Analysis of polymorphism of strawberry genotypes (Fragaria L.) according to the strawberry red root spot resistance gene RPF1 for identification of strawberry forms promising for breeding and horticulture

Red root spot (Phytophthora fragariae var. fragariae Hickman) is one of the most important strawberry diseases in the temperate climate zone. Identification of forms, carrying resistance genes, is an important stage in breeding programs aimed at obtaining red root spot resistant strawberry varieties. Diagnostic DNA markers of target genes will increase reliability of identification and efficiency of strawberry breeding for the creation of resistant genotypes. The purpose of this study is analysis of polymorphism of wild species of Fragaria L. genus and strawberry varieties (F. × ananassa Duch.) according to the strawberry red root spot resistance gene Rpf1 using molecular markers. The research sunjects were the wild species F. orientalis Los., F. moschata Duch., F. ovalis Rydb., F. virginiana Duch. ssp. platypetala and strawberry varieties (F. × ananassa Duch.) of different ecological and geographic origin. Total genomic DNA of strawberry was extracted from the fresh leaves using the Puchooa method. To assess the allelic state of Rpf1 gene, SCAR-R1A marker (linked to the Rpf1 dominant allele) and OPO-16C marker (linked to the rpf1 recessive allele) have been used. SCAR-R1A marker was identified in wild species F. virginiana Duch. ssp. platypetala (vegetation region: British Columbia, Canada), pineapple strawberry varieties Bylinnaya and promising selected forms 62-41 (Bylinnaya × Feyyerverk), 65-17 and 65-24 (Olimpiyskaya nadezhda × Bylinnaya). These genotypes are characterized by heterozygous Rpf1rpf1 genotype according to Rpf1 gene (both markers are present in genotype) and can be used as red root spot resistance source in marker-assisted selection. In the remaining studied genotypes of strawberry, SCAR-R1A marker was not detected, which presumably indicated their homozygous recessive genotype rpf1rpf1 according to Rpf1 gene. The research results can be useful for breeders of strawberry and researchers of plant biodiversity of p. Fragaria.

Genomic investigation of the strawberry pathogen Phytophthora fragariae indicates pathogenicity is associated with transcriptional variation in three key races

ABSTRACTThe oomycete Phytophthora fragariae is a highly destructive pathogen of cultivated strawberry (Fragaria × ananassa), causing the root rotting disease, ‘red core’. The host-pathogen interaction has a well described gene-for-gene resistance relationship, but to date neither candidate avirulence nor resistance genes have been identified. We sequenced a set of American, Canadian and UK isolates of known race type, along with three representatives of the closely related pathogen of the raspberry (Rubus idaeus), Phytophthora rubi, and found a clear population structure, with a high degree of nucleotide divergence seen between some race types and abundant private variation associated with race types 4 and 5. In contrast, between isolates defined as UK races 1, 2 & 3 (UK1-2-3) there was no evidence of gene loss or gain; or the presence of insertions/deletions (INDELs) or Single Nucleotide Polymorphisms (SNPs) within or in proximity to putative pathogenicity genes could be found associated with race variation. Transcriptomic analysis of representative UK1-2-3 isolates revealed abundant expression variation in key effector family genes associated with pathogen race; however, further long read sequencing did not reveal any long range polymorphisms to be associated with avirulence to race UK2 or UK3 resistance, suggesting either control in trans or other stable forms of epigenetic modification modulating gene expression. This work reveals the combined power of population resequencing to uncover race structure in pathosystems and in planta transcriptomic analysis to identify candidate avirulence genes. This work has implications for the identification of putative avirulence genes in the absence of associated expression data and points towards the need for detailed molecular characterisation of mechanisms of effector regulation and silencing in oomycete plant pathogens.

Utilisation of immunochemical methods for detection of Colletotrichum spp. in strawberry

Four polyclonal and two monoclonal antibodies were prepared and tested to detect a quarantine pathogen of strawberry – Colletotrichum acutatum. Only one of them, polyclonal IgG K91, was sensitive enough to recognize the pathogen. This antibody was genus-specific and did not cross-react with several other fungal pathogens of strawberry (Phytophthora fragariae, P. cactorum, Verticillium albo-atrum, Botrytis cinerea, Pythium ultimum). Four techniques, PTA-ELISA, dot blot, immunoprint and immunofluorescent microscopy were used to test the specifity and sensitivity of antibodies. After artificial infection of strawberry (cvs Elsanta, Vanda, and Kama), Colletotrichum acutatum was detected by PTA-ELISA, dot blot and immunoprint in roots, crowns, petioles and fruits in the latent stage of the disease. For reliable detection in the latent stage it is recommended to use at least two of the mentioned techniques.

Methods for detection of Phytophthora fragariae var. rubi on raspberry

Phytophthora fragariae var. rubi (Wilcox & Duncan), a causal agent of raspberry root rot, is a serious soil-borne pathogen listed by EPPO as an A2 quarantine pest. Root samples were collected from badly diseased raspberry plants showing a variety of characteristic and often dramatic symptoms during surveys carried out in western Serbia in 2002. Identification of the causal agent was performed in collaboration work with the Scottish Crop Research Institute (S.C.R.I.), Dundee, UK. Necrotic roots were plated on selective French bean agar (incorporating ampicilin, ryfamicin, bavistin and hymexasol). Detection of isolates was based on cultural and morphological features compared with referent cultures. DNA was extracted directly from the sampled roots using extraction buffer (200 mM Tris-HCl pH 8.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS), purified by multi spin separation columns [Thistle Scientific (Axygen)] or in 24:1 mixture of chlorophorm-isoamyl alcohol and amplified by nested PCR (ITS 4 and DC 6 for first round, DC 1 and DC 5 for second round). Diluted DNA extracts were also amplified by conventional PCR with modified 'universal' Phytophthora primers (ITS 6, ITS 7 and ITS 8, Cooke et al., 2000) and digested with Msp1. Digestion patterns of the universal primers PCR product from infected roots matched those of Scottish strains. P. fragariae var. rubi occurred on 8 out of 14 sites. Our results indicate that nested PCR (ITS 4 and DC 6 for first round, DC 1 and DC 5 for second round) or digestion of the 'universal' Phytophthora primers PCR product for detection of P. fragariae var. rubi are more sensitive and less time-consuming and therefore recommended for use.