Screening of Apple Cultivars for Scab Resistance in Kazakhstan

: Scab, caused by Venturia inaequalis , is the most destructive fungal disease of apple world-wide. Apple scab incidence was studied in apple orchards in the south and southeast of Kazakhstan, including the Almaty, Zhambyl, and Turkestan regions, during 2022 and 2023. Disease incidence was higher in the Zhambyl region than in the Turkestan and Almaty regions in both years. The field evaluation suggested that 19 genotypes showed resistance to apple scab. Molecular screening was carried out using eight gene-specific molecular markers (AM19, CH05e03, OPL19, Hi07f02, AL07, K08, HB09, and CH02f06). The results of the molecular screening revealed that in 38 of the 45 studied cultivars, which included 11 Kazakh cultivars and 34 foreign cultivars, the Rvi ( Rvi2 , Rvi4 , Rvi5 , Rvi6 , Rvi8 , Rvi9 , Rvi11 , Rvi14 , and Rvi15 ) resistance genes were amplified. Resistance genes such as Rvi2 , Rvi4 , Rvi6 , and Rvi9 are still useful for breeding, but their use is recommended only in extended pyramids of multiple resistance genes. Several cultivars will be strong candidates for further breeding programs against apple scab and for the pyramiding of scab resistance genes in new cultivars.


Introduction
Apples (Malus domestica L.) are among the most important fruit crops grown worldwide.The apple cultivation area in Kazakhstan is 35.7 thousand hectares, or 75% of the total field area used for the production of stone and pome fruit crops [1].The southern and southeastern regions of Kazakhstan have the most favorable climatic conditions for growing fruit crops, in particular apple trees.To enhance the economic efficiency of apple cultivation, a critical factor is the expansion of resistant cultivars.Resistant apple cultivars fulfill requirements such as productivity, fruit quality, and resistance to diseases for sustainable agriculture production [2].
In many regions of Kazakhstan, apple scab, caused by the ascomycete fungus Venturia inaequalis, is the most serious disease of apple [3].Temperate regions with humid climates are highly favorable to this disease.In cases of severe infection, production losses of up to 70% have been reported [4,5].Most of the commercial apple cultivars are susceptible to this disease, and growers must spray fungicides several times within a season [6][7][8][9].Applying so many treatments raises ecological problems and consumer health concerns, in addition to the direct financial costs for growers.Pathogen resistance to fungicides has become a challenging problem in the control of diseases and has threatened the performance of some commercial fungicides [9].Although some fungicides can still be effective against their target pathogens [10,11], the application of additional disease control strategies, such as sanitary measures in orchards, is essential.For example, leaf litter management [12][13][14][15] helps reduce disease severity and the risk of fungicide degradation.Furthermore, planting scab-resistant cultivars provides scab resistance in the long term to facilitate more sustainable apple production.
Knowledge of the pathogenicity and virulence factors needed for fungal infection is important because it represents the targets that will allow researchers to identify and deploy resistance genes against these microorganisms [16].Phenotypically, the effects of resistance genes against V. inaequalis (Rvi) have been shown to cover a continuum from complete immunity to near-susceptibility depending on the genetic background, pathogen, and environment [17].Currently, identifying the cultivars of apple crops that are resistant to the scab pathogen is a priority task [18].
Developing and cultivating a new apple cultivar takes a long time and involves many steps because of the biology of fruit plants.Apple trees take up to 8-10 years to fully mature, even when using early-fruiting initial forms.This limits the study of the source material using traditional genetic methods, which require not only the first seed hybrid generation but also the second and third [19,20].A cultivated species with resistance against diseases provides an excellent opportunity to identify resistant genotypes and to use biodiversity to counter existing problems in fruit production.
Advances in technology have greatly improved the efficiency and accuracy of molecular markers, making them essential tools in plant breeding programs.Their origin, location in the genome, and the determined degree of resistance to the disease have been established [21].Molecular markers have been developed for the most common resistance genes, which allows the identification of genotypes with target genes, their deployment in new plants, and targeted selection [22,23].
Twenty non-allelic genes that determine resistance to various scab races have been identified in apple trees, and highly informative DNA markers have been developed for most of them [24].Molecular markers make it possible to evaluate hybrid families of seedlings for resistance in the very initial stages of plant development, significantly reducing the time needed to assess this important breeding trait.
According to a new nomenclature proposed by Bus et al. [17,25], apple scab resistance genes are named Rvik (R refers to the resistance gene, vi refers to Venturia inaequalis, and k refers to the differential host), and the corresponding Avr genes of the pathogen are named avrRvik.
Currently, 20 scab resistance genes have been identified [17,26].The new and old names of the apple resistance genes, along with their differential hosts, are listed as Rvi1 , and V d3 [31].
Major genes such as Rvi2 [32][33][34], Rvi4 [34][35][36], and Rvi9 [21] have also been identified from different cultivars.The Rvi2 and Rvi4 genes have been mapped in the same linkage group (LG-2) at the distal end [21].Molecular markers such as SSRs and SNPs have been identified and reported in several studies to detect these resistance genes [17,18,27,30,[32][33][34]. The Rvi5 gene was shown to be responsible for resistance in Malus micromalus and Malus atrosanguinea 804 by Dayton and Williams [37].Patocchi et al. [38] used a genome scanning approach (GSA) for the identification of the molecular markers associated with this gene.They developed the SSR marker Hi07h02, which is closely linked with Rvi5 on LG-17 at the distal end [28].Recently, with the use of the apple genome as a reference, a 228 kb region likely containing the Rvi5 gene was identified [39].Rvi6 was the first scab resistance gene identified from a wild relative (M.floribunda) of apples.This gene remains the most widely studied and characterized scab resistance gene in apples.It generally conditions a chlorotic reaction in resistant segregants.The molecular marker Al07 linked with this gene is positioned at 1.1 cM [40,41] and is closely linked on LG-1 [21].The SCAR marker AL07 is located 0.2 cM from the gene [42].The allele Rvi6 is determined by the presence of a 466 bp expected amplification product, while susceptible cultivars form a 724 bp product.The presence of both fragments indicates a heterozygous state for this gene [43].Later, AM19 was found closer to a resistance gene than AL07 and was used for chromosome walking of the BAC library of 'Florina'.The resistance conferred by this gene is influenced by the gene environment [44].A new Rvi8 gene was discovered in M. sieversii accession W193B by Bus et al. [33].It is closely linked with the Rvi2 gene on LG-2 at the lower end [21].It was further observed that Rvi8 is overcome by race 8 of the V. inaequalis isolate NZ188B.2.In another study [27], the marker OPL19 SCAR was found to be closely related to both genes.OPL19-SCAR was initially used to identify the Rvi2 gene in the apple genome [32].However, a separate scab resistance factor, Rvi8, was subsequently identified in the vicinity of the Rvi2 gene.It was found that the target product of the marker OPL19-SCAR-a 433 bp fragment-is amplified in carriers of the genes Rvi2 and Rvi8, demonstrating different degrees of resistance against artificial infection by individual scab races.According to the data, the two resistance genes (Rvi8 and Rvi2) are not dependent on one another [33].The Rvi11 gene was identified in M. baccata by Dayton and Williams [45] and was mapped to the same LG (LG-2) [21].Gygax et al. [46] developed the first molecular marker linked to this gene.Three SSR markers, namely CH02c06, CH05e03, and CH03d01, were developed.The Rvi11 gene has been mapped at about 0.6 cM [46].Rvi15 was identified from the accession GMAL 1473, a clone of R12740-7A (Russian seedling) [47,48].It was mapped on LG-2 (at the proximal end) [21] using the progeny of a cross between 'Idared' and GMAL 2473.Two closely associated markers were identified: CH02c02a and CH02f06 [36,47].This is the most promising resistance gene that can be incorporated relatively quickly into a new cultivar in combination with other scab resistance genes for durable resistance [49].It should be noted that depending on the parent forms, some seedlings that do not carry known resistance genes may be resistant to the pathogen due to the presence of other genetic determinants of resistance.As a result, three putative toll interleukin1 receptor-NBS-LRR resistance genes, namely, Rvi15-A, Rvi15-B, and Rvi15-C, were identified in this region.
Nowadays, virulent isolates have been shown to exist for most of the scab resistance genes used in apple breeding, including some carrying multiple virulence factors [17,50,51].These findings highlight the need to breed for durable resistance.One way to achieve durable resistance is to pyramid multiple scab resistance genes in a cultivar, and it is desirable to combine several genetic factors that control immunity in one genotype [17].
This study presents the results of a molecular genetic analysis of apple cultivars to identify Rvi2, Rvi4, Rvi5, Rvi6, Rvi8, Rvi9, Rvi11, and Rvi15 genes that are promising for further breeding, determining their resistance to scab and the incidence of apple scab in the most important apple growing areas of Kazakhstan.

Disease Monitoring
Eight apple orchards in the Almaty, Turkestan, and Zhambyl regions (Table 1) were monitored for the incidence of apple scab through phytopathological studies in 2022 and 2023.A total of 45 apple cultivars, which included 11 Kazakh cultivars and 34 foreign cultivars, were investigated in the current study.

Field Evaluation
The phytopathological assessment was conducted from 10 June to 30 August in 2022 and 2023 to study the incidence of apple scab.The number of trees per cultivar for disease evaluation is presented in Table 1.The susceptible cultivars 'Golden Delicious' and 'Idared' served as positive controls.Scab incidence was defined as the percentage of infected leaves (infected leaves/all leaves).A leaf was considered infected if there were matte, olive green-to-black-colored lesions on it, indicating active sporulation [52].

Collection of Plant Materials, DNA Extraction, and Detection of Rvi Genes with Molecular Markers
Three leaf samples from each of three trees per cultivar were collected from apple orchards located in the Almaty, Zhambyl, and Turkestan regions and from the pomological garden of the Kazakh Fruit and Vegetable Research Institute.DNA was isolated from fresh leaves of apple cultivars.For the identification of the scab resistance genes Rvi2/Rvi8, Rvi2/Rvi4/Rvi9/Rvi11, Rvi5, Rvi11, Rvi14, and Rvi15, the following markers were applied: OPL19, CH05e03, Hi07h02, K08, HB09, and CH02f06, respectively.AL07 and AM19 SCAR markers were used to identify Rvi6 [17, 18,27,30,43,46,53,54].
Extraction was conducted using the method of Doyle et al. [55] with a modification that included an additional purification of the samples.A modified CTAB method was used by introducing an additional component, polyvinylpyrrolidone (1%), into the composition of the lysis buffer, which provided a DNA yield of sufficient purity for PCR amplification.
The negative control for the studied loci was the cultivar 'Golden Delicious'.Primer sequences and their sizes are shown in Table 2.The primers used in this study were synthesized by Sigma-Aldrich (Darmstadt, Germany).A 15 µL PCR reaction mixture contained 20 ng of genomic DNA, 1.5 µL of dNTPs, 2.5 µL of MgCL 2 , 10 µL of each primer, 1 µL of Taq polymerase, and 2.5 µL of 10x Taq buffer (+(NH 4 ) 2 SO 4 , -KCL).All PCR components were manufactured by Thermo Fisher Scientific, Waltham, MA, USA.Amplification was carried out in a thermal cycler according to the following programs:

Data analysis
Data analysis was performed based on the results of molecular analysis.The visualization of PCR products was achieved through electrophoresis.The banding pattern of each amplified PCR product was scored as "+", indicating the presence of resistance, or "−", indicating the absence of the resistance gene.Genetic distance was evaluated through Popgen software (version 1.32, Yeh et al., 2000 [56]) by calculating the Dice coefficient [57].This calculated index was used to develop the Unweighted Pair Group Method with Arithmetic Mean (UPGMA).The dendrogram was drawn in Molecular Evolutionary Genetics Analysis (MEGA software, version 11, Tamura et al., 2021 [58]).

Field Evaluation of Apple Cultivars for Scab Resistance
Apple scab did not develop in many of the orchard-year combinations included in this study (Tables 3-5).Specifically, the susceptible control 'Idared' developed disease only in 3 of 14 orchard-year combinations that included this cultivar.Similarly, the other susceptible control 'Golden Delicious' developed disease only in 10 of 16 orchard-year combinations.Based on the limited disease development, conclusions about phenotypic host resistance in the test cultivars must be interpreted with caution.Among the test cultivars, 'Maksat', 'Kamila', 'Diana', 'Saltanat', 'Korey', 'Mutsu', 'Talgarskoye', 'Tulpan', 'Williams Pride', 'Piros', 'Honeycrisp', 'SuperChief', SQ159 (Natyra), 'Modi', 'Golden Resistant', and 'Prima' did not develop scab in our trials.However, among these cultivars, only 'Korey' was tested in more than two orchard-year combinations.Several other cultivars developed low levels of disease incidence (5% or less), with the caveat of low disease pressure across most trials as explained above.
UPGMA cluster analysis based on the presence or absence of resistance genes revealed that SQ159 (Natyra) and 'Williams Pride' formed a distinct cluster.The remaining 43 cultivars formed two large subclusters, one with 20 cultivars and the other with 23 cultivars.Several genotypes clustered together closely, including Malus sieversiii and 'Golden Delicious' (Figure 1).

Discussion
Progress in fruit breeding strongly depends on the availability of a rich diversity of genetic resources [3].
Up to now, V. inaequalis isolates have commonly been used in genetic experiments for their known specific ability to overcome one of the apple scab resistance genes represented in the differential host set [17].The identification of differential hosts with monogenic resistances will assist in the monitoring of pathogen populations to determine the potential of specific Rvi genes, currently the main sources of resistance in apple breeding.The Rvi9 gene generally conditions a chlorotic reaction in resistant segregants.Caffier [60].In our study, this gene was found in six Kazakh local cultivars ('Ainur', 'Aigul', 'Kamila', 'Saltanat', 'Talgarskoe', and Voskhod').The Rvi15 gene provides full resistance to apple scab [48], and there are no reports yet on the breaking of this resistance.In the present study, this gene was identified in four Kazakh local cultivars ('Ainur', 'Maksat', 'Kamila', and 'Talgarskoe').
'Aport' is the most used cultivar in Kazakh apple breeding as a donor of the taste qualities of fruits and frost resistance, and 'Aport' has the scab resistance genes Rvi2 + Rvi8 and Rvi11 [61].The results of our previous research on 'Aport' x M. sieversii scion-rootstock combinations showed resistance to powdery mildew and scab at the beginning of fruiting over 3 years [62].
Based on foliar resistance reactions, apple R genes can be grouped into three predominant resistance classes exhibiting distinctive resistance responses: the classical hypersensitive response (HR), in which fungal growth is normally terminated very rapidly on penetration, e.g., conditioned by Rvi4, Rvi5 [63] 1) is predominant in the European V. inaequalis population [65], which explains the highly susceptible status of cv.'Golden Delicious'.On the other hand, resistance genes in our study such as Rvi2, Rvi4, Rvi6, and Rvi9 are still useful for breeding, but their use is recommended only in extended pyramids of ≥3 resistance genes [66].
Many breeding programs worldwide are aiming at breeding for durable disease resistance against apple scab [67].Until now, most of the scab-resistant cultivars that have been released carry only Rvi6 [66].However, the value of the resistance mediated by Rvi6 is weakened by the occurrence of the avrRvi6 races of the pathogen in Europe [65] and the US [10], which can break the resistance of Rvi6.Therefore, the use of single R genes of durable resistance is not effective in the long term, which suggests a combination of different R genes for new cultivars, as carried out in pyramidization breeding programs [3].Several Rvi genes described to date, including Rvi5, Rvi11, Rvi12, Rvi14, and Rvi15, confer durable resistance to scab; therefore, they are of special interest for resistance breeding [66].The obtained data are important for identifying new donors for optimizing key stages of the breeding process for long-term resistance to the pathogen by pyramiding target genes.

Figure 1 .
Figure 1.UPGMA (Unweighted Pair-Cluster Method using Arithmetic Averages) dendrogram based on the presence or absence of apple scab resistance genes in 45 apple cultivars or accessions assessed in southern and southeastern Kazakhstan.

Figure 1 .
Figure 1.UPGMA (Unweighted Pair-Cluster Method using Arithmetic Averages) dendrogram based on the presence or absence of apple scab resistance genes in 45 apple cultivars or accessions assessed in southern and southeastern Kazakhstan.

Table 1 .
Experimental sites included in this study.
* Site names are used in the following tables and text to identify the respective breeding programs.** Number of trees used for disease assessment.

Table 2 .
Characteristics of molecular markers for apple scab resistance genes.

Table 3 .
Results of monitoring apple genotypes for scab disease incidence (%) in the Turkestan region.

Table 4 .
Results of monitoring apple genotypes for scab disease incidence (%) in the Almaty region.
* "-" means that these genotypes were not grown in the Almaty region.

Table 5 .
Results of monitoring apple genotypes for scab disease incidence (%) in the Zhambyl region.

Table 6 .
Results of the identification of scab resistance genes of apple genotypes.