FURTHER INSIGHTS INTO MALUS FUSCA FIRE BLIGHT RESISTANCE

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1 Journal of Plant Pathology (2017), 99 (Special issue), Edizioni ETS Pisa, FURTHER INSIGHTS INTO MALUS FUSCA FIRE BLIGHT RESISTANCE O.F. Emeriewen 1, K. Richter 2, M.-V. Hanke 1, M. Malnoy 3 and A. Peil 1 1 Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Institute for Breeding Research on Fruit Crops, Pillnitzer Platz 3a, Dresden, Germany 2 Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Institute for Resistance Research and Stress Tolerance, Erwin-Baur-Str. 27, 06484, Quedlinburg, Germany 3 IASMA- Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach, 1, San Michele all Adige, Trentino, Italy SUMMARY The apple wild species accession Malus fusca MAL0045 had been found to be resistant to fire blight in artificial inoculation trials with Erwinia amylovora strain Ea222_ JKI. Consequently, using a population derived by crossing MAL0045 with Idared, the corresponding fire blight resistance locus of M. fusca (Mfu10) was mapped on chromosome 10 explaining up to 66% of phenotypic variation at a logarithm of the odd (LOD) ratio of 31.0 with the strain Ea222_JKI. Moreover, the very aggressive strain Ea3049 only minimally affected MAL0045 but significantly affected the population although could not overcome the resistance of Mfu10. To further understand the resistance mechanism of M. fusca, we evaluated resistance of the original mapping population, comprised of 134 individuals, to E. amylovora strain ZYRKD3-1 which causes the breakdown of the resistance of M. robusta 5 (Mr5) and the Mr5 fire blight resistance QTL on LG3. Our results showed that the major QTL of M. fusca on LG10 could still be detected at the same exact position with a higher effect on fire blight resistance, indicating that ZYRKD3-1 has no effect on Mfu10, although the mean percent lesion length of the population was almost doubled compared with Ea222_JKI. Keywords: Erwinia amylovora, Mfu10, QTL mapping, phenotyping, ZYRKD3-1. Corresponding author: O.F. Emeriewen ofere.emeriewen@julius-kuehn.de INTRODUCTION Fire blight is a disease that poses serious threat to apple (Malus domestica) production worldwide. Most commercial cultivars are susceptible to this disease caused by the bacterial pathogen Erwinia amylovora (Burrill) Winslow et al. The pathogen invades blossoms, fruits, leaves, stems, woody branches and rootstocks crown, causing blight to susceptible hosts (Peil et al., 2009). Economic losses as a result of fire blight epidemics are enormous as they run in estimated millions of Euros and Dollars in Europe and the US, respectively. Fire blight is quite difficult to manage. Pome fruit growers employ techniques such as application of antagonists or antibiotics, removal of infected tissues, or the complete removal of infected trees (Norelli et al., 2003; Malnoy et al., 2012). The use of antibiotics such as streptomycin is effective for fire blight control in North America, but is generally banned in many European countries. Unfortunately, in many regions, streptomycin resistant E. amylovora has emerged, thus requiring alternative means of disease control. The use of fire blight resistant cultivars is a feasible alternative control measure since varying degrees of resistance to the disease could be found in Malus. Resistant wild Malus species accessions that are donors of quantitative trait loci (QTLs) are known including Malus robusta 5 (Mr5; Peil et al., 2007), M. fusca MAL0045 (Emeriewen et al., 2014), M. floribunda 821 and the ornamental cultivar Evereste (Durel et al., 2009), and M. arnoldiana (Emeriewen et al., 2017). Further, mild resistance has been reported in cultivars like Fiesta (Calenge et al., 2005; Khan et al., 2006) and Florina (Le Roux et al., 2010). However, fire blight resistance is strain dependent (Norelli and Aldwinkle, 1986). For instance, the accession M. robusta 5, which was resistant to E. amylovora strain Ea222 (Peil et al., 2007), was shown to be susceptible to some more aggressive strains (Peil et al., 2011; Vogt et al., 2013; Wöhner et al., 2014). Consequently, the highly aggressive Canadian strain Ea3049 and the E. amylovora avrrpt2 EA mutant strain ZYRKD3-1 cause the complete breakdown of the fire blight resistance QTL of Mr5 located on linkage group 3 (LG3), and the underlying gene, FB_MR5 (Fahrentrapp et al., 2013; Broggini et al., 2014). On the other hand, Vogt et al. (2013) showed that the

2 46 Resistance of Mfu10 to ZYRKD3-1 strain Journal of Plant Pathology (2017), 99 (Special issue), Fig. 1. Responses of M. fusca (MAL0045) and Idared to the avrrpt2 EA mutant strain ZYRKD3-1. M. fusca accession MAL0045 was resistant to both strains. The corresponding fire blight resistance QTL of M. fusca (Mfu10) was mapped on LG10 explaining up to 66% of phenotypic variation using strain Ea222_JKI (Emeriewen et al., 2014). Although the resistance donor, MAL0045, was only minimally affected by the very aggressive Canadian strain Ea3049 in subsequent phenotypic evaluation, the progenies derived by crossing MAL0045 and Idared were significantly affected (Emeriewen et al., 2015). Nevertheless, Mfu10 was not broken down by this strain (Emeriewen et al., 2015); a stark contrast with the situation in Mr5 where the resistance QTL was completely broken down, thus confirming the difference in resistance mechanisms of both Malus accessions. Not much is known about the resistance/susceptibility of other Malus QTLs to different aggressive strains of E. amylovora. In order to establish durable resistance against fire blight, pyramiding of resistance QTLs is highly recommended. Hence, it is essential to identify genotypes that are resistant to highly aggressive strains of E. amylovora. To further understand the resistance mechanism of Mfu10 and its donor MAL0045, we carried out another phenotypic evaluation of the progeny population of MAL0045 Idared using the resistance-breaking avrrpt2 EA mutant strain of Mr5 ZYRKD3-1. MATERIALS AND METHODS Plant materials. The original mapping population (ID: 05210) of 134 individuals derived from a cross between the fire blight resistant M. fusca accession MAL0045 of the JKI Malus wild species collection and the susceptible M. domestica cultivar Idared previously reported by Emeriewen et al. (2014), remained the same. The progeny is being maintained in the orchard. Artificial shoot inoculation and evaluation of disease incidence. For phenotypic evaluation, up to 10 replicates of scions for each progeny individual and the parents, M. fusca MAL0045 and Idared, were grafted on rootstock M9. Plants were grown in the greenhouse and actively growing shoots with a minimum length of 25 cm were inoculated in spring by cutting the two youngest leaves with a pair of scissors dipped into bacterial suspension of the avrrpt2 EA mutant strain ZYRKD3-1 at a concentration of 10 9 cfu/ ml. The inoculated plants remained in the greenhouse at o C (day), 20 o C (night), and 85% air humidity. Disease incidence was recorded 28 days post inoculation (dpi). Percent lesion length (PLL) was determined as ratio of the length of shoot necrosis to the length of the total shoot. Statistical analysis and mapping of the fire blight resistance locus. Pearson s correlations of the phenotypic data of ZYRKD3-1, Ea222_JKI (Emeriewen et al., 2014) and Ea3049 (Emeriewen et al., 2015) were calculated using SAS Enterprise Guide 4.3 (SAS Institute Inc, Cary, NC, USA). Marker-phenotype association was determined by Kruskal-Wallis analysis using MapQTL 5 (Van Ooijen, 2004). Interval and multiple QTL mapping were also performed using MapQTL 5. RESULTS Phenotypic evaluation and statistical analysis. Since the graftings of 19 of the 134 individuals of the original mapping population were not suitable for phenotyping, only 115 progeny individuals together with their parents were screened for resistance/susceptibility to fire blight using the avrrpt2 EA mutant strain ZYRKD3-1. Results of phenotypic evaluation showed that whereas an average percent lesion length of 97.6 was recorded for the susceptible parent Idared, 1.9% was recorded for the resistant MAL0045 accession (Fig. 1). The mean PLL for all 115 progenies was 38.5 with a median of In general, 16 progenies showed 0%, 13 progenies recorded < 1.0% while 49 progenies recorded > 50% lesion length. The pairwise comparisons of the PLLs obtained after inoculation of the progenies with ZYRKD3-1 and Ea222_JKI (Emeriewen et al., 2014) as well as ZYRKD3-1 and Ea3049 (Emeriewen et al., 2015) are presented in Fig. 2. Phenotypic results were significantly correlated with r = 0.78 for Ea222_JKI/ZYRKD3-1 and r = 0.68 for ZYRKD3-1/Ea3049. Nevertheless, the mean PLL of 38.5% obtained with ZYRKD3-1 is considerably higher than the mean PLLs of 22.6, 9.4 and 9.0% obtained for this same M. fusca Idared progeny in three respective years of phenotypic evaluation with Ea222_JKI (Emeriewen et al., 2014). However, the mean PLL is substantially lower than the mean PLL of 62.4% obtained with Ea3049 (Emeriewen et al., 2015). Mapping of the resistance locus. The genetic map of M. fusca (Emeriewen et al., 2014) was used as a template

3 Journal of Plant Pathology (2017), 99 (Special issue), Emeriewen et al. 47 a) b) Fig. 2. Pairwise comparison of PLL of M. fusca Idared progenies after inoculation with E. amylovora strains Ea222_JKI, Ea3049 and ZYRKD3-1. PLL: percent lesion length; PC: Pearson s correlation coefficient; RG: regression line function. Table 1. Kruskal-Wallis analysis of linkage group 10 of Malus fusca after inoculation with the avrrpt2 EA mutant strain ZYRKD3-1. Locus Map position K PLL of alleles in: Coupling Repulsion * CH02b ** CH02c *** CH03d *** FR481A *** FRM *** FR149B *** FR367A *** *** ** K, value of Kruskal-Wallis analysis (significance levels: *= 0.05, **= 0.005, ***= ). Locus and map position as in Emeriewen et al. (2014); PLL: percent lesion length. for marker-phenotype association analysis (Kruskal-Wallis analysis) and for QTL mapping. For mapping analyses, the average PLL of replicates of each genotype were used as numerical traits. Kruskal-Wallis analysis revealed that only markers mapping on LG10 showed any significant correlation with fire blight resistance (Table 1). The markers in this group with the strongest correlation with resistance levels as indicated by the highest K values are FR481A and FRM4 with 73.5 and 72.9, respectively (Table 1). The differences of PLLs for individuals inheriting alleles of SSRs on LG10 in coupling with resistance and repulsion against resistance are also presented in Table 1. The greatest difference (ca. 64%) could be observed for SSR markers FR481A and FRM4. Nevertheless, for SSR markers, CH03d11 and FR367A (K = 61.9 and 58.4, respectively), a high difference between PLLs for coupling and repulsion (ca. 60% and 59%, respectively) was observed. Quantitative trait locus (QTL) mapping detected one QTL with a significant logarithm of the odd (LOD). This QTL located at the exact position as Mfu10 (Emeriewen et al., 2014) explained about 89% of the phenotypic variation with a LOD of 36 (Fig. 3). The closest markers to the QTL peak are FR481A and FRM4 (1.53 cm apart) Fig. 3. LOD score plot, threshold and percent phenotypic variation explained (% Expl.) of the trait necrosis along LG10 of Malus fusca using the phenotypic data of progenies of M. fusca Idared inoculated with the mutant strain ZYRKD3-1. and bracketed by two other SSR markers, CH03d11 and FR149B. DISCUSSION The Type III secretion (T3SS) pathogenicity island (PAI) possessed by Erwinia amylovora encodes hypersensitive response and pathogenicity (hrp) genes which deposits effector proteins into hosts, thereby causing fire blight in susceptible species (Khan et al., 2012). The AvrRpt2 EA effector protein is an example of the many identified effectors and is known to be a homolog of the AvrRpt2 effector protein of Pseudomonas syringae (Zhao et al., 2006). Recently, the role of AvrRpt2 EA in the Malus robusta 5- E. amylovora host-pathogen system (Vogt et al., 2013) was examined by sequencing the avrrpt2 EA gene and inoculating Mr5 with different wild type strains of E. amylovora as well as an avrrpt2 EA mutant strain called ZYRKD3-1 (Zhao et al., 2006). In describing the gene-for-gene relationship between Mr5 and E. amylovora, Vogt et al. (2013) identified a single nucleotide polymorphism (SNP) in the avrrpt2 EA gene which is responsible for virulence on Mr5

4 48 Resistance of Mfu10 to ZYRKD3-1 strain Journal of Plant Pathology (2017), 99 (Special issue), by means of an exchange of cysteine (C-allele) amino acid to serine (S-allele) at position 156 of the sequence. Resultantly, the resistance of Mr5 itself and the fire blight resistance QTL located on LG3 were broken down by strains of E. amylovora possessing the S-allele of the avrrpt2 EA (Peil et al., 2011; Vogt et al., 2013) as well as the avrrpt2 EA mutant strain, ZYRKD3-1 (Wöhner et al., 2014) but not by the C-allele strains. In the meantime, the M. fusca fire blight resistance QTL (Mfu10) was identified after a population of M. fusca Idared progeny was inoculated with a C-allele strain (Vogt et al., 2013), Ea222_JKI (Emeriewen et al., 2014). Thereafter, fire blight resistance of this population was re-evaluated with the highly aggressive Canadian strain Ea3049 which carries the S-allele that overcomes the resistance of Mr5. This S-allele strain could not overcome the resistance of M. fusca accession, MAL0045, and ultimately could not breakdown Mfu10 (Emeriewen et al., 2015). However, Mfu10 was significantly affected by Ea3049 since it explained only 41% of the phenotypic variation with this strain (Emeriewen et al., 2015), but hitherto explained 66% of the phenotypic variation with Ea222_JKI (Emeriewen et al., 2014). Since the absence of the avrrpt2 EA gene appeared to have played a contributory role in the virulence of ZYRKD3-1 on Mr5, it became necessary to investigate any such similar role in M. fusca by phenotyping the M. fusca progeny using the avrrpt2 EA mutant strain, ZYRKD3-1. The phenotypic evaluation using ZYRKD3-1 revealed a higher mean PLL on the progeny (38.5%) compared to the means recorded in three different years (22.6, 9.0, 9.6%) using the C-allele strain Ea222_JKI (Emeriewen et al., 2014). Wöhner et al. (2014) recorded even higher mean PLL (71.4) for the Idared Mr5 progeny and a lower correlation between both strains (r = 0.17) than in the current study (r = 0.78). Furthermore, the mean PLL recorded with this mutant strain is substantially lower than the mean PLLs of 62.4% for the M. fusca Idared progeny (Emeriewen et al., 2015), and 80.4% for the Idared Mr5 progeny (Wöhner et al., 2014), respectively using the S-allele strain, Ea3049. Nevertheless, the correlation between Ea3049 and ZYRKD3-1 (r = 0.68) in the current study is higher than the correlation (r = 0.13) of both strains reported by Wöhner et al. (2014). Kruskal-Wallis and QTL analyses performed with the M. fusca genetic map (Emeriewen et al., 2014) as template together with the phenotypic data obtained with ZYRKD3-1, showed that only markers on LG10 correlated significantly with fire blight resistance levels with a QTL of significant LOD score being detected. Multiple QTL mapping performed with FR481A set as cofactor showed that Mfu10 could explain 89% of the phenotypic variation at a LOD of 35.2 (Fig. 3). The position of the QTL remained the same as previously reported using Ea222_JKI (Emeriewen et al., 2014) and Ea3049 (Emeriewen et al., 2015). Interestingly however, the effect of the QTL using ZYRKD3-1 is higher than previously reported. This is the complete opposite of the situation in Mr5 where this mutant strain completely broke down the resistance of Mr5 itself (Vogt et al., 2013) as well as the resistance QTL of Mr5 located on LG3 (Wöhner et al., 2014). This result is further confirmation that the mechanisms of resistance of M. fusca and Mr5 are different. Furthermore, it supports the assumption that other genetic factors appear to contribute to the resistance of M. fusca, possibly the presence of more than one resistance gene, given the two clusters of differential response of progenies to the highly aggressive Ea3049 and the mutant avrrpt2 EA strain shown in Fig. 2b. The type of discrepancy between the reaction of M. fusca (MAL0045) and the M. fusca Idared progeny to Ea3049, where the resistance donor parent was only minimally affected but the progeny significantly affected (Emeriewen et al., 2015), was not found in the current study. What was clear, however, was that individuals inheriting alleles in coupling with Mfu10 showed in average a 36% lesser PLL than the progenies with alleles in repulsion (Emeriewen et al., 2015). This variation in PLL was higher in the current study with individuals inheriting the 156 bp allele of FRM4 (in coupling with Mfu10), showing an average PLL of ca. 64 lesser than progenies inheriting the 166 bp allele (in repulsion) of the same SSR marker. Therefore, genotypes resistant to both Ea3049 and ZYRKD3-1 strains are strong candidates for pyramiding to obtain durable resistance. ACKNOWLEDGEMENTS This research is funded by Deutsche Forschungsgemeinschaft (Project Number-: AOBJ: ). REFERENCES Broggini G.A.L., Wöhner T., Fahrentrapp J., Kost T.D., Flachowsky H., Peil A., Hanke M.-V., Richter K., Patocchi A., Gessler C., Engineering fire blight resistance into the apple cultivar Gala using the FB_MR5 CC-NBS-LRR resistance gene of Malus robusta 5. 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5 Journal of Plant Pathology (2017), 99 (Special issue), Emeriewen et al. 49 Emeriewen O.F., Richter K., Hanke M.-V., Malnoy M., Peil A., The fire blight resistance QTL of Malus fusca (Mfu10) is affected but not broken down by the highly virulent Canadian Erwinia amylovora strain E2002A. European Journal of Plant Pathology 141: Emeriewen O.F., Peil A., Richter K., Zini E., Hanke M-V., Malnoy M., Fire blight resistance of Malus arnoldiana is controlled by a quantitative trait locus located at the distal end of linkage group 12. European Journal of Plant Pathology doi: /s Fahrentrapp J., Broggini G.A.L., Kellerhals M., Peil A., Richter K., Zini E., Gessler C., A candidate gene for fire blight resistance in Malus robusta 5 is coding for a CC-NBS-LRR. Tree Genetics and Genomes 9: Khan M.A., Duffy B., Gessler C., Patocchi A., QTL mapping of fire blight resistance. Molecular Breeding 17: Khan M.A., Zhao Y.F., Korban S.S., Molecular mechanisms of pathogenesis and resistance to the bacterial pathogen Erwinia amylovora, causal agent of fire blight disease in Rosaceae. Plant Molecular Biology Reporter 30: Le Roux P.M.F., Khan M.A., Broggini G.A.L., Duffy B., Gessler C., Patocchi A., Mapping of quantitative trait loci for fire blight resistance in the apple cultivars Florina and Nova Easygro. Genome 53: Malnoy M., Martens S., Norelli J.L., Barny M., Sundin G.W., Smiths T.H.M., Duffy B., Fire Blight: Applied genomic insights of the pathogen and host. Annual Review of Phytopathology 50: Norelli J.L, Aldwinckle H.S., Differential susceptibility of Malus spp. cultivars Robusta 5, Novole, and Ottawa 523 to Erwinia amylovora. Plant Disease 70: Norelli J.L., Jones A.L., Aldwinkle H.S., Fire blight management in the twenty-first century using new technologies that enhance host resistance in apple. Plant Disease 87: Peil A., Garcia-Libreros T., Richter K., Trognitz F.C., Trognitz B., Hanke M.V., Flachowsky H., Strong evidence for a fire blight resistance gene of Malus robusta located on linkage group 3. Plant Breeding 126: Peil A., Bus V.G.M., Geider K., Richter K., Flachowsky H., Hanke M.V., Improvement of fire blight resistance in apple and pear. International Journal of Plant Breeding 3: Peil A., Flachowsky H., Hanke M.-V., Richter K., Rode J., Inoculation of Malus robusta 5 progeny with a strain breaking resistance to fire blight reveals a minor QTL on LG5. Acta Horticulturae 986: Van Ooijen J.W., MapQTL 5 Software for the mapping of quantitative trait loci in experimental populations. Plant Research International, Wageningen, the Netherlands. Vogt I., Wöhner T., Richter K., Flachowsky H., Sundin G.W., Wensing A., Savory E.A., Geider K., Day B., Hanke M.V., Peil A., Gene-for-gene relationship in the host-pathogen system Malus robusta 5-Erwinia amylovora. New Phytologist 197: Wöhner T.W., Flachowsky H., Richter K., Garcia-Libreros T., Trognitz F., Hanke M-V., Peil A., QTL mapping of fire blight resistance in Malus robusta 5 after inoculation with different strains of Erwinia amylovora. Molecular Breeding 34: Zhao Y., He S.Y., Sundin G.W., The Erwinia amylovora avrrpt2 EA gene contributes to virulence on pear and Avr- Rpt2 EA ss recognized by Arabidopsis RPS2 when expressed in Pseudomonas syringae. Molecular Plant-Microbe Interactions 19: Received February 3, 2017 Accepted May 2, 2017

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