Red Stele Root Rot Research Articles

Identification of <i>Rca2</i> and <i>Rpf1</i> resistance alleles in strawberry breeding forms of intervarietal hybrid origin

Background. The creation of varieties with genetic resistance to fungal pathogens is one of the most important directions in strawberry breeding. A promising direction of accelerated selection of valuable specimens is molecular screening for the presence in their genotype of diagnostic DNA markers, linked to target genes of agrobiological traits. Objectives of the work were determination of the allelic state of the Rca2 (resistance to anthracnose) and Rpf1 (resistance to red stele root rot) genes in selected strawberry forms to identify genotypes resistant to Colletotrichum acutatum Simmonds and Phytophthora fragariae var. fragariae Hickman.Material and methods. The object of research was breeding forms of strawberries of intervarietal hybrid origin. As a result of 22 crossing combinations, 61 selection forms were obtained, in which the allelic composition of resistance genes was studied. The Rca2 anthracnose resistance allele was identified by means of the STS-Rca2_240 marker. The Rpf1 red stele root rot resistance allele was identified with the help of SCAR-R1A marker. Results. At least one of the two genes was identified in 20 selected strawberry forms (32.8%). The STS-Rca2_240 marker was identified in 15 strawberry seedlings (22.9%) from six hybrid combinations ‘Vicoda’ × ‘Roxana’, ‘Florence’ × ‘Faith’, ‘Asia’ × ‘Aprica’, ‘San Andreas’ × ‘Monterey’, ‘Alisa’ × ‘Quicky’, ‘Quicky’ × ‘Olympia’). The SCAR-R1A marker was identified in five strawberry seedlings (8.2%) from four hybrid combinations (‘Privlekatelnaya’ × ‘Bylinnaya’, ‘Feyerverk’ × ‘Bylinnaya’, ‘Bylinnaya’ × ‘Feyerverk’, ‘Bylinnaya’ × ‘Olimpiyskaya nadezhda’). It has been established that all strawberry selected forms with identified marker fragments for resistance genes are characterized by a heterozygous combination of alleles.Conclusions. Promising sources of resistance alleles are strawberry selected forms: 2/1-24, 2/1-34 (‘Quicky’ × ‘Olympia’), 3/9-5 (‘Florence’ × ‘Faith’), 3/12-2, 3/12- 9 (‘Alisa’ × ‘Quicky’), 4/7-10, 4/7-19, 4/7-20 (‘Asia’ × ‘Aprica’), 5/2-26, 5/2-32 (‘San Andreas’ × ‘Monterey’), 9/ 1-11, 9/1-12, 9/1-13, 9/1-32, 9/1-37 ‘Vicoda’ × ‘Roxana’) in case of Rca2 gene; 72-24, 72-71 (Privlekatelnaya × Bylinnaya), 69-29 (‘Feyerverk’ × ‘Bylinnaya’), 62-41 (‘Bylinnaya’ × ‘Feyerverk’), 61-15 (‘Bylinnaya’ × ‘Olimpiyskaya nadezhda’) in case of Rpf1 gene.

Genes and Loci Controlling the Resistance of Strawberry (F. ananassa Duch.) to Pathogens

Strawberry (F. ànanassa) is one of the most popular and widespread berry crops in the world. Strawberry diseases are the main limiting factor that seriously affects the yield of plants and leads to economic losses. Most strawberry cultivars are very sensitive to pathogenic fungi and bacteria, such as Colletotrichum acutatum (anthracnose root), Colletotrichum gloeosporioides (crown rot), Fusarium oxysporum fragariae (fusarium wilt), Phytophthora fragariae (red stele root rot), Verticillium dahlia (verticillium wilt), Botrytis cinerea (grey mould) and Xanthomonas fragariae (angular leaf spot). Currently, due to the rapid development of molecular genetics in general, and in particular knowledge related to the organization of genomes of agricultural crops, the use of DNA technologies in strawberry breeding has become possible. To identify disease resistance loci, the most popular approach was to map the loci of quantitative traits (QTL). Accurate evaluation of heritability and the number, location and effects of loci controlling traits are the basis for strategic decisions in breeding programs. The purpose of this review is to present up-to-date data on known genes and quantitative trait loci (QTL) that control the resistance to the most common strawberry pathogens.

Analysis of the inheritance of the marker SCAR-R1A, linked to the Rpf1 red stele root rot resistance gene, in strawberry hybrid progeny

Background. Resistance to pathogens is an important breeding trait of a cultivar. Red stele root rot (Phytophthora fragariae var. fragariae Hickman) is a dangerous root disease. Revealing the patterns of resistance inheritance and identifying promising genotypes is an important stage in the development of strawberry cultivars resistant to red stele root rot. The purpose of the study was to identify patterns of inheritance for the SCAR-R1A marker, linked to the Rpf1 red stele root rot resistance gene, in the strawberry hybrid combinations.Materials and methods. The target materials were the strawberry cultivars ‘Bylinnaya’, ‘Olimpiyskaya Nadezhda’, ‘Privlekatelnaya’ and ‘Feyyerverk’, and hybrid seedlings of the cross combinations Bylinnaya × Olimpiyskaya Nadezhda, Bylinnaya × Feyyerverk, Olimpiyskaya Nadezhda × Bylinnaya, Privlekatelnaya × Bylinnaya, and Feyyerverk × Bylinnaya. The Rpf1 gene was identified with the marker SCAR-R1A.Results and conclusion. For the hybrid combination Bylinnaya × Olimpiyskaya Nadezhda, the percentage of seedlings with an Rpf1 resistance allele was 33.3%. For the combination Bylinnaya × Feyyerverk, their percentage was 37.2%; for Olimpiyskaya Nadezhda × Bylinnaya, 39.4%; for Privlekatelnaya × Bylinnaya, 39.6%; and for Feyyerverk × Bylinnaya, 36.2%. The average percentage of seedlings with an Rpf1 allele for the studied combinations was 37.1%. Assessment of the compliance between the observed segregation and theoretical one according to the χ2 criterion confirmed the monogenic character of the studied trait and the Mendelian ratio of inheritance frequencies for the marker fragments of the Rpf1 gene as 1 : 1. Therefore, all identified seedlings with an Rpf1 allele are characterized by a heterozygous genotype. Strawberry hybrids promising for breeding were identified: 62-41 (Bylinnaya × Feyyerverk), 65-17, 65-24 (Olimpiyskaya Nadezhda × Bylinnaya), and 69-29 (Feyyerverk × Bylinnaya).

Assessment of the adaptive potential of strawberry genotypes based on the development of endophytic microbiota and identified R-genes

The paper presents the results of a study of the strawberry genotypes (Fragaria L.) by characteristics of endophytic microbiota and genetic determinants of resistance to fungal pathogens. Highly adaptive genotypes were identified: F. virginiana Duch. ssp. platypetala, F. orientalis Los., F. ovalis Rydb., F. moschata Duch., 298-19-9-43, Urozhaynaya CGL, Flora, Privlekatelnaya and Troitskaya. These strawberry forms are characterized by a high frequency of testing of bacterial microbiota, which suppresses endophytic fungal pathogens. The wild strawberry F. virginiana Duch. ssp. platypetala is characterized by red stele root rot resistance (Rpf1 gene). Strawberry varieties Borovitskaya and Elianny are characterized by anthracnose resistance (Rca2gene).

Marker controlled screening of resistant to red stele root rot (Rpf1 gene) strawberry selected forms

The results of the analysis of allelic polymorphism of the Rpf1 red stele root rot resistance gene in strawberry hybrid forms using diagnostic DNA markers were shown. The recessive homozygous genotype (rpf1rpf1) was identified in strawberry seedlings 932-29 (F. virginiana subsp. platypetala (Rydb.) Staudt × Feyerverk), 933-4 (F. virginiana subsp. platypetala (Rydb.) Staudt × Rubinovyy kulon), 62-7, 62-23 (Bylinnaya × Feyerverk), 65-2, 65-15 (Olimpiyskaya nadezhda × Bylinnaya) and 35-16 (922-67 × Maryshka). The heterozygous state of the Rpf1 gene was identified in strawberry forms 69-29 (Feyerverk × Bylinnaya), 62-41 (Bylinnaya × Feyerverk), 72-24, 72-71 (Privlekatelnaya × Bylinnaya), 65-17, 65-24 (Olimpiyskaya nadezhda × Bylinnaya), which allows them to be used in breeding for resistance to P. fragariae var. fragariae as valuable initial forms.

Analysis of strawberry promising varieties and selected forms by resistance to red stele root rot using molecular markers

The analysis of the allelic state of the Rpf1 red stele root rot gene in 14 promising foreign strawberry varieties and 6 selected forms of the I.V. Michurin FSC breeding was completed. The Rpf1 gene in a heterozygous state was identified in strawberry forms 61-15 (Bylinnaya × Olimpiyskaya nadezhda), 69-29 (Feyerverk × Bylinnaya), and 72-71 (Privlekatelnaya × Bylinnaya), which makes it possible to recommend them for involvement in breeding work to create resistant to P. fragariae var. fragariae strawberry varieties. Strawberry varieties Lebedushka, Elianny, Florence, Malwina, Monterey, Polka, Verona, Vima Tarda Asia, Chamora Turusi, Clery, Flamenco, Salsa and Symphony, and selected forms 56-5 (Gigantella Maxim × Privlekatelnaya), 69-42 (Feyerverk × Bylinnaya) and 35-16 (922-67 × Maryshka) have a recessive homozygous genotype.

Marker-assisted screening of promising forms in the strawberry breeding

The results of the use diagnostic DNA-markers in the breeding of strawberry (Fragaria × ananassa Duch.) were shown. The carriers of target alleles of red stele root rot resistance (F. virginiana Duch. ssp. platypetala, Bylinnaya, 69-29 (Feyerverk × Bylinnaya), 72-24 and 72-71 (Privlekatelnaya × Bylinnaya)), anthracnose resistance (Borovitskaya, Sudarushka, Elianny, Troubadour, 933-4 (F. virginiana Duch. ssp. platypetala × Rubinovyy kulon)), high mesifurane content in fruits (F. orientalis Los., F. moschata Duch., F. virginiana Duch. ssp platypetala, Lastochka, Torpeda, Flora, Samson, 932-29 (F. virginiana Duch. ssp. platypetala × Feyerverk), 56-5 (Gigantella × Privlekatelnaya)) and γ-decalactone content in fruits (F. orientalis Los., F. moschata Duch., F. ovalis Rydb., Bylinnaya, Kupchikha, Sonata, Vima Tarda) were identified. These genotypes are valuable initial forms for involvement in the breeding process to improve the strawberry assortment.

Молекулярно-генетический анализ отборных и элитных форм земляники по генам резистентности <i>Rpf1</i> и <i>Rca2</i>

Исследования проведены в 2019 – 2020 гг. в Тамбовской области. В качестве биологических объектов использованы перспективные отборные и элитные формы земляники садовой селекции Федерального научного центра им. И. В. Мичурина. Показаны результаты молекулярно-генетического анализа отборных и элитных форм земляники по генам устойчивости к фитофторозному увяданию Rpf1 и антракнозу Rca2. Маркер SCAR-R1A, сцепленный с геном Rpf1, выявлен у форм 62-41 (Былинная × Фейерверк), 69-29 (Фейерверк × Былинная) (предполагаемый генотип по гену устойчивости — Rpf1rpf1). Маркер STS-Rca2_240, сцепленный с геном Rca2, выявлен у отборной формы 933-4 (Fragaria virginiana Duch. ssp. platypetala × Рубиновый кулон) (предполагаемый генотип по гену устойчивости — Rca2rca2). У остальных изученных генотипов земляники маркеры SCAR-R1A и STS-Rca2_240 не выявлены (предполагаемый генотип по локусам устойчивости — rpf1rpf1rca2rca2). Для получения перспективных форм земляники садовой, совмещающих в генотипе гены устойчивости к антракнозной чёрной гнили (Rca2) и фитофторозному увяданию (Rpf1), необходимо дальнейшее проведение целенаправленной селекционной работы с привлечением в гибридизацию идентифицированных источников целевых аллелей генов резистентности.

Development and evaluation of a recombinase polymerase amplification assay for rapid detection of strawberry red stele pathogen

Phytophthora fragariae causes drastic damage in strawberry crops. P. fragariae infects strawberry roots and causes red stele root rot. Although P. fragariae is a quarantine organism, its spread in Finland continues as more and more fields contract the disease. The spread can be halted through developing rapid and reliable detection assays. We have developed a rapid recombinase polymerase amplification (RPA) assay for P. fragariae targeting the Phytophthora mitochondrial DNA intergenic atp9-nad9 marker. The assay is DNA-extraction free and capable of detecting as low as 10 fg of P. fragariae genomic DNA. We found the assay reliable for diagnosing field plants when samples are adequately collected. We also applied the RPA assay to the detection of the pathogen in the soil through coupling the assay with baiting with the host plant. The results suggest that if only a small number of samples are analysed, the baiting results will not be reliable.

Analysis of strawberry genetic collection (Fragaria L.) for Rca2 and Rpfl genes with molecular markers

Strawberry (Fragaria x ananassa Duch.) varieties are susceptible to many fungal diseases. Identification of forms, carrying resistance genes, is an important stage in breeding programs leading to resistant varieties. The use of molecular markers allows to determine with high reliability the presence of the necessary genes in the genome and to identify promising forms. Some of the common strawberry's diseases, causing significant damage to strawberry plantations, are anthracnose (Colletotrichum acutatum Simmonds) and red stele root rot (Phytophthora fragariae var. fragariae Hickman). Dominant Rca2 gene is involved in monogenic resistance to C. acutatum pathogenicity group 2. Rpf1, Rpf2, Rpf3 genes are determined in monogenic resistance to red stele root rot. The purpose of this study was molecular genetic testing genotypes of genus Fragaria L. to identify carriers of Rca2 allele anthracnose resistance and Rpf1 allele red stele root rot resistance. The objects of study were the wild species of the genus Fragaria L. and strawberry varieties (Fragaria x ananassa Duch.) of different ecological and geographic origin. To assess allelic state Rca2 anthracnose resistance gene the dominant SCAR marker STS-Rca2_240 was used, was linked to the resistance gene Rca2 with a genetic distance of 2.8 cM. Rpf1 gene red stele root rot resistance was identified with the dominant SCAR marker R1A, was linked to the resistance gene Rpf1 with a genetic distance of 3.0 cM. The resistant allele of the marker STS-Rca2_240 was identified in the Laetitia variety (Rca2Rca2 or Rca2rca2 genotype), which allows us to recommend it as a promising source in breeding for anthracnose resistance. The other studied forms have homozygous recessive state of the marker STS-Rca2_240 (putative genotype rca2rca2). The resistant allele of the marker SCAR-R1A in the varieties and wild species of strawberry under study is absent, which presumably indicates their homozygous recessive genotype of Rpf1 gene (rpf1rpf1).

Development of SCAR Markers Linked to a Phytophthora fragariae Resistance Gene and Their Assessment in European and North American Strawberry Genotypes

Two dominant sequence characterized amplified region (SCAR) markers (linked at 3.0 cM, coupling phase) were constructed for the strawberry (Fragaria ×ananassa Duch.) gene Rpf1. This gene confers resistance to red stele root rot, caused by the soil-born fungus Phytophthora fragariae Hickman var. fragariae. The SCAR markers were developed originally from the sequence of RAPD OPO-16C(438) that is linked in repulsion phase to the Rpf1 allele. This SCAR primer set produced multiple bands in the resistant test progeny and in some of the susceptible progeny; therefore, new SCARs were developed based on the sequence differences among these bands. These new SCARs were linked in coupling phase to the Rpf allele and mapped to the same location as the original RAPD OPO-16C(438). The SCAR markers, as well as some additional RAPD markers known to be linked to Rpf1, were shown to be highly conserved in linkage to the gene based on examination of 133 European and North American Fragaria L. sp. cultivars and breeding selections. These flanking RAPD and SCAR-PCR markers can be used in breeding programs for the selection of red stele (Rpf1) resistance.

Molecular Markers Linked to Phytophthora fragariae Resistance Genes in Strawberry

The commercial strawberry (Fragaria × ananassa Duch.) is susceptible to red stele root rot caused by the fungus Phytophthora fragariae Hickman var. fragariae. Characteristics of the disease are the reddened steles and “rat tail” appearance of the roots. Infected plants are dwarfed, exhibit wilting, and eventually die. Red stele resistance in strawberries and virulence of P. fragariae behave according to the gene-for-gene system. Resistance genes and their corresponding avirulence genes have been identified by screening plant roots for infection. The goal of this international research project with CPRO-DLO, The Netherlands, is to develop highly specific molecular markers for various Rpf resistance genes (Rpf1, Rpf2, Rpf3, and Rpf6) that confer resistance against P. fragariae. Bulked Segregant Analysis was used to identify RAPD and AFLP markers putatively linked to P. fragariae resistance genes. The bulked DNAs representing subsets of three F1 populations that segregated monogenically for either resistance or susceptibility to P. fragariae. The map of these Rpf loci was generated using JoinMap®. The RAPD markers linked to the resistance genes are being converted into SCAR markers, while the AFLP markers will be used as probes for the detection of P. fragariae. The application of the molecular markers linked to resistance genes will have direct applicability to strawberry breeding programs. Marker-facilitated selection of these resistance genes would allow an efficient means in the screening and selection of plant material containing these genes and help in the pyramiding for resistance to P. fragariae. Genetic improvement of the strawberry by the ability to pyramid resistance genes will contribute to the productivity of the strawberry industry worldwide.

Identification of RAPD markers linked to a Phytophthora fragariae resistance gene (Rpf1) in the cultivated strawberry

Bulked segregant analysis (BSA) was used to identify seven random amplified polymorphic DNA (RAPD) markers linked to the Rpf 1 gene. Rpf 1 confers resistance to Phytophthora fragariae var. fragariae, the causal agent of red stele root rot in Fragaria spp. The bulked DNAs represented subsets of a F1 population obtained from the cross Md683×Senga Sengana which consisted of 60 plants and segregated in a 1:1 ratio for resistance or susceptibility to race 2.3.4 isolate NS2 of P. fragariae. Seven markers were shown to be linked to Rpf 1 and were generated from four primers; five of these markers were in coupling phase and two in repulsion phase with respect to the gene. A linkage map of this resistance gene region was generated using JoinMap 2.0TM. The manner in which Rpf 1 and the linked markers co-segregated indicated that they are inherited in a disomic fashion. These markers could enable gene pyramiding and marker-assisted selection of resistance genes in strawberry breeding programmes.

Annapolis and Cornwallis strawberries

Annapolis and Cornwallis are short-day, fresh market strawberry (Fragaria × ananassa Duch.) cultivars with resistance to red stele root rot caused by Phytophthora fragariae Hickman. Annapolis, which produces large fruit, is early ripening. Cornwallis, which ripens in the early to mid-season, may have some processing applications. Key words: Fragaria × ananassa, fruit breeding, Phytophthora fragariae

‘Lester’ Strawberry

Abstract ‘Lester’ strawberry (Fragaria × ananassa Duchesne) was bred and selected to overcome a serious fault (red stele root rot susceptibility) of its productive, high-quality ‘Raritan’ parent, a major cultivar in the northeastern United States. ‘Lester’ offers strawberry growers of this region a cultivar that is resistant to red stele root rot, has attractive, good quality fruit, and ripens 2 to 3 days before ‘Raritan’. The cultivar was named for Lester W. Greeley of Farmington, N.H., who recently retired from the USDA, where he was associated with small grain, vegetable, and small fruit improvement programs for a period spanning more than 30 years. Greeley first noted the consistent plant and fruit production and high fruit recovery (often >90% marketable) of the selection later named in his honor.

The U.S. Department of Agriculture Strawberry Breeding Program1

Abstract The U.S. Department of Agriculture strawberry breeding program has introduced 61 cultivars from 6 breeding locations in its 61-year history. Fifteen to 20 of these cultivars have been widely grown, and 5 of the recent releases show exceptional promise. From its inception, the program has been aimed at producing high-quality, multipurpose cultivars with disease resistance and broad regional adaptation. Because of disease pressures, genetic resistance has been emphasized, especially for red stele root rot. The USDA has become the principal source of red stele-resistant germplasm for breeding pruposes, and has constantly refined and improved its disease-screening techniques. Red stele and Verticillium resistance have now been incorporated into everbearing strawberries adapted to the eastern United States. Investigations concerning virus detection and eradication and the propagation of virus-free stocks have led to state virus-free plant certification programs, which assure the public of production of the cleanest possible strawberry nursery stocks. The work of the USDA and cooperating state stations has played a significant role in the improvement of strawberry cultivars and the development of modern strawberry production in the United States.

‘Tribute’ and ‘Tristar’ Everbearing Strawberries1

Abstract ‘Tribute’ and ‘Tristar’ are everbearing strawberry cultivars (Fragaria × ananassa Duch.) that fruit in spring, summer, and fall (as indicated by the prefix “Tri-”). They are the first everbearing strawberries bred for culture in the eastern United States which combine resistance to red stele root rot, incited by Phytophthora fragariae Hickman, with a high degree of tolerance to verticillium wilt, incited by Verticillium albo-atrum Reincke & Berth. Their generally consistent good health, productivity, and fruit quality under a number of cultural systems suggest that these cultivars will offer new opportunities to the strawberry home gardener and commercial grower alike.

‘Scott’ Strawberry1

Abstract ‘Scott’ is a vigorous and productive strawberry cultivar (Fragaria X ananassa Duch.) with firm, large fruit. Named for Donald H. Scott, former USDA strawberry breeder known internationally for breeding disease-resistant strawberries, this is the latest in a series of cultivars resistant to red stele root rot and bred for culture in the eastern United States by the U. S. Department of Agriculture and the University of Maryland.

Reaction of Strawberry Seedlings Inoculated with Race Composites of Phytophthora fragariae Hickman1

Abstract In a study of breeding of strawberry (Fragaria × ananassa Duch.) for resistance to red stele root rot caused by Phytophthora fragariae Hickman, two different composites of races of the fungus were used to screen seedlings, one with and one without race A-5. In the absence of race A-5, all resistant parents produced between 28.6 to 72.0% resistant seedlings depending upon parental combinations. When race A-5 was included in the composite, two breeding selections that were derived from Fragaria chiloensis Yaquina A clone transmitted more resistance to their seedlings than three other parents in a diallel comparison. Mean squares and variance components were higher for general than for specific combining ability. Resistance is partially dominant for the races involved in this study.

Mass Screening of Young Strawberry Seedlings for Resistance to Phytophthora fragaria Hickman1

Abstract Young strawberry (Fragaria × ananassa Duch.) seedlings were screened over a 4-year period for resistance to a composite of races of the red stele root rot causal organism, Phytophthora fragariae. A mixture of mycelia of 5 races of the organism, agar and water used as a root dip at the time of planting into sand on 6- to 8-week-old seedlings gave uniform results in the elimination of susceptible seedlings approximately five months later. The technique lends itself to efficient and effective mass screening of strawberry seedlings for relative resistance to P. fragariae.