Fourteen primers were designed specific to SNP (G fi C mutation) in the cytochrome b gene of Venturia inaequalis, corresponding to G143A substitution related to strobilurin resistance. Specificity of the primers and amplification efficiency were preliminarily tested in conventional PCR at different annealing temperatures. The performance of several preselected primer sets was verified in real-time PCR with SYBR Green I dye. Finally, two primer sets (Ô2WtÕ and Ô5MtÕ) were successfully applied for discrimination between wild-type and mutated allele. Different sensitivity of the detection of homologous and non-homologous DNA corresponded to the difference in Ct values equalled 10.5 for Ô2WtÕ and 12.0 for Ô5MtÕ primer set. Primers specific to Mycosphaerella fijensis cytochrome b sequence (Wille et al. 2002) were applied for additional control of PCR inhibitors. The reliability of the new method was evaluated and verified using two series of reference DNA dilutions and artificial mixtures of both types of DNA. Developed real-time PCR assay was applied to measure the ratio of mutated to non-mutated allele in field samples, collected in Poland in 2009, using two series of reference DNA in every run. The measured mutation level for the samples derived from orchards with conventional chemical control was very high (50-100%). For two populations originating from one organic orchard, the measured level of mutation was 1% and 46%. The combination of molecular and traditional tests for the evaluation of mutation level and monitoring of resistance levels in orchards is recommended.
Among the fungal diseases of apple trees, serious yield losses are due to an apple scab caused by Venturia inaequalis. Protection against this disease is based mainly on chemical treatments, which are currently very limited. Therefore, it is extremely important to introduce cultivars with reduced susceptibility to this pathogen. One of the important sources of variability for breeding is the process of polyploidization. Newly obtained polyploids may acquire new features, including increased resistance to diseases. In our earlier studies, numerous tetraploids have been obtained for several apple cultivars with ‘Free Redstar’ tetraploids manifesting enhanced resistance to apple scab. In the present study, tetraploids of ‘Free Redstar’ were assessed in terms of phenotype and genotype with particular emphasis on the genetic background of their increased resistance to apple scab. Compared to diploid plants, tetraploids (own-rooted plants) were characterized with poor growth, especially during first growing season. They had considerably shorter shoots, fewer branches, smaller stem diameter, and reshaped leaves. In contrast to own-rooted plants, in M9-grafted three-year old trees, no significant differences between diplo- and tetraploids were observed, either in morphological or physiological parameters, with the exceptions of the increased leaf thickness and chlorophyll content recorded in tetraploids. Significant differences between sibling tetraploid clones were recorded, particularly in leaf shape and some physiological parameters. The amplified fragment length polymorphism (AFLP) analysis confirmed genetic polymorphism of tetraploid clones. Methylation-sensitive amplification polymorphism (MSAP) analysis showed that the level of DNA methylation was twice as high in young tetraploid plants as in a diploid donor tree, which may explain the weaker vigour of neotetraploids in the early period of their growth in the juvenile phase. Molecular analysis showed that ‘Free Redstar’ cultivar and their tetraploids bear six Rvi genes (Rvi5, Rvi6, Rvi8, Rvi11, Rvi14 and Rvi17). Transcriptome analysis confirmed enhanced resistance to apple scab of ‘Free Redstar’ tetraploids since the expression levels of genes related to resistance were strongly enhanced in tetraploids compared to their diploid counterparts.
Field resistance to white pine blister rust (WPBR) (Cronartium ribicola J. C. Fischer) was investigated on 53 black currant (Ribes nigrum L.) genotypes (cultivars and breeding selections) in 1998 and 1999. Uredia did not form on the black currant `Titania' and 17 advanced selections during field evaluations made at the Experimental Orchard at Dabrowice, near Skierniewice, Poland.
Bacterial canker, caused by Pseudomonas syringae pv. syringae and pv. morsprunorum, is one of the most important diseases of stone fruit trees (Prunus spp.). The pathogen infects buds, flowers, fruitlets, leaves and shoots, from which the disease spreads to the branches, boughs and trunks, causing necrosis and cankers. The efficacy of different chemical and biological products for the control of bacterial canker on stone fruit trees was tested in 2018–2021. The experiments were conducted in sour cherry, plum and sweet cherry orchards in central Poland. Foliar application of the tested preparations was performed three times a season. The biological efficacy of the tested products in the control of bacterial canker was evaluated on sour cherry on the basis of infected leaves and fruits and on plum and sweet cherry on the basis of infected leaves. The highest efficacy was observed for products containing various forms of copper—copper oxide, copper oxychloride and copper hydroxide—as well as fertilizers with copper gluconate and the fungicide Luna Care 71.6 WG (fluopyram and fosetyl-Al). However, the biological preparations were significantly less effective. The conducted studies showed that preparations based on copper gluconate can be a valuable alternative to typical copper fungicides.
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