Scab resistance in ‘Geneva’ apple is conditioned by a resistance gene cluster with complex genetic control

Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most severe diseases of apple worldwide. It is the most studied plant–pathogen interaction involving a woody species using modern genetic, genomic, proteomic and bioinformatic approaches in both species. Although ‘Geneva’ apple was recognized long ago as a potential source of resistance to scab, this resistance has not been characterized previously. Differential interactions between various monoconidial isolates of V. inaequalis and six segregating F1 and F2 populations indicate the presence of at least five loci governing the resistance in ‘Geneva’. The 17 chromosomes of apple were screened using genotyping‐by‐sequencing, as well as single marker mapping, to position loci controlling the V. inaequalis resistance on linkage group 4. Next, we fine mapped a 5‐cM region containing five loci conferring both dominant and recessive scab resistance to the distal end of the linkage group. This region corresponds to 2.2 Mbp (from 20.3 to 22.5 Mbp) on the physical map of ‘Golden Delicious’ containing nine candidate nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) resistance genes. This study increases our understanding of the complex genetic basis of apple scab resistance conferred by ‘Geneva’, as well as the gene‐for‐gene (GfG) relationships between the effector genes in the pathogen and resistance genes in the host.     

Phenolic compounds in apple leaves after infection with apple scab

Leaves of the scab-susceptible apple (Malus domestica) cultivar Golden Delicious were harvested from May to August 2008 and 2009. Some leaves were healthy and some infected with fungus Venturia inaequalis. The phenolic compounds were analysed in healthy leaves, infected leaves and in the scab spot tissue. In comparison to healthy leaves, the infected leaves showed higher contents of hydroxycinnamic acid, flavanols and phloridzin, while lower contents on procyanidins, quercetins and phloretin. The total amount of phenolic compounds in the infected tissue was 10 to 20 % higher than in the healthy leaves. Accumulation of phenolic compounds is a post-infection response, and probably their further transformation is a prerequisite for plant resistance.     

Infection and Stroma Formation by Venturia inaequalis on Apple Leaves with Different Degrees of Susceptibility to Scab

Conidia of Venturia inaequalis germinated and formed appressoria on all leaves independently of age and origin within the first 15 hours after inoculation. On the youngest expanded leaves of the apple variety Golden Delicious 80% of the conidia developed stromata within the first 24 hours, sporulation occurred after 8 days. On the second leaf (counted from the top of the twig) stroma formation was slowed down compared to the first and sporulation occurred after 10 days only. On the third leaf the fungus rarely formed stromata and never sporulated. The fourth leaf already showed complete ontogenetic resistance. In the diploid variety Priscilla the gene Vf, which confers resistance against scab to apple leaves, was expressed as reduced stroma formation without sporulation, as in the case of ontogenetic resistance. In the triploid variety Sir Prize with the gene Vf, stroma formation was retarded and colonization and sporulation occurred later on the first leaf, similarly to the reaction of the second leaf of variety Golden Delicious.     

Quantification of polyphenolic compounds and relative gene expression studies of phenylpropanoid pathway in apple (Malus domestica Borkh) in response to Venturia inaequalis infection

Apple (Malus domestica Borkh) is rich in phenolic compounds, which may enhance resistance to scab disease caused by Venturia inaequalis. In present study, apple cv. Golden Delicious was used for estimation of total phenols, flavonoids and quantification of six individual phenolic compounds. between control vs inoculated samples at different inoculation stages. The relative gene expression of phenylalanine ammonia lyase, chalcone synthase and flavanone 3 hydroxylase increased and polyphenolic compounds were constitutively upregulated at different post-inoculation stages. Data suggest that synthesis and accumulation of polyphenols is closely related with disease resistance against Venturia inaequalis. This study may play a vital role in understanding and finding out the governing mechanisms of scab resistance.     

Enzyme activity of the phenylpropanoid pathway as a response to apple scab infection

The study was performed on apple trees, ‘Golden Delicious' cv., which is a scab-susceptible cultivar. The phenolic content of apple fruit was determined in different parts of the peel. The phenolic compounds were analysed in the scab spot, in the tissue around the spot and in the healthy tissue. We determined the concentration of various phenolic compounds and related enzyme activities. Infection with the Venturia inaequalis fungus enhanced the metabolism of phenolic compounds at the scab spot, around the spot and in healthy peel. Compared with the healthy tissue and the tissue around the spot, the scab spot showed higher enzyme activity for all tested enzymes, except for dihydrochalcone 2′-O-glucosyltransferase, which had lower activity in the scab spot. In comparison to the healthy peel, the scab spot showed up to 3.4 times more hydroxycinnamic acids, up to 1.1 times more dihydrochalcones and up to 1.4 times more flavan-3-ols. In contrast, the healthy peel showed up to 1.6 times more flavonols than the scab spot.     

Interaction between Venturia inaequalis and Apple Callus Tissue Cultures: an Electron Microscopic Study

Untrastructural interactions between Venturia inaequalis and callus cultures from scab susceptible and resistant apple varieties, were similar. Host cell wall changes, appositions, and invagination of host plasmamembrane at sites of close contact with fungal hyphae were regularly observed. The ultrastructural observations are described and discussed. The host cell alterations as well as many fungal structures corresponded to those known in young leaves of susceptible apple varieties.     

Virulence and Molecular Diversity of Venturia inaequalis in Commercial Apple Growing Regions in Kashmir

Seventy‐one isolates of Venturia inaequalis collected from commercial apple growing areas of Kashmir were characterized on international differential apple hosts and analyzed by Random Amplified Polymorphic Microsatellites (RAMS), PCR–RFLP and sequencing of rDNA for elucidation of variability. Virulence analysis on a differential set categorized them into four pathogenic races, viz. (0), (1), (2) and (1,2) in the first time comprehensive molecular analysis of this in India and especially from Jammu and Kashmir, a north‐western Himalayan state of India. Race groups (0), (1), (2) and (1,2) contained isolates from diverse areas without specificity to any geographical zone or region. Cluster analysis of the RAMS and PCR–RFLP revealed a high genotypic diversity within V. inaequalis isolates. Three major clusters were obtained and the isolates could not be categorized on the basis of either their geographical distribution or the cultivar from which they were isolated. amova analysis of pathogen populations at regional or race level revealed high diversity within the populations. Pairwise F_ST comparisons between the populations revealed less genetic differentiation, thereby indicating existence of frequent gene flow in Kashmir. The 24 rDNA sequences of V. inaequalis showed high haplotype diversity of 0.938 and 0.40 nucleotide diversity. Again clustering at regional or race level detected greater part of variability within groups than among groups, thereby indicating high diversity in V. inaequalis populations in Kashmir valley.     

Vf scab resistance of Malus

The apple production in temperate regions with spring rains, the Scab caused by the fungus Venturia inaequalis is the most important constraint. To produce spotless apples and avoid damage that develops during storage, growers apply fungicide on a regular or weather-determined basis. All major apple cultivars are highly susceptible to this disease. To limit the need for fungicide applications, apple breeders are currently introgressing disease resistance from wild Malus accessions into commercial lines. The first attempts to do this were made 100 years ago. As apples are self-incompatible, pseudo-backcrossing is used to eliminate unwanted traits from wild Malus and select new cultivars that are attractive to both producers and consumers. This process, from the first cross of a commercial cultivar with a wild, disease-resistant Malus, is extremely long due to apple’s long juvenile phase, the need for more than seven backcross steps and the high heterozygosity of this genus. Therefore, most of today’s scab-resistant cultivars rely on a single introduction of scab resistance from Malus floribunda 821, referred to as Vf. In this paper, we trace the history of Vf from its initial identification through its use in breeding and commercial production. We sum up the literature describing how and where Vf resistance has been overcome by new pathotypes of V. inaequalis. Finally, we describe the current knowledge of the genes behind Vf resistance, its mode of action and the use of Vf genes in gene technology.     

Interaction between natural and synthetic fruit odor influences response of apple maggot flies to visual traps

In order to improve perimeter trapping for apple maggot fly behavioral control, we designed a set of experiments which aimed to reach a better understanding of the nature of the interaction between the natural host odor released by susceptible and low‐susceptibility apple cultivars, and an artificial host odor currently employed as a lure along with visual traps for apple maggot fly, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), perimeter trapping programs. The response of apple maggot flies to lured and unlured visual traps deployed in different rectangular arrays of susceptible and low‐susceptibility apple cultivars (two central trees of a particular cultivar surrounded by four perimeter trees of the same or a different cultivar) was evaluated over 2 years under field conditions. In uniform blocks of susceptible (Tidemann Red, Jersey Mac) or low‐susceptibility (Marshall McIntosh) cultivars, lured traps recovered a significantly greater proportion of the total capture than unlured traps, irrespective of lure position (center or perimeter trees). Unlured traps on central susceptible apple cultivars (Red Astrachan, Gala, Fuji) recovered a significantly greater proportion of the total capture than unlured traps on surrounding low‐susceptibility cultivars (Marshall McIntosh, Paula Red, Red Delicious, and Golden Delicious). Placing the lures near traps on low‐susceptibility cultivar trees surrounding unlured traps on central susceptible cultivar trees reduced apple maggot fly visits to traps on central trees, but the latter still recovered a similar proportion of the total capture as lured traps on perimeter trees. By contrast, placing the lures near traps on central susceptible cultivar trees surrounded by unlured traps on low‐susceptibility cultivar trees allowed lured central traps to receive a significantly greater proportion of the total capture than unlured perimeter traps. We conclude that the synthetic and natural host odor of susceptible cultivars interact additively in attracting apple maggot flies to visual traps, and that, when given the choice, traps and lures should be deployed on preferred rather than on less preferred cultivar trees. Implications for trap deployment strategies for tephritid monitoring and control are discussed in the light of our findings.     

Screening apples for OPD20/600 using sequence-specific primers

Apple scab, caused by Venturia inaequalis (Cke.) Wint., is the most serious disease of apple trees in many areas of the world. Resistance to V. inaequalis, derived from the small-fruited species Malus floribunda 821, is determined by a major dominant gene, Vf. Using random decamer primers, we identified a RAPD marker, OPD20/600, which is linked to the Vf gene. OPD20/600 was then cloned and sequenced. Sequence-specific primers based on the marker were used to further screen M. floribunda 821, 7 scab-susceptible apple cultivars, 10 scab-resistant apple cultivars, and 28 scab-resistant Coop selections. The sequence-specific primers allowed identification of polymorphisms of OPD20/600 based on the presence or absence of a single band. The advantages of sequence-specific primers over decamer primers for developing genetic markers are discussed.     

Identification of a leucine-rich repeat receptor-like serine/threonine-protein kinase as a candidate gene for <Emphasis Type="Italic">Rvi12 (Vb)</Emphasis>-based apple scab resistance

Apple scab caused by Venturia inaequalis is the most important fungal disease of apples (Malus × domestica). Currently, the disease is controlled by up to 15 fungicide applications to the crop per year. Resistant apple cultivars will help promote the sustainable control of scab in commercial orchards. The breakdown of the Rvi6 (Vf) major-gene based resistance, the most used resistance gene in apple breeding, prompted the identification and characterization of new scab resistance genes. By using a large segregating population, the Rvi12 scab resistance gene was previously mapped to a genetic location flanked by molecular markers SNP_23.599 and SNP_24.482. Starting from these markers, utilizing chromosome walking of a Hansen’s baccata #2 (HB2) BAC-library; a single BAC clone spanning the Rvi12 interval was identified. Following Pacific Biosciences (PacBio) RS II sequencing and the use of the hierarchical genome assembly process (HGAP) assembly of the BAC clone sequence, the Rvi12 resistance locus was localized to a 62.3-kb genomic region. Gene prediction and in silico characterization identified a single candidate resistance gene. The gene, named here as Rvi12_Cd5, belongs to the LRR receptor-like serine/threonine-protein kinase family. In silico comparison of the resistance allele from HB2 and the susceptible allele from Golden Delicious (GD) identified the presence of an additional intron in the HB2 allele. Conserved domain analysis identified the presence of four additional LRR motifs in the susceptible allele compared to the resistance allele. The constitutive expression of Rvi12_Cd5 in HB2, together with its structural similarity to known resistance genes, makes it the most likely candidate for Rvi12 scab resistance in apple.     

Expression profiling in HcrVf2-transformed apple plants in response to Venturia inaequalis

Apple scab resistance is one of the most well-characterized plant–pathogen interactions in a woody plant species. While the HcrVf2 gene from the wild apple Malus floribunda 821 has proved capable of conferring scab resistance to the susceptible cv. Gala after genetic transformation, its identification represents only the first step in understanding the molecular mechanisms and, hence, the network of genes underlying the defence response. We used a PCR-based suppression subtractive hybridization to identify apple genes that are differentially expressed after Venturia inaequalis inoculation. Subtractive hybridization was performed between cDNA from challenged leaves of HcrVf2-resistant transgenic Gala and susceptible cv. Gala plants. A library of 523 unigenes was constructed and characterized by assigning a putative function via comparison with public databases. This set of pathogen-modulated apple genes includes many defence-related genes and is therefore an important source of information for understanding the molecular basis of the Malus–V. inaequalis interaction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional analysis and expression profiling of <Emphasis Type="Italic">HcrVf1</Emphasis> and <Emphasis Type="Italic">HcrVf2</Emphasis> for development of scab resistant cisgenic and intragenic apples

Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (P_MdRbc) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar ‘Gala’ was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. ‘Santana’.     

Isolation of 21 new polymorphic microsatellite loci in the phytopathogenic fungus Venturia inaequalis

Twenty‐one new polymorphic microsatellite markers were isolated in the phytopathogenic fungus Venturia inaequalis, the causal agent of apple scab. An enrichment protocol was used to isolate microsatellite loci and the level of polymorphism was assessed on 44 European isolates. All loci were polymorphic with an average of 9.1 alleles per locus (range 2–24). Tests of cross‐species amplifications suggest that at least some of these microsatellites could be used in different species, mainly Spilocaea pyracanthae and S. eriobotryae.     

A detailed linkage map around an apple scab resistance gene demonstrates that two disease resistance classes both carry the V f gene

A detailed genetic map has been constructed in apple (Malus x domestica Borkh.) in the region of the v _f gene. This gene confers resistance to the apple scab fungus Venturia inaequalis (Cooke) Wint. Linkage data on four RAPD (random amplified polymorphic DNA) markers and the isoenzyme marker PGM-1, previously reported to be linked to the v _f gene, are integrated using two populations segregating for resistance to apple scab. Two new RAPD markers linked to v _f (identified by bulked segregant analysis) and a third marker previously reported as being present in several cultivars containing v _f are also placed on the map. The map around v _f now contains eight genetic markers spread over approximately 28 cM, with markers on both sides of the resistance gene. The study indicates that RAPD markers in the region of crab apple DNA introgressed with resistance are often transportable between apple clones carrying resistance from the same source. Analysis of co-segregation of the resistance classes 3A (weakly resistant) and 3B (weakly susceptible) with the linked set of genetic markers demonstrates that progeny of both classes carry the resistance gene.This work was supported in part by grants from the New Zealand Foundation for Research Science and Technology (FoRST) Programme 94-HRT-07-366 and ENZA New Zealand (International)     

Polyphenol metabolism of developing apple skin of a scab resistant and a susceptible apple cultivar

During fruit development, the concentration of main polyphenols (flavonols, flavanols, dihydrochalcones, hydroxycinnamic acids, anthocyanins) and the activities of related enzymes (phenylalanine ammonia lyase, chalcone synthase/chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, flavonol synthase, peroxidase) were monitored in apple (Malus domestica Borkh.). The seasonal survey was performed at five different sampling dates and included the healthy peel of the resistant cultivar ‘Florina’ and healthy peel, scab symptomatic spot and the tissue around the infected spot of the susceptible cultivar ‘Golden Delicious’. From all enzymes tested, chalcone synthase/chalcone isomerase had the highest activity in both cultivars, while phenylalanine ammonia lyase had the lowest. The healthy peels of the susceptible and the resistant cultivar did not show differences in the accumulation of the main polyphenol groups present in the apple skin. However, in the resistant cultivar ‘Florina’, an increase of polyphenol enzyme activities could be observed in late stages of fruit development, which seems to be related to the anthocyanin accumulation in ripe fruits. Significant differences in the polyphenol metabolism were observed in the three different tissues of the susceptible cultivar ‘Golden Delicious’. Increased concentrations of hydroxycinnamic acids, dihydrochalcones and flavan-3-ols were found in the scab symptomatic spots and surrounding tissues. Phenylalanine ammonia-lyase, dihydroflavonol 4-reductase, flavanone 3-hydroxylase and peroxidase showed higher activities in the scab symptomatic spot compared to other analysed tissues, whereas the activities of other enzymes remained unchanged. Highest induction of polyphenol accumulation after scab infection was observed in early developmental stages, whereas enzyme activities were increased in later stages.