (Fisher, 1922) was used for statistical analysis of symptom and PCR data. P values < 0·05 and < 0·001 reflect significant and highly significant differences, respectively.

Plant DNA isolation, PCR detection and real-time PCR analysis

Total DNA was extracted from WDV-agroinoculated or insect-inoculated wheat plants with GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer’s instructions. DNA (approx 10 ng) was used as a template for each PCR. To test the quality of templates, control PCRs were carried out with the primer pair 25S_for/25S_rev (5′-TGG GTT TAG ACC GTC GTG AGA CAG GTT-3′ and 5′-AGC AAG GCC ACT CTG CCA CTT ACA A-3′, respectively) designed to amplify the wheat 25S rDNA gene (GenBank accession X07841.1). The PCR programme for 25S rDNA gene amplification was: 95°C for 2 min, 20 or 30 cycles of 95°C for 20 s, 62°C for 15 s, 70°C for 7 s, and a final extension for 5 min at 70°C. The samples were tested for WDV infection using the PCR primer pair WDV_Rep_for/WDV_Rep_rev (5′-CGC CTT GGA CTC TCT TCG CAC-3′ and 5′-ATA TAC TAG TGA CGG ATA GAC CAT TCA AAC G-3′, respectively) designed using the WDV-[Enkoping1] Rep gene sequence (GenBank accession AJ311031.1). The PCR programme for WDV amplification was: 95°C for 2 min, 18, 20 or 30 cycles of 95°C for 20 s, 55°C for 15 s, 70°C for 7 s, and a final extension step for 5 min at 70°C. For agroinoculated plant samples, the number of cycles suitable for detection of replicated WDV DNA (rather than WDV sequences present in any remaining Agrobacterium inoculum) was determined using primers that would amplify the kanamycin (kan) gene present on the binary vector. The primers were: kan5′ (5′-ATG ATT GAA CAA GAT GGA TTG CAC GC-3′) and kan 3′ (5′-AGA AGA ACT CGT CAA GAA GGC GAT AG-3′), with a PCR programme of 95°C for 2 min, and then 20 or 30 cycles of 95°C for 20 s, 65°C for 15 s, 70°C for 15 s, and a final extension step for 5 min at 70°C. KOD Hot Start DNA Polymerase (Novagen) was used for all amplifications. PCR products were visualized by electrophoresis through a 2% agarose 1 ×  TBE gel and staining with ethidium bromide.

Real-time PCR analysis was performed using the RotorGene RG-3000 PCR machine and the Power SYBR Green PCR Master Mix (Applied Biosystems). The results were analysed with Rotorgene software (Corbett Research). WDV_RepQfor and WDV_RepQrev (5′-AAT TCC TCA GCA TGG TTT GC-3′ and 5′-TGT AAG TGG CAA CTG GGT CA-3′, respectively) primers were used for WDV amplification. Fluorescence intensities of WDV-specific products were normalized to those of 25S ribosomal DNA-specific products generated with the 25SQfor/25SQrev (5′-GGG TTT AGA CCG TCG TGA GA-3′ and 5′-ATT GTG TGA ATC AAC GGT TCC-3′, respectively) primer pair. Real-time PCR values can be quantified accurately when the copy number of the normalization control gene is comparable with the copy number of the test gene. However, because WDV copy numbers can vary considerably between infected lines, the real-time PCR assay should be regarded only as semi-quantitative.

Results and discussion

Agroinoculation-based WDV resistance tests

The WDV susceptibility of the four Hungarian winter wheat cultivars and control wheat plants was first tested by agroinoculation. All plants survived inoculation and, based on symptoms, the plants were grouped into three categories; healthy (H: no symptoms), mild (M: mild chlorosis or occasional streaks without dwarfing) or with strong symptoms (S: chlorosis or streaking and dwarfing). Agroinoculated Mv Dalma and Mv Vekni plants rarely developed symptoms; 41 of 43 Mv Dalma plants and 34 of 36 Mv Vekni plants were designated as healthy and the remaining two plants of each cultivar were placed in category M. No category S Mv Dalma or Mv Vekni plants were obtained. In contrast, 7 of 30 Mv Emese, and 9 of 36 Mv Regiment plants were placed in the M or S categories and for these cultivars, three (Mv Emese) or four (Mv Regiment) agroinoculated plants exhibited dwarfing (category S; Table 1). The susceptibility of the Hungarian cultivars was compared to wheat cvs Piko, Trintella and Chinese Spring, previously shown to be susceptible to WDV agroinoculation (M. Smith and M.I. Boulton, John Innes Centre, Norwich, UK, unpublished data). These control plants developed WDV symptoms at a similar frequency to Mv Emese and Mv Regiment (Table 1) although dwarfing, chlorosis and streaking were more severe for Trintella and Chinese Spring, and four of the Chinese Spring plants died within 30 days of inoculation. For all cultivars it was not clear whether the chlorosis or streaking identified in plants of category M were viral symptoms or were a result of damage following agroinoculation.

To correlate symptoms with WDV infection, the DNA from selected plants was subjected to 20 and 30 cycles of PCR with WDV-specific primers. Sixteen plants of each of the Hungarian wheat cultivars were analysed, including all of the plants designated as S or M, and the remainder were randomly selected healthy ones. The control wheat cultivars were not studied further. After 30 cycles of PCR no products were obtained from any of the Mv Dalma plants and only two Mv Vekni plants (one categorized as M and one as H) were PCR positive. In contrast, eight of the Mv Emese and eight Mv Regiment plants gave WDV products at 30 cycles (data not shown). However, several of these samples (four Mv Emese, five Mv Regiment and one Mv Vekni) also gave products after 30 cycles of PCR using kan primers specific for the binary vector, indicating that Agrobacterium inoculum was present in, or on, the plants and that approximately half of the WDV positive results could be incorrect. In contrast, after 20 cycles of PCR, vector-specific (kan) products were not amplified from any plant (data not shown) suggesting that WDV fragments amplified at 20 cycles should derive only from replicating virus. Thus, 20 cycles of WDV-specific PCR were used to confirm WDV infection, thereby preventing the identification of false positives caused by amplification of inoculum-derived WDV sequence. Importantly, after 20 cycles, no WDV products were amplified from the Mv Dalma plants and only one Mv Vekni plant (categorized as M) was PCR positive. In contrast, five Mv Emese and four Mv Regiment plants were PCR positive (Table 1). The three Mv Emese plants categorized as S were WDV PCR positive but the four plants assessed as M did not yield products. However, two plants assessed to be healthy produced WDV products. For Mv Regiment two of the four category S plants produced WDV-specific products. Of the five category M plants tested, two were WDV positive. None of the healthy plants gave products (Table 1). These data show that it is important to confirm infection phenotypes using a virus-specific diagnostic test. Taken together, the PCR analysis showed that Mv Dalma plants were not infected by agroinoculation and Mv Vekni plants were infected only at very low frequency (1/16 plants). In contrast, Mv Emese and Mv Regiment became infected at approximately the frequency obtained for other WDV susceptible wheat lines (M. Smith and M. Boulton, unpublished data).

The results of the agroinoculation assays suggest that Mv Dalma and Mv Vekni are less susceptible to WDV than Mv Emese and Mv Regiment plants. However, it cannot be discounted that the efficiency of Agrobacterium T-DNA transfer varies between plant cultivars, therefore the WDV susceptibility of all four Hungarian wheat cultivars were also tested by natural (leafhopper vector) transmission.

Leafhopper transmission-based WDV resistance tests

To determine whether the WDV resistance of Mv Dalma and Mv Vekni identified using agroinoculation was maintained following leafhopper transmission, plants were exposed to viruliferous P. alienus vectors. The infection results (number and phenotypes) for each cultivar at 4 weeks after inoculation were consistent for all three replicated experiments and therefore the data were combined. Furthermore, vector survival was similar for all cultivars in all experiments and visual examination of leafhopper behaviour did not reveal any plant preference. As Table 2 shows, all 15 Mv Regiment and 16 of the 17 vector treated Mv Emese plants developed symptoms (symptoms were categorized as previously: H- healthy, M-mild and S-with strong symptoms). Most of these plants showed strong symptoms (14 Mv Regiment and 13 Mv Emese plants were assessed as category S) and only one plant (Mv Emese) was classified as H. In contrast, none of 19 Mv Dalma or 19 Mv Vekni plants showed symptoms (Table 2, Fig. 1) suggesting that Mv Dalma and Mv Vekni are resistant to WDV following transmission by P. alienus. However, the symptomless phenotype of the Mv Dalma and Mv Vekni plants could be due to complete resistance (the plants cannot be infected) or because these plants are infected without showing symptoms.

Table 2.   Number of plants showing symptoms and infection rates (detected by PCR analysis) following leafhopper transmission of the Czech isolate of Wheat dwarf virus (WDV-[CZ]) to wheat (Triticum aestivum) cultivars
Plant cultivarNumber of plants with symptomsaPhenotype distribution (H/M/S)bPCR positives at 30 cyclescPCR positives at 20 cyclesc
  1. aNumber of plants with symptoms/number of plants tested. Symptoms were evaluated 4 weeks after agroinoculation. Differences between Mv Dalma-Mv Vekni and Mv Emese-Mv Regiment are highly significant (Fisher’s exact test, < 0·001).

  2. bH: healthy symptomless plants, M: plants with mild symptoms, S: plants with strong symptoms.

  3. c30, or 20, cycles of PCR were carried out to detect low, or high, virus levels, respectively. Number of plant samples that produced WDV-specific products/number of plants tested. At 30 cycles, differences between Mv Dalma-Mv Vekni and Mv Emese-Mv Regiment are significant (Fisher’s exact test, < 0·01), while at 20 cycles, differences between Mv Dalma-Mv Vekni and Mv Emese-Mv Regiment are highly significant (Fisher’s exact test, < 0·001).

Mv Dalma0/1919/0/010/195/19
Mv Vekni0/1919/0/011/193/19
Mv Emese16/171/3/1317/1716/17
Mv Regiment15/150/1/1415/1514/15
Figure 1.

 Typical phenotypes of Hungarian wheat (Triticum aestivum) plants following vector transmission of the Czech isolate of Wheat dwarf virus (WDV-[CZ]). Psammotettix alienus leafhoppers transferred from WDV-[CZ] infected wheat plants were used to transmit virus to Hungarian winter wheat cultivars and the symptoms were assessed 4 weeks later. Typical Mv Emese and Mv Regiment plants show stunting (upper panel). Chlorosis, mottling and streaking were clearly visible on the leaves of all Mv Regiment plants (bottom panel), but less clear on most Mv Emese leaves. Mv Dalma plants remained symptomless.

To determine whether Mv Dalma and Mv Vekni plants were infected without symptoms, and if so, to compare the amount of WDV present in these plants and in the susceptible Mv Regiment and Mv Emese cultivars, DNA from all vector treated plants of all four Hungarian cultivars were subjected to WDV PCR assays. Two WDV-specific PCRs were carried out for each vector treated plant using 30 and 20 cycles to detect very low, or moderate to high, virus titres, respectively. WDV could be detected in all vector treated Mv Regiment and Mv Emese plants using 30 cycles of PCR, and almost all accumulated WDV to high titres (14/15 Mv Regiment and 16/17 Mv Emese gave products after 20 cycles of PCR; Table 2, Fig. 2). In contrast, WDV could be detected in only 5/19 Mv Dalma and 3/19 Mv Vekni plants after 20 cycles, and after 30 cycles only around half gave WDV-specific PCR products (Table 2, Fig. 2). It is likely that the data obtained using 30 cycles of PCR is correct for most plants subjected to leafhopper transmission because although WDV can be excreted in the honeydew of leafhoppers, sampled leaves had emerged after leafhoppers were removed from the plants, thereby limiting the likelihood of contamination. Statistical analysis of these data showed significant difference (< 0·01) between the infection rates of Mv Dalma and Mv Vekni compared to Mv Emese and Mv Regiment. These data suggest that Mv Dalma and Mv Vekni cultivars are resistant (or tolerant) to WDV.

Figure 2.

 PCR analysis to detect WDV infection in the Hungarian cultivars of winter wheat (Triticum aestivum) following leafhopper transmission of the Czech isolate of Wheat dwarf virus (WDV-[CZ]). Three replicate transmission tests were carried out, the PCR results of the last infection are shown. 25s rDNA PCR was used as a control, and then 30 or 20 cycles of WDV test PCRs were carried out. H: healthy symptomless plants, M: plants with mild symptoms and S: plants with strong symptoms. Plants D1–3, E1–3, R1–3 and V1–3 were used for the real-time PCR assays shown in Table 3.

To compare the levels of WDV in selected vector infected plants, real-time PCR assays were conducted. Plants were selected from the third transmission experiment where WDV-specific products were detected in some Mv Vekni and Mv Dalma plants after 20 cycles of PCR, and thus these were likely to represent the plants containing the highest titres of WDV (Fig. 2). Three WDV PCR positive plants of each cultivar (D1–D3 for Mv Dalma, E1–E3 for Mv Emese, V1–V3 for Mv Vekni and R1–R3 for Mv Regiment) were selected. Plants D1, D2, D3 and V1 were chosen because they produced WDV products after 20 cycles of PCR. In addition, two Mv Vekni (V2 and V3) samples that were PCR positive only at 30 cycles were selected. Strong WDV bands had been produced at 20 cycles from most Mv Regiment and Mv Emese plants. Three Mv Regiment plants (R1–3), and two Mv Emese plants (E1 and E3) which were PCR positive at 20 cycles, and a further sample (E2) that was PCR positive only at 30 cycles were chosen for real-time PCR studies (Fig. 2). Thus, E2, the only Mv Emese plant that was PCR positive only at 30 cycles, was likely to represent the Mv Emese plant containing the lowest titre of WDV. Importantly, real-time PCR analysis (Table 3) confirmed that WDV accumulated to much lower levels in the infected Mv Dalma or Mv Vekni plants than in the Mv Regiment or Mv Emese plants. Mv Dalma D1 contained the highest WDV level among the Mv Dalma and Mv Vekni plants, and therefore the relative WDV levels of other plants were compared to those of plant D1 (Table 3). WDV levels were between 8·4 and 71 times higher in Mv Regiment, and 33 and 44 times higher in the E1 and E3 Mv Emese plants, than in the D1 plants. In line with the result of previous PCR tests, E2 was an exceptional Mv Emese plant (Fig. 2), which contained very low levels of WDV. Very low amounts of WDV were present in all three Mv Vekni (V1–V3) and the remaining two Mv Dalma (D2 and D3) samples (Table 3). Indeed, the WDV levels in these five plants were in the range of 100–10 000 times lower than those identified in five of the six Mv Regiment and Mv Emese samples tested. Considering that, based on non-quantitative PCR (Fig. 2, Table 2), the V1–3 and D1–3 plants of Mv Vekni and Mv Dalma contained the highest WDV titres, whereas the R1–3 and E1 and E3 plants contained WDV levels representative of the majority of Mv Regiment and Mv Emese plants (Fig. 2), it is clear that infected Mv Dalma and Mv Vekni plants generally accumulate significantly lower levels of WDV than infected Mv Emese or Mv Regiment plants.

Table 3.   The relative amount of WDV DNA in selected plant samples following leafhopper transmission of the Czech isolate of Wheat dwarf virus (WDV-[CZ]) to wheat (Triticum aestivum) cultivars
Plant sampleaRelative WDV valueb
  1. aThree plants of each genotype were analysed.

  2. bWDV amount was assessed by real-time PCR. WDV values were obtained following normalization of the real-time PCR values to the internal control (25S rDNA gene) values.

  3. Relative WDV values were calculated compared to the highest WDV value obtained in Mv Dalma (plant 1) which was designated as 100%.

Mv Dalma 
 1100
 27
 33
Mv Emerse 
 14400
 21
 33300
Mv Regiment 
 11200
 27100
 3840
Mv Vekni 
 13
 21
 32

Taken together, the results of the symptom assessment and PCR studies confirm that Mv Dalma and Mv Vekni plants are partially resistant to WDV in glasshouse conditions. Fewer Mv Dalma and Mv Vekni plants become infected even under strong virus inoculation pressure and despite the use of plants at growth stages previously shown to be optimal for WDV infection (Lindblad & Sigvald, 2004). Moreover, when Mv Dalma and Mv Vekni plants are infected, virus accumulates only to a very low titre compared with Mv Emese and Mv Regiment and infections are symptomless.

The results of the WDV agroinoculation and vector transmission assays were in agreement; for both procedures fewer Mv Vekni and Mv Dalma plants were infected compared to Mv Regiment and Mv Emese. Agroinoculation could be an attractive way to screen plants for WDV susceptibility since it can be carried out in the glasshouse without the need for large scale insectary facilities and the unpredictability of sporadic disease outbreaks in the field. It is particularly appropriate for studying resistance of wheat to WDV since European wheat isolates show a high level of sequence identity (Ramsell et al., 2008; Kundu et al., 2009) and therefore the use of cloned WDV is unlikely to limit its validity. However, before the WDV agroinoculation technique can be used as a routine screening procedure its efficiency must be improved because only 20–30% of the plants of the susceptible control lines (Chinese Spring and Trintella) showed clear infection with symptoms (category S) (Table 1). It is likely that by using alternative inoculation methods (Redinbaugh et al., 2001; Boulton, 2008) and optimized Agrobacterium strains the efficiency of WDV agroinoculation could be improved.

Agroinoculation delivers viral DNA rather than requiring transmission of encapsidated virus, and therefore if used in conjunction with insect transmission studies, allows dissection of the resistance mechanism. For example, plants that can be infected by agroinoculation, but not by leafhoppers, may possess resistance against unencapsidation of the virus or against leafhopper feeding. Thus, when both inoculation techniques are used it is possible to differentiate plant lines with resistance to the vector, rather than to virus replication or virus movement. This information allows knowledge-based breeding towards the pyramiding of multiple resistance genes, an approach that should result in a durable WDV resistance.

The data presented in this study, showing that under laboratory and glasshouse conditions Mv Vekni and Mv Dalma possess resistance to WDV, correlate well with previous field observations that these two cultivars developed limited WDV symptoms. These data strongly suggest that these cultivars represent the first identified sources of WDV resistance in wheat and are likely to provide an important resource for future wheat breeding programs. Moreover, as Mv Dalma and Mv Vekni have very different pedigrees (G. Vida, Martonvásár, Hungary, personal communication) they may have different resistance determinants, potentially allowing their resistance factors to be combined. Interestingly, WDV resistance breeding programs have not been conducted in Hungary. However, as WDV has emerged as the most important viral wheat pathogen in Hungary (Pribék et al., 2006), it is likely that disease outbreaks during pedigree selection of Mv Dalma and Mv Vekni cultivars could lead to unintentional WDV resistance selection. Thus WDV resistance determinants could also be present in other Hungarian breeding lines and cultivars.

In the current study Mv Vekni and Mv Dalma were resistant to agroinoculation with WDV DNA, and there were no differences between insect survival and behaviour on these cultivars compared with Mv Regiment and Mv Emese, suggesting that in the resistant cultivars virus replication or movement is targeted, rather than insect feeding. This contrasts with the WDV tolerance identified in the barley cultivar Post, where virus concentration was not reduced although infected plants showed less dwarfing and had increased yields (Habekußet al., 2009). Further studies are required to clarify the basis of the WDV resistance traits. Mapping populations are being generated by crossing Mv Vekni with WDV susceptible lines (G. Vida, Martonvásár, Hungary unpublished results) to study the inheritance of WDV resistance and to map the resistance determinants.

Acknowledgements

This research was supported in Hungary by grants from the OTKA (K60102) and from the National Office for Research and Technology (GAK ‘TRIPATOL’). D. Silhavy was supported by the Bolyai János Scholarship. A. Hangyáné Benkovics is a graduate student of Corvinus University of Budapest (‘Program of Viticulture’ at the Doctoral School of Horticulture). M. Boulton and I. Bedford were supported by grant-in-aid from the Biotechnology and Biological Sciences Research Council (BBSRC). We thank B. Hangya and L. Hiripi for advice on statistical analysis and real-time PCR, respectively. Virus vector acquisition and transmission tests were undertaken within the high containment area of the John Innes Centre’s Entomology facility, under DEFRA FERA licences PHL 185E/5884 and 185E/6067. WDV clones were held, and agroinoculation carried out according to DEFRA licence PHL185E/6182(10/2009).

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