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Article

Evaluation of Fire Blight Resistance of Eleven Apple Rootstocks Grown in Kazakhstani Fields

by
Gulshariya Kairova
1,
Alexandr Pozharskiy
2,
Nurzhan Daulet
1,
Maxim Solomadin
1,
Nurlan Sandybayev
1,
Marina Khusnitdinova
2,
Gulnaz Nizamdinova
2,
Zagipa Sapakhova
3 and
Dilyara Gritsenko
2,*
1
Plant Protection and Quarantine Department, Kazakh National Agrarian Research University, Almaty 050010, Kazakhstan
2
Laboratory of Molecular Biology, Institute of Plant Biology and Biotechnology, Almaty 050040, Kazakhstan
3
Breeding and Biotechnology Laboratory, Institute of Plant Biology and Biotechnology, Almaty 050040, Kazakhstan
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(20), 11530; https://doi.org/10.3390/app132011530
Submission received: 26 August 2023 / Revised: 17 October 2023 / Accepted: 17 October 2023 / Published: 21 October 2023
(This article belongs to the Section Agricultural Science and Technology)
Browse Figures
Versions Notes

Abstract

:
Clonal rootstocks of apple trees are one of the main components of intensive horticulture, and they play an important role in regulating tree growth, yield, fruit quality, and resistance to biotic and abiotic stresses. In the two-year monitoring survey reported here, eleven rootstocks mainly used for cultivar grafting in Kazakhstan were assessed for fire blight resistance, taking genetic background into consideration. The loci FB_F7 on LG7 and FB_MR5 and RLP1 on LG3 were analyzed on the basis of SCAR and SNP markers. The rootstocks ‘Geneva 41’, ‘62-396’, and ‘Geneva16’, bearing resistant alleles for five markers (AH0JFXM, AH21B92, AH4AAGA, AH5I8MI, and AH6R6SQ), four markers (AH0JFXM, AH21B92, AH4AAGA, and AH5I8MI), and one (AH4AAGA) SNP marker, respectively, were not infected during the monitoring period despite weather conditions in 2022 being favorable for the development and spread of Erwinia amylovora. No connection was identified between the presence of a resistant allele for the AE10-375 marker and fire blight resistance responses. This is the first study to evaluate the fire blight resistance of foreign and local rootstocks grown in the Kazakhstan southern region.

1. Introduction

Apple is one of the most economically important pome fruit crops in the world. In Kazakhstan, the fields occupied by apple gardens amount to 35.7 thousand hectares, which is 75% of the total field area used for the propagation of stone and pome fruit crops in the country, according to data from the Bureau of National Statistics Agency for strategic planning and reforms of the Republic of Kazakhstan (2023).
The pandemic bacterial disease infecting pome fruit is fire blight, which can cause devastating yield losses [1,2]. The causal agent of fire blight is the enterobacterium Erwinia amylovora (Burrill) Winslow et al. [3]. This pathogen can infect flowers, fruits, shoots, woody tissues, and rootstock crowns of the apple tree, resulting in tree death in cases of severe infection [4].
The control of fire blight epidemics is crucial not only for commercial fruit production but also for biodiversity safety. The distribution of fire blight in wild populations of apples is an especially important concern [5].
The propagation of resistant host plants is considered the most effective strategy for managing fire blight, especially since quantitative trait loci (QTLs) linked to fire blight resistance have been identified in several wild and cultivated Malus species, with phenotypic variance ranging from 15 to 83% [6,7,8,9,10,11]. However, the introduction of resistance genes from wild species into apple varieties is a time-consuming process; indeed, it can take decades due to the long generation times of apple trees and multiple pseudo-backcrosses [12] needed to eradicate the undesirable fruit qualities of wild apples, such as small size and bitter or astringent flavors. Because of this, most commercially successful apple cultivars are still susceptible to fire blight [13].
Resistant wild accessions are likewise used in rootstock breeding programs worldwide. The fire blight-resistant rootstocks of the Geneva series G.11, G.41, G.202, G.214, G.890, G.935, and G.969 [14] are descendants of the wild apple Malus × robusta 5 (Mr5) that bears a resistant QTL on LG3 [15,16]. Nevertheless, the moderately resistant M.7, the Budagovskij series B.9 and B.118, and the very susceptible Malling series M.26 and M.9 [16] are widely used in the grafting of commercial cultivars due to their high-quality horticultural traits.
Molecular methods enable the rapid and reliable identification of resistant genotypes for marker-assisted selection [17]. Identification of loci associated with fire blight resistance in apples is made possible by SSR [15], SCAR [18], and SNP [19] markers. The use of whole-genome sequencing (WGS) offers a great opportunity to not only confirm the presence of all resistant alleles but also to discover new ones [20,21]. Nevertheless, WGS is expensive and, therefore, not yet widely used in the screening of plant genotypes for breeding.
The recently developed SNP panel for the identification of disease resistance and horticultural traits in apples was validated by screening 240 phenotyped individuals of Malus × domestica Borkh to use in marker-assisted selection [19]. This panel includes five SNP markers of loci on LG3 that explain up to 80% of phenotypic variation in fire blight resistance [15]. These five SNP and SCAR markers for evaluating the presence of loci FB_F7 on LG7 [10] were used in the present study for testing eleven rootstocks commonly used for commercial apple grafting in Kazakhstan. An investigation of the connection between genetic background and environmental conditions was the main purpose of our two-year monitoring survey, which enabled us to distinguish whether rootstocks growing in the climate conditions of the Kazakhstan southern region are either susceptible or resistant to fire blight.

2. Materials and Methods

2.1. Disease Monitoring, Sampling, and DNA Isolation

Eleven types of apple rootstocks, including two local ones, were investigated in the current study (Table 1). These rootstocks are widely used for scion grafting in Kazakhstani commercial gardens. Eight out of the eleven researched apple rootstocks are included in the state register list of tested and approved rootstocks for cultivation in the southern region of Kazakhstan. This register list consists of seven clonal rootstocks: 62-396, M9, MM 106, Arm 18, B.7-35, B.16-20, and Zhetysu 5, as well as a seed rootstock, Malus sieversii. All registered rootstocks were included in the present work. Additionally, Geneva 16, Geneva 41, and B.9 fields were evaluated for fire blight occurrences as commonly known disease-resistant rootstocks [14,22].
For all rootstock types, the average size of growing fields was at least 100 square meters. Most of the studied rootstocks were planted in the pomological garden of Talgar field (Almaty region); only M9 and MM106 types were grown in two different fields (Table 1). Fields typically had an age range of nine to eleven years. The Talgar (Tal), Agricultural Innovations Limited(AIL), Shelek, Kazakhstan, and Sady Zhetysu Trade (SZT), Almaty region, Kazakhstan fields are located in the Almaty region; the Devrosh (Dev) field is located in the Zhambyl region. The apple rootstocks in the Almaty region were grown in the same climate zone; for the fields in the Almaty region, the meteorological station collecting the climate data was located in Esik city. The fourth field was in the city of Merke, in the Zhambyl region, which experiences a dry climate with sharp fluctuations in temperature, according to data from the National Hydrometeorological Service of Kazakhstan (2021 and 2022). The meteorological station in Merke was used to collect the climate data for the Dev field.
The fields were treated using the systemic fungicide Rayok (Avgust) and the insecticide Movento® Energy (Bayer, Leverkusen, Germany) in the early spring of 2021 and 2022. Chemicals that act against fire blight were not applied.
The rootstock plants were monitored in the field to identify the presence of fire blight infection during 2021 and 2022, taking environmental conditions into consideration.
Disease distribution index (DDI) was assessed in mid-July using a 5-point scale according to the following classification: 0—asymptomatic; 1—sporadic symptoms of fire blight in apple rootstock field, less than 10% of affected trees; 2–10% to 20% of rootstock trees affected by pathogen; 3–20% to 50% of the rootstock trees affected by pathogen; 4–50% to 100% of trees damaged by infection [31]. DDI was calculated by dividing fire blight-affected trees on 1,000 randomly examined trees in the field multiplied by 100. The disease severity index (DSI) [31] was calculated using the following formula:
DSI = (fire blight shoots/total shoots) × 100
Seed rootstocks of Malus sieversii were represented by different genotypes collected from the Tian Shan mountains. The researched fields of rootstocks were predominantly located in the southern region of the country.
DNA isolation for genetic and pathogen testing was conducted using ten samples of every rootstock type according to the CTAB protocol [32]. Detection of E. amylovora was performed by testing rootstock bark and shoots using the real-time PCR method described by Gottsberger [33].

2.2. Weather Data Acquisition

Surface meteorological observations at Esik and Merke stations during 2021 and 2022 were carried out at 18:00, 21:00, 00:00, 03:00, 06:00, 09:00, 12:00, and 15:00 Greenwich Mean Time (hereafter referred to as SGV). Observations of the intensity and development of atmospheric processes and phenomena, including air temperature, precipitation, atmospheric pressure, wind, and the relative humidity of the air, were carried out every second month according to the meteorological database RSE “Kazhydromet” available at https://www.kazhydromet.kz/en/meteo_db (accessed on 6 February 2023). The data were collected following the protocols developed by the World Meteorological Organization [34].

2.3. SCAR Marker Amplification

In the present work, two markers were used that are associated with fire blight resistance: AE10-375 and GE-8019 [10]. For each DNA sample, 60 ng DNA was amplified in a 25 µL reaction mix containing 1 × Taq buffer (750 мM; Tris HCl, pH 8.8, 200 мM; (NH4)2SO4, 0.1% Tween 20), 2.5 мM; MgCl2, 0.2 мM; dNTPs, 0.2 мM; of each of the respective primers, and 1 unit Taq polymerase (Thermo Scientific, Waltham, MA, USA). The PCR cycling conditions for every marker are described in Table 2.
The amplification products were analyzed based on electrophoresis in a 1.5% agarose gel in TAE buffer.

2.4. SNP Genotyping

The fire blight resistance loci FB_MR5 [35,36] and RLP1 [9] were analyzed in the present rootstock types based on the 5 SNP markers described in Table 3. These markers have previously been validated by screening 240 phenotyped individuals Malus × domestica Borkh [19]. The genotyping procedure was conducted according to the protocol described by Chagné et al. The design of primers and probes was implemented using the Custom Taqman® Assay Design Tool with Taqman assay ID (Table 3).
The analysis of the results was carried out using both the QuantStudio™ 12K Flex v1.2 and the Taqman® Genotyper v1.7.1 software (Thermo Fisher Scientific, Waltham, MA, USA). The autocalled genotypes were manually examined by verifying the real-time trace and the endpoint call [19].

3. Results

3.1. Observing and Tracking the Occurrence of Fire Blight in Rootstock Fields

Disease monitoring was conducted in four different fields of eleven rootstock types for two years (2021–2022), considering the weather conditions.
The DDI in 2021 was slightly higher in the ‘B.9’ and ‘Zh.5’ fields compared to other rootstocks, with the index of 7.1% and 6.3% respectively. Less than 5% of disease distribution was observed in remaining rootstock types. Considering the results of DDI in 2022, the highest DDI was observed in the ‘B.9’ field, with a disease prevalence of 16.9%. According to the DDI scale, the field received a rating of 2, which corresponded to the brown polygon (Figure 1, Table 4). ’62-396’, ‘G.41’, and ‘G.16’ showed zero prevalence of disease and corresponded to light green polygons. The DDI among other rootstocks remained below 10% and indicated green-brown polygons (Figure 1).
Evaluation of the DDI results showed that fire blight occurrence was nearly twice as high in 2022 than in 2021 (Table 4). However, no severe infection was detected in any of the fields in 2022 because any trees identified as pathogen-positive based on qPCR testing were immediately eliminated.
The maximum DSI was less than 5%. Real-time PCR analysis was conducted using the primers hpEaF and hpEaR and the probe hpEaP targeting E. amylovora chromosomal DNA [33]. All apple rootstocks exhibiting fire blight symptoms were analyzed by qPCR testing. The maximum DSI value of 1.87 was recorded for the B9 rootstock in the AIL field. DDI values of 1 were obtained for eight rootstocks except 62-396, G.41, and G.16, all of which showed zero DDI (Figure 1).
A strong correlation between levels of infection and weather conditions has previously been demonstrated. Researchers have found that temperature, humidity, and precipitation are the most important factors for disease development [37,38]. The temperature and air humidity in early spring are crucial because bacteria overwintering in cankers begin to spread by insects, birds, rain, wind, and equipment. Wounds in shoots occur during mechanical treatments of trees, and roots act as a gateway for the entrance of pathogens into rootstocks [16,39].
In the current study, the highest percentage of disease distribution was recorded in 2022 and was accompanied by increased precipitation and air humidity during springtime (Figure 2).
Compared with 2021, slight increases in temperature of +4.2 and +3.9 °C were recorded in April of 2022 in the Almaty and Zhambyl regions, respectively. Humidity was also at least 10% higher in the spring of 2022 in both regions, with precipitation at twice the level in 2021.

3.2. Genetic Determinants Influencing Resistance to Erwinia amylovora in Apple Rootstocks

Eleven types of apple rootstocks, developed by crossing genetically dissimilar parents, were analyzed to reveal the carriers of resistant alleles to E. amylovora, which causes fire blight in apple trees.
The SNP markers AH0JFXM, AH21B92, AH4AAGA, AH5I8MI, and AH6R6SQ—for confirming fire blight-resistant loci situated on LG3—and the SCAR markers AE10-375 and GE-8019—to verify the presence of the pathogen-resistant locus FB_F7—were analyzed in the present study. We identified the resistant allele of SCAR marker AE10-375 in rootstocks ‘MM106’, ’62-396’, and ‘Geneva 16’ (Table 5 and Table S1, Figure 3 and Figure 4). The resistant form of GE-8019 was not found to be present in the analyzed samples.
The presence of dominant alleles of the markers AE10-375 and GE-8019, 375 and 397 bp in length, respectively, are indicative of resistant genotypes, as previously shown in the progeny of two crosses between the cultivar ‘Fiesta’ and either the ‘Discovery’ or ‘Prima’ cultivars [6,10]. Those genotypes with high resistance to the pathogen have previously been shown to harbor resistance alleles for both markers, in contrast to less-resistant genotypes bearing a resistance allele for only one of the two markers [10]. In the present study, no specimens were found to bear resistance alleles for both markers.
The researched SNP markers were identified in the ‘Geneva 41’, ‘Geneva 16’, and ‘62-396’ rootstocks (Figure 4, Table 5 and Table S1). G.41 harbors resistant alleles for all markers because the apple rootstock is a progenitor of Malus × robusta ‘Robusta 5’, which is the origin of LG3 resistance [19]. The resistant form of the four markers (except AH6R6SQ) were identified in the ‘62-396’ rootstock, which is used only rarely for grafting of commercial cultivars in Kazakhstan compared with ‘MM106’ and ‘M9’. There was no evidence that the ‘MM106’ and ‘M9’ rootstocks carry any of the resistant forms of SNP markers, and only MM106 was positive for the allele of SCAR marker AE10-375. G.16 was found to harbor the resistant allele for marker AH4AAGA. None of the resistant forms of the researched markers were identified in the ‘Arm18’, ‘B9’, Zh.5, ‘M9’, ‘B.7-35’, M. siversii, and ‘B16-20’ rootstocks. M. siversii, randomly collected from the field, was represented by 10 different genotypes.
The local rootstocks Zh.5 and especially M. siversii are starting to become more popular in Kazakhstan because they are much better adapted to local environmental conditions. However, the genetic background of local apple rootstocks has still not been fully characterized; indeed, it has been barely researched to date. Consequently, the local breeding of pome fruit crops is based mainly on phenotypic data.
The two-year survey showed that the rootstocks G.41, G.16, and ‘62-396’ demonstrated the best resistance to fire blight despite outbreaks in neighboring plots of other rootstocks in both 2021 and 2022. It is important to mention that G.16 carries the resistant allele for only one SNP marker; nevertheless, its DSI was zero. Fire blight outbreaks were recorded for ‘MM106’, positive for marker AE10-375, and non-marker resistant rootstock types in the fields (Table 4). The greater spread of infection occurring in 2022 is because of increased air humidity, temperature, and precipitation. Our assessment is that the natural spread of the pathogen among the fields and of resistance of rootstocks to E. amylovora was not fully fledged because infected trees were immediately eliminated following identification based on PCR testing.

4. Discussion

Clonal rootstocks of apple trees are one of the main components of intensive horticulture, and they play an important role in regulating tree growth, yield, fruit quality, and resistance to biotic and abiotic stresses [40,41]. Since 1917, when apple rootstock breeding began at the East Malling Station, the development of improved rootstock types in many traits has continued. In the process of apple rootstock breeding, it is crucial to initially focus on horticultural characteristics, such as grafting compatibility, reproductive ability, and dwarfism, and their effects on scion growth. Regarding the area for planting apple trees, significant attention should be focused on both environmental factors and biological factors such as viruses, bacteria, nematodes, and pests [42].
European countries have mostly focused on the breeding and planting of dwarf rootstocks, which have advantageous characteristics in terms of production, fruit quality, resistance to diseases and pests, and competition with weeds for water and nutrients [43]. In Eastern Europe, farmers have additionally sought to breed rootstocks with cold tolerance and easy propagation [44]. In North America and Canada, the focus has been mainly on dwarfism and freezing tolerance [29]. In Kazakhstan, farmers have valued rootstock types that have advantageous characteristics in fruit quality and cold tolerance. Only recently, they have sought to propagate disease-resistant rootstocks. The main diseases spread in apple gardens in Kazakhstan are fire blight [5,43,45], apple scab [46,47], powdery mildew [48,49], and diseases of viral origin [50,51,52].
Because fire blight is widespread in Kazakhstan, farmers have aimed to cultivate apple trees comprising rootstock and scion that both have pathogen resistance.
LG3 [15], LG12 [7,53], and LG10 [8], derived from wild apples, are loci that have been allocated and confirmed as conferring fire blight resistance. Moreover, the QTL FB_F7, identified on LG7 of the domesticated apple cultivar ‘Cox’s Orange Pippin’, has been described as the most promising for breeding because of its moderate and stable response to disease [10,18]. Since the QTLs on LG3 and FBF7 explain a significant level of phenotypic variation, ranging from 67% to 83% and 35% to 40%, respectively, they were selected for testing using already validated markers [9]. Moreover, the local rootstocks were derived from genotypes of wild apples, such as M. sieversii and M. niedzwetzkyana, in which FBF7 has been previously identified [54,55]. Clearly, the local and Russian rootstocks were not extensively tested for other markers associated with resistance to fire blight.
In the present work, ‘MM106’, which was positive for marker AE10-375, was infected in both 2021 and 2022, whereas G.41, which was negative for QTL FB_F7 and positive for five SNP markers associated with LG3, was resistant. The presence of resistance alleles for both markers AE10-375 and GE-8019 in the genotypes has previously been shown to indicate high resistance to the fire blight pathogen [10], but the rootstock types analyzed here were found to be negative for marker GE-8019. A similar level of spreading and severity of disease was observed for both positive and negative AE10-375 rootstock types; however, rootstocks additionally bearing resistant alleles for SNP markers, such as G.16 and ‘62-396’, showed no sign of infection in the fields.
The resistance of G.41, G.16, and B.9 to fire blight was previously demonstrated in field surveys by grafting different cultivar scions [30,56,57,58]. In the current study, a restrained distribution of disease was shown only for the G.41, G.16, and ’62-396’ fields, while the B.9 field was the most infected. The susceptibility of B.9 might be explained on the basis of non-grafted rootstocks [59], considering that resistance in the field was only confirmed for grafted B.9 in an early study [30]. Further investigations should be carried out in fields with both grafted and non-grafted B.9 rootstock.
A recent study on the genetic background of resistance of rootstock ‘62-396’ revealed the presence of QTL FB_F7 and an additional resistant allele tested by SSR marker CH-F7-FB1 [60]. However, information about the resistance of rootstock to fire blight in greenhouses or in fields was not provided. In the current study, as a result of our two years of field monitoring, we demonstrated for the first time that ‘62-396’ carries four resistant alleles of five SNP markers and is resistant to fire blight. We assume that Mr5 was included in the breeding process during the development of ‘62-396’ [61]. Resistant loci on LG3 and LG7 cause the ‘62-396’ rootstock to be of interest to current breeding programs and grafting because these loci explain up to 80% [61] and 46% [10,18] of the phenotyping variation, respectively.
Despite the enormous potential of wild apples, the local rootstocks Zh.5 and M. sieversii were found to be susceptible and to lack the investigated markers. Because M. sieversii is represented by different genotypes, the evaluation of resistance to fire blight of the species is not possible, particularly when genotype by environment interactions significantly contribute to the resistance [62,63]. Diverse levels of fire blight resistance among M. sieversii accessions have also been previously reported [13,23,25,26,27,28,44].
Two years of monitoring enabled us to distinguish whether rootstocks were susceptible or resistant to fire blight when grown in the climate conditions of the Kazakhstan southern region. Among the eleven studied rootstocks, we found G.41, G.16, and ‘62-396’ to be the most promising for propagation and grafting because they were not infected during the monitoring period despite the weather conditions in 2022 being favorable for the development and spreading of E. amylovora. All other rootstocks showed higher levels of infection in 2022 compared with 2021.
The present study is the first to evaluate the fire blight resistance of foreign and local rootstocks in the conditions of the Kazakhstan southern region, considering their genetic background and climate conditions. Previous works have mostly focused on testing M9 and MM106, which were introduced to the fields of Kazakhstan many years ago.

5. Conclusions

We conclude that the rootstocks G.41, G.16, and ‘62-396’ are the most promising for propagation, breeding, and grafting in Kazakhstan because they were found to be most resistant to fire blight based on the results of the present study in which their genetic background and the local climate conditions were considered. The productive characteristics of these rootstocks are comparable to those recognized in the worldwide dwarf rootstocks M9 and MM106. However, the local rootstock Zh.5 and M. sieversii were both found to be susceptible and exhibit a lack of resistance alleles, as tested by SNP genotyping and SCAR amplification.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app132011530/s1, Table S1. SNP genotyping data, genotype matrix.

Author Contributions

Writing—Original draft preparation, funding acquisition project administration, G.K.; Software, A.P.; Methodology and investigation, N.D., M.K. and G.N.; Data curation, M.S.; Formal analysis, N.S.; Visualization and supervision and resources, Z.S.; Conceptualization and writing—review and editing, D.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research and APC were funded by the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan (Grant No. AP09259636 “Study of the genetic resistance of promising Apple varieties and rootstocks to a dangerous disease-fire blight using SNP markers”).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interests.

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Applsci 13 11530 g001
Figure 1. Distribution of fire blight among eleven rootstock types on four fields in 2022. DDI-disease distribution index.
Figure 1. Distribution of fire blight among eleven rootstock types on four fields in 2022. DDI-disease distribution index.
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Figure 2. Weather conditions for two-year monitoring of apple rootstocks in the Almaty and Zhambyl regions.
Figure 2. Weather conditions for two-year monitoring of apple rootstocks in the Almaty and Zhambyl regions.
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Figure 3. SNP-genotyping by five markers associated with resistant loci to fire blight.
Figure 3. SNP-genotyping by five markers associated with resistant loci to fire blight.
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Figure 4. Resistance of apple rootstocks with respect to genetic background. The green color indicates resistant rootstocks; light orange indicates susceptible MM106.
Figure 4. Resistance of apple rootstocks with respect to genetic background. The green color indicates resistant rootstocks; light orange indicates susceptible MM106.
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Table 1. Main characteristics of clonal apple rootstocks.
Table 1. Main characteristics of clonal apple rootstocks.
DesignationParentage and OriginShort DescriptionField *References
62-39613–14 × “Paradise Budagovskij”, Michurinsk State Agrarian University, Russia. Approved for use in the southern region of Kazakhstan since 1997. Dwarfing. The rootstock is char acterized by high resistance to frost, cold, and drought. Cultivars grafted on this rootstock are early-bearing and productive. In the nursery, it provides a high yield of seedlings.1[23]
M9Source is not confirmed, East Malling Research Station, England. Approved for use in the southern region of Kazakhstan since 1961.Dwarfing. It is characterized by a shallow root system. Cultivars grafted on this rootstock require physical support and do not regularly bear fruit. The rootstock is not sufficiently cold-resistant for planting in northern regions of Kazakhstan. 1; 3[24]
MM 106Northern Spy × M.1, East Malling Research Station, England. Approved for use in 1961 in the southern region of Kazakhstan.Semi-dwarfing. The rootstock is not sufficiently drought- and frost-resistant. Cultivars grafted on this rootstock begin bearing fruit in 4–5 years.3; 4[24]
Arm 18Free pollination of M9. Scientific Center of Viticulture Fruit-Growing and Wine-Making, Armenia. Approved for use since 1997 in the southern region of Kazakhstan.Dwarfing. The rootstock is highly frost-resistant, productive in the nursery, and shows excellent compatibility in rootstock–scion interactions.1[25]
B.7-35M4 × M-9, Buynaksk Experimental Production Station, Russia. Approved for use in 1991 in the southern region of Kazakhstan.Dwarfing. The rootstock is characterized by high winter hardiness, productivity in the nursery, robust growth and development, and rootstock–scion compatibility.1[26]
B.16-20M;4 × M-9, Buynaksk Experimental Production Station, Russia.
Approved for use in 1991 in the southern region of Kazakhstan.		Semi-dwarfing. The rootstock is characterized by high winter hardiness and productivity.1[27]
Zhetysu 5
(Local breeding)		The crossing of a semi-dwarf rootstock 57-146 with Malus niedzwetzkyana by open pollination, Kazakh Research Institute of Fruit Growing and Viticulture, Kazakhstan. Approved for use since 2009 in the southern region of Kazakhstan.Semi-dwarfing. The rootstock is characterized by rapid development, drought resistance, and sufficient production of mother plants. Fruit-bearing occurs 5 years after planting of cultivars grafted onto this rootstock.1[28]
Malus sieversiiFree pollination of wild apple.
Approved for use in 1961 in the southern and eastern regions of Kazakhstan.		Vigorous. Seedling rootstock is included on the Red List of rare and endangered plant species. The rootstock is drought- and frost-resistant.3Not published
Geneva 41
(G 41)		Malling 27 × M. robusta ‘Robusta 5’, Cornell University, USA.Dwarfing. The rootstock is highly resistant to fire blight and cold. 2[14,22,29]
Geneva 16 (G 16)Ottawa 3 × Malus floribunda, Cornell University, USA.Dwarfing. Resistant to fire blight and scab but susceptible to powdery mildew and latent viruses. Productive and drought-resistant.2[14,22,29]
B.9 M8 × Red standard, Michurinsk State Agrarian University, Russia.Dwarfing. The rootstock is highly frost-resistant. Cultivars grafted onto this rootstock begin bearing fruit in 3–4 years.2[30]
Note *—1. Kazakh Scientific Research Institute of Fruit and Vegetable Growing, Talgar, Almaty region. 2. Agricultural Innovations Limited, Almaty region. 3. Sady Zhetysu Trade, Almaty region. 4. Devrosh, Zhambyl region.
Table 2. Marker, F-forward and R-reverse primer sequences, PCR cycling program.
Table 2. Marker, F-forward and R-reverse primer sequences, PCR cycling program.
Gene, LocusMarkerPrimer Sequence (5′–3′)PCR Cycling Program
F7 QTLAE10-375CTGAAGCGCACGTTCTCC-F
CTGAAGCGCATCATTTCTGATAG-R		1× 95 °C—3 min, 35× (95 °C—40 s; 60 °C—40 s; 72 °C—60 s), 1× 72 °C—10 min.
F7 QTLGE-8019TTGAGACCGATTTTCGTGTG-F
TCTCTCCCAGAGCTTCATTGT-R		1× 95 °C—3 min, 35× (95 °C—40 s; 60 °C—40 s; 72 °C—60 s), 1× 72 °C—10 min.
Table 3. Description of the five single-nucleotide polymorphisms (SNPs) analyzed in rootstock types.
Table 3. Description of the five single-nucleotide polymorphisms (SNPs) analyzed in rootstock types.
SNP MarkerLG *Gene/Locus NameSNP ID Taqman Assay IDSNP TypeReference
FB-MR5-K353MR5FB-MR5-NZsnEH034548_K35AH0JFXMG/T[35]
FB-MR5-R2493MR5FB-MR5-NZsnEH034548_R249AH21B92A/G[35]
FB-MR5-rp16k15_M1063MR5FB-MR5-rp16k15_M106AH4AAGAA/C[35]
RLP1a3RLP1RLP1aAH5I8MIC/A[9]
RLP1b3RLP1RLP1bAH6R6SQA/T[9]
* LG—linkage group.
Table 4. Disease distribution index values in 2021 and 2022.
Table 4. Disease distribution index values in 2021 and 2022.
RegionRootstock20212022
Almaty62-39600
M91 (3.2%)1 (7.1%)
Apm 181 (5.4%)1 (8.5%)
G.4100
G.1600
B.91 (7.1%)2 (16.9%)
Zh.51 (6.3%)1 (9.7%)
B.7-351 (4.1%)1 (7.4%)
B.16-201 (2.7%)1 (5.2%)
M. siversii1 (3.1%)1 (6.8%)
Almaty, Turkistan MM1061 (2.2%)1 (5.0%)
Table 5. Results of SCAR amplification and SNP genotyping.
Table 5. Results of SCAR amplification and SNP genotyping.
Rootstock NameSCAR MarkersSNP Markers
AE10-375 GE-8019 AH0JFXMAH21B92AH4AAGAAH5I8MIAH6R6SQ
62-396375 bp -G/TA/GA/CC/AA/A
M9--T/TG/GC/CC/CA/A
Apm 18--T/TG/GC/CC/CA/A
G.41--G/TA/GA/CC/AA/T
G.16375 bp -T/TG/GA/CC/CA/A
B.9--T/TG/GC/CC/CA/A
Zh.5--T/TG/GC/CC/CA/A
B.7-35--T/TG/GC/CC/CA/A
B.16-20--T/TG/GC/CC/CA/A
M. siversii--T/TG/GC/CC/CA/A
MM106375 bp -T/TG/GC/CC/CA/A
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Kairova, G.; Pozharskiy, A.; Daulet, N.; Solomadin, M.; Sandybayev, N.; Khusnitdinova, M.; Nizamdinova, G.; Sapakhova, Z.; Gritsenko, D. Evaluation of Fire Blight Resistance of Eleven Apple Rootstocks Grown in Kazakhstani Fields. Appl. Sci. 2023, 13, 11530. https://doi.org/10.3390/app132011530

AMA Style

Kairova G, Pozharskiy A, Daulet N, Solomadin M, Sandybayev N, Khusnitdinova M, Nizamdinova G, Sapakhova Z, Gritsenko D. Evaluation of Fire Blight Resistance of Eleven Apple Rootstocks Grown in Kazakhstani Fields. Applied Sciences. 2023; 13(20):11530. https://doi.org/10.3390/app132011530

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Kairova, Gulshariya, Alexandr Pozharskiy, Nurzhan Daulet, Maxim Solomadin, Nurlan Sandybayev, Marina Khusnitdinova, Gulnaz Nizamdinova, Zagipa Sapakhova, and Dilyara Gritsenko. 2023. "Evaluation of Fire Blight Resistance of Eleven Apple Rootstocks Grown in Kazakhstani Fields" Applied Sciences 13, no. 20: 11530. https://doi.org/10.3390/app132011530

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