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WRKY transcription factors are important players in plant regulatory networks, where they control and integrate various physiological processes and responses to biotic and abiotic stresses. Here, we analysed six rose genomes of 5 different species (Rosa chinensis, R. multiflora, R. roxburghii, R. sterilis, and R. rugosa) and extracted a set of 68 putative WRKY genes, extending a previously published set of 58 WRKY sequences based on the R. chinensis genome. Analysis of the promoter regions revealed numerous motifs related to induction by abiotic and, in some cases, biotic stressors. Transcriptomic data from leaves of two rose genotypes inoculated with the hemibiotrophic rose black spot fungus Diplocarpon rosae revealed the upregulation of 18 and downregulation of 9 of these WRKY genes after contact with the fungus. Notably, the resistant genotype exhibited the regulation of 25 of these genes (16 upregulated and 9 downregulated), while the susceptible genotype exhibited the regulation of 20 genes (15 upregulated and 5 downregulated). A detailed RT–qPCR analysis of RcWRKY37, an orthologue of AtWRKY75 and FaWRKY1, revealed induction patterns similar to those of the pathogenesis-related (PR) genes induced in salicylic acid (SA)-dependent defence pathways in black spot inoculation experiments. However, the overexpression of RcWRKY37 in rose petals did not induce the expression of any of the PR genes upon contact with black spot. However, wounding significantly induced the expression of RcWRKY37, while heat, cold, or drought did not have a significant effect. This study provides the first evidence for the role of RcWRKY37 in rose signalling cascades and highlights the differences between RcWRKY37 and AtWRKY75. These results improve our understanding of the regulatory function of WRKY transcription factors in plant responses to stress factors. Additionally, they provide foundational data for further studies. [ABSTRACT FROM AUTHOR]

Resistance responses in the leaves of eight rose genotypes, Knock Out (highly resistant), Arka Nishkant (moderately resistant), R. multiflora (highly susceptible), Arka Swadesh (highly susceptible), IIHRR 13-4 (susceptible), Arka Parimala (susceptible), R. indica (susceptible) and IIHRR 4-15-12 (moderately susceptible), exhibiting varied levels of resistance against black spot were investigated post artificial inoculation with black spot pathogen, Diplocarpon rosae. There was consistent increase in the activities of defense related enzymes such as catalase, peroxidase, polyphenol oxidase, superoxide dismutase and phenylalanine ammonia lyase and other defense related secondary metabolites like phenols and flavonoids at different phases of black spot progression and increase was high in resistant genotypes Knock Out and Arka Nishkant. The peak activity of defense enzymes and high concentration of other metabolites was witnessed during early stages of infection in the resistant genotypes while it was during later phase in the susceptible genotypes. These results suggested that the faster and stronger activation of defense system is associated with the resistance against black spot in the rose genotypes. [ABSTRACT FROM AUTHOR]

Summary: Black spot disease, which is caused by the ascomycete Diplocarpon rosae, is the most severe disease in field‐grown roses in temperate regions and has been distributed worldwide, probably together with commercial cultivars. Here, we present data indicating that muRdr1A is the active Rdr1 gene, a single‐dominant TIR‐NBS‐LRR (Toll/interleukin‐1 receptor‐nucleotide binding site‐leucine rich repeat) (TNL)‐type resistance gene against black spot disease, which acts against a broad range of pathogenic isolates independent of the genetic background of the host genotype. Molecular analyses revealed that, compared with the original donor genotype, the multiple integrations that are found in the primary transgenic clone segregate into different integration patterns in its sexual progeny and do not show any sign of overexpression. Rdr1 provides resistance to 13 different single‐spore isolates belonging to six different races and broad field mixtures of conidia; thus far, Rdr1 is only overcome by two races. The expression of muRdr1A, the active Rdr1 gene, leads to interaction patterns that are identical in the transgenic clones and the non‐transgenic original donor genotype. This finding indicates that the interacting avirulence (Avr) factor on the pathogen side must be widespread among the pathogen populations and may have a central function in the rose–black spot interaction. Therefore, the Rdr1 gene, pyramided with only a few other R genes by sexual crosses, might be useful for breeding roses that are resistant to black spot because the spread of new pathogenic races of the fungus appears to be slow. [ABSTRACT FROM AUTHOR]

Background: Black spot is one of the most severe and damaging diseases of garden roses. We present the draft genome sequence of its causative agent Diplocarpon rosae as a working tool to generate molecular markers and to analyze functional and structural characteristics of this fungus. Results: The isolate DortE4 was sequenced with 191x coverage of different read types which were assembled into 2457 scaffolds. By evidence supported genome annotation with the MAKER pipeline 14,004 gene models were predicted and transcriptomic data indicated that 88.5% of them are expressed during the early stages of infection. Analyses of k-mer distributions resulted in unexpectedly large genome size estimations between 72.5 and 91.4 Mb, which cannot be attributed to its repeat structure and content of transposable elements alone, factors explaining such differences in other fungal genomes. In contrast, different lines of evidences demonstrate that a huge proportion (approximately 80%) of genes are duplicated, which might indicate a whole genome duplication event. By PCR-RFLP analysis of six paralogous gene pairs of BUSCO orthologs, which are expected to be single copy genes, we could show experimentally that the duplication is not due to technical error and that not all isolates tested possess all of the paralogs. Conclusions: The presented genome sequence is still a fragmented draft but contains almost the complete gene space. Therefore, it provides a useful working tool to study the interaction of D. rosae with the host and the influence of a genome duplication outside of the model yeast in the background of a phytopathogen. [ABSTRACT FROM AUTHOR]

Black spot disease caused by the hemibiotrophic ascomycete Diplocarpon rosae is the most devastating disease of field-grown roses. Although resistance to black spot is an important trait for rose breeding, little information on the diversity of the pathogen is currently available. To date, a number of single-spore isolates have been characterized based on a set of test genotypes of the host. In this study, six polymorphic simple sequence repeat ( SSR) markers for D. rosae were developed and their potential application to single-spore isolates collected from a wide range of geographic locations within and outside Europe discussed. Populations of the fungus were then analysed in different German rose collections/populations. Gene diversity was highest in older rose collections managed without fungicide application, and lowest in the two- to three-year-old testing sites of German rose breeders. Additional analyses of a global collection of single samples and populations revealed no unique alleles from any of the locations, indicating that global trading of rose varieties has led to an admixture of the pathogen. [ABSTRACT FROM AUTHOR]

The study was an evaluation of the efficacy of Atpolan 80 EC (76% mineral oil SN) in the protection of ornamental plants against some pathogens - the causal agents of powdery mildew, leaf spot and rust. Depending on the target pathogen, Atpolan 80 EC was used at concentrations from 0.5 to 2% in spray treatments carried out 4 to 9 times at 7-day intervals. At each of the test concentrations, Atpolan 80 EC, used as a curative treatment of roses, significantly inhibited the development of the pathogens responsible for powdery mildew and black spot. In the control of powdery mildew (Sphaerotheca pannosa var. rosae), Atpolan 80 EC sprayed on some rose varieties 4 times at 0.5 and 0.75% showed an efficacy of 96% and 98%, respectively, while at higher concentrations completely inhibited the development of disease symptoms. Its efficacy was from 74.4 to 100%, depending on the concentration, in the protection of Rosa canina 'Fenders', a rootstock for roses. When used as a remedy to control black spot (Diplocarpon rosae), its efficacy after 6 spray treatments was from 31.5% to 85.1%, depending on the concentration used and the susceptibility of the rose variety to the pathogen. However, after 9 treatments its effectiveness was found to decrease. When used curatively on chrysanthemums, Atpolan 80 EC significantly inhibited the development of symptoms of white rust caused by Puccinia horiana. After spraying chrysanthemum plants 4 times with the oil at a concentration of 2% there was a 2.5 times reduction in the number of telia forming per leaf, and, depending on the concentration, it caused 16.6 to 100% of telia to disintegrate. There was no evidence of phytotoxicity of Atpolan 80 EC towards the plant species and varieties tested. [ABSTRACT FROM AUTHOR]

Micromycetes inhabiting the leaves of 20 cultivars of climbing roses (Rosa L.), grown in Botanic Garden of the Jagiellonian University in Cracow was investigated in the three successive years of research. Sixty-five taxa of of micromycetes was recorded with a few species dominating: Alternaria alternata, Epicoccum nigrum, Pestalotia rosae, Penicillium brevicompactum and Sordaria fimicola, accompanied by various other microfungi. A high abundance of rose black spot caused by Diplocarpon rosae was also observed. The affected leaves revealed advancing necrosis, substantially enhancing at the end of the growing season. Defoliation took place from June to October. Micromycetes inhabiting the leaves of climbing roses in Botanic Garden of the Jagiellonian University in Cracow considerably deteriorated the decorative aspect of the plants. [ABSTRACT FROM AUTHOR]

The article focuses on a study which investigated the efficacy of seven commercially available biostimulants against the foliar pathogens Guignardia aesculi, leaf blotch of horsechestnut and Diplocarpon rosae black spot of roses. Biostimulants reportedly improve a plant's health, growth, and protection. Results revealed that none of the biostimulant products tested met an adequate degree of pathogen control to warrant placement of or supplementation with traditional synthetic fungicides.

Previous studies have shown the role of melanized appressoria in the pathogenicity of various fungi. Diplocarpon rosae is a worldwide outdoor fungal pathogen of rose plants causing black spot disease of rose leaves. To fully understand how this fungus colonizes its host, which is critical for the development of an efficient and sustainable disease management program, we studied the fungal (especially the appressoria) structures of D. rosae in detail at an early stage of infection. Using both microscopic and biochemical analyses, we observed strong melanized appressoria formation localized at the point of D. rosae penetration, which forms the pathogen-plant interface. Treatment of infected plants with melanin biosynthesis inhibitors (MBIs) prevented melanization of D. rosae appressoria and positively correlated with significant reductions in black spot disease symptoms, suggesting that melanization of appressoria might be a critical factor for the pathogenicity of D. rosae. Our findings were confirmed and validated by the lack of melanized appressorial ring formation on an artificial surface and on a D. rosae-non host plant system, Arabidopsis thaliana. Our findings suggest that localized melanization of appressoria is a crucial factor for the pathogenicity of D. rosae and treatment of the fungus with MBIs seems to be a promising disease management alternative for black spot disease of roses. [ABSTRACT FROM AUTHOR]

The interactions of four pathotypes of Diplocarpon rosae with 34 species and hybrids of Rosa were compared in an ongoing search for criteria of potential relevance to rose breeding. There were greater similarities in the resistance-susceptibility interactions of these roses to the pathotypes DA1, CW1 and ZM1, than to a fourth pathotype, DA2. Among the species roses, only those of the section Cinnamomeae were susceptible to DA2 and this susceptibility was transmitted in several cases to hybrid progeny. CW1 had the greatest pathogenicity of the four pathotypes. In a cross between R. rugosa cv. Scabrosa, which was resistant and R. rugosa f. alba, which was susceptible to all pathotypes, the progeny were tested for resistance to pathotypes DA1, CW1 and ZM1. Each of the 20 progeny was susceptible to all three pathotypes. This shows that, unlike the well characterized Rdr1 gene for resistance to D. rosae, the resistance of R. rugosa cv. Scabrosa is not determined by a dominant major gene. The diploid hybrid, R. rugosa cv. Martin Frobisher × cv. Mistress Quickly, was resistant to all four pathotypes, but an induced tetraploid of this hybrid was susceptible to all pathotypes. The relevance of these findings to the breeding of roses for resistance to black spot disease is discussed. [ABSTRACT FROM AUTHOR]

Black spot disease of rose, incited by the fungus Diplocarpon rosae, is found worldwide and is the most important disease of garden roses. A gene-for-gene interaction in this pathosystem is evidenced by the presence of pathogenic races of D. rosae and the previous discovery of a dominant resistance allele at the Rdr1 locus in the diploid Rosa multiflora. The objective of the present study was to genetically analyze resistances to North American black spot races 3, 8, and 9 previously reported in tetraploid roses. Resistance to North American races 3 and 8 segregated 1:1 in multiple F1 populations, indicating that both are conferred by dominant alleles at single loci and are present in simplex (Rrrr) configuration. Gene pyramiding was demonstrated by combining both resistances into single genotypes. Resistance to race 9 was partial and segregated in a quantitative fashion. Analysis of these populations with microsatellite markers previously developed for Rdr1 revealed that the gene conferring race 3 resistance resides within the same R gene cluster as Rdr1. Race 8 resistance segregated independently and is, therefore, a novel locus for black spot resistance in rose which we have named Rdr3. NBS and LRR profiling were used in a bulked segregant analysis to identify a marker 9.1 cM from Rdr3, which was converted to a SCAR marker form for marker-assisted breeding. [ABSTRACT FROM AUTHOR]

Black spot disease, incited by the fungus Diplocarpon rosae Wolf, is the most important disease of roses ( Rosa hybrida L.) in the outdoor landscape. Though partial resistance exists in cultivated germplasm, the genetic basis of this trait has not yet been elucidated. Six diploid and six tetraploid rose cultivars were crossed in two factorial combining ability arrays. Whole plant and detached leaf inoculation methods were used to assess partial resistance under two different disease pressures using a characterized single-spore isolate. Parents from both arrays had significant general combining ability effects across multiple inoculation methods and environments. Specific combining ability was not significant for either array. Parent per se performance was highly correlated with progeny performance on a family mean basis. High positive correlations among whole plant and detached leaf inoculation methods indicate that detached leaf assays can substitute for whole plant assays. Based on these results, a breeding strategy including parental selection and early, among-family selection is proposed. To our knowledge, this is the first investigation of combining ability for disease resistance in rose. [ABSTRACT FROM AUTHOR]

Black spot disease is an important disease of roses with worldwide occurrence. It is caused by Diplocarpon rosae, an ascomycetous fungus and its control relies on fungicides. The effects of strobilurins and triazoles on D. rosae development within the host and on disease symptoms have not been well studied. Strobilurins completely inhibited germination of conidia when applied protectively 1 week before inoculation or on the same day with the inoculum. They were, however, not effective in eradicating the disease when applied after the fungus was established in the host. Triazoles reduced the germination rate of the conidia when applied protectively and they inhibited disease symptom development when applied after the fungus was established in the host but before symptom expression. Application of triazoles after symptom development suppressed further development of the disease, but in the case of treatment with myclobutanil yellowing and defoliation still occurred. Post-infection application of triazoles led to the apparent breakdown of subcuticular mycelia, intercellular mycelia, and hyphae in the epidermal cells, while the effects of strobilurins were limited to the subcuticular mycelia. Triazoles were more effective than strobilurins because they are more systemic. [ABSTRACT FROM AUTHOR]

An attempt was made to study the biocontrol efficacy of antagonistic microorganisms in phylloplane of rose cv. Edward to manage the black spot (Diplocarpon rosae) disease. Eight antagonistic microorganisms were tested in vivo against the black spot pathogen. Among these, Trichoderma viride and Pseudomonas fluorescens pf1 reduces the mycelial growth significantly. These two biocontrol agents were evaluated for their ability to induce defense-related enzymes and chemicals in plants. Increased activity of phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenoloxidase (PPO) and total phenolics were recorded in all the biocontrol agents treated leaves. P. fluorescens Pf1 recorded early and increased synthesis of the entire defense-related enzymes and total phenol within 6 days. The application of biocontrol agents induced the defense-related enzymes involved in phenyl propanoid pathway in addition to direct antagonism, which collectively contribute for enhanced resistance against invasion of Diplocarpon rosae in rose. [ABSTRACT FROM AUTHOR]

Blackspot disease, caused by the fungus Diplocarpon rosae Wolf, is the most serious disease problem of roses worldwide. From a breeding perspective, the documented occurrence of races in the pathosystem necessitates the characterization of the race structure for the pathogen, identification of resistance genes in rose germplasm, and determination of the nature of genetic resistance to breed stably resistant rose cultivars. The objective of this study was to characterize the diversity of eastern North American races of D. rosae and to simultaneously determine the degree to which partial resistances existed in rose hosts. Twelve rose genotypes were inoculated with 14 single-conidial isolates collected from eastern North America. Three distinct D. rosae races were identified based on presence/absence of infection in the differential array. There were five distinct reaction patterns across pathogen isolates among the 12 rose genotypes. The partial resistance components of leaf area with symptoms, lesion length and incubation period were evaluated for all inoculated plants. Variation for expression of partial resistance was noted, and partial resistance appears to behave in a race-specific manner for some rose genotypes. Early defoliation differed between genotypes and may prove to be another component of partial resistance. [ABSTRACT FROM AUTHOR]

Blackspot, caused byDiplocarpon rosae, is the most severe and ubiquitous disease of garden roses, but information is lacking about genotype-specific forms of resistance and susceptibility of the host. Macro- and microscopic analyses of 34 rose genotypes with a defined monoconidial culture black spot inoculum identified susceptible and resistant rose genotypes and further genotype-specific subdivisions, indicating the presence of partial forms of resistance and different resistance mechanisms. In total, eight interaction types were characterized, five representing compatible (types 1–5) and three representing incompatible interactions (types 6–8). The incompatible interactions were characterized by the lack of any visible fungal structures beneath the cuticle (type 8), single-cell necroses (type 7) or necroses of larger cell clusters (type 6), the latter two types with penetration hyphae and haustoria in epidermal cells. [ABSTRACT FROM AUTHOR]

Treatment of rose shoots with 50 µM acibenzolar-S-methyl (BTH) resulted in increased protection against Diplocarpon rosae. This was accompanied by the induction and accumulation of a set of extracellular proteins as shown by SDS-PAGE and 2D-PAGE. Some of these proteins have been identified as PR-1, PR-2, PR-3 and PR-5 proteins by immunoblot analysis probed with tobacco antisera against PR-1c, PR-N, PR-Q and PR-S protein. Most of the extracellular proteins activated by BTH were also induced and found to accumulate in leaves upon infection with Diplocarpon rosae. However, their accumulation was much more pronounced in BTH-pretreated leaves than in water-pretreated leaves upon a challenge inoculation with D. rosae, particularly, the 15 kD PR-1, 36 and 37 kD PR-2 proteins. They may be more important in the expression of disease resistance. [ABSTRACT FROM AUTHOR]

Blackspot is a serious disease on cultivated roses and resistance is rare; however, some wild Rosa species exhibit a high level of resistance. Disease incidence in field-grown offspring from three seedling families was evaluated as percentage of infected leaves (regardless of the size and number of spots) on each plant. Dolly, Queen Elizabeth and Friesia were used as pistillate parents and the resistant germplasm L83 (derived from the amphidiploid R. × kordesii) was the staminate parent. Assessments were performed during 3 years. Relative tolerance was seen in 71, 54 and 87%, respectively, of the individuals from the three different seedlings families. The infection pattern corresponded well to environmental factors such as precipitation. The importance of timing and duration of assessments over several years, as well as the correlation between blackspot infection and winter hardiness, are discussed. [ABSTRACT FROM AUTHOR]

Differentiation into physiological races of Diplocarpon rosae, which causes blackspot on wild and cultivated roses, was investigated with single conidial isolates of the pathogen. Infection experiments with a simple excised leaf assay demonstrated differential interactions between single isolates and a set of 10 test rose genotypes. Differential reactions could be observed among rose varieties as well as between and within a wild rose species. Accordingly, five different physiological races can be identified among the 15 isolates tested. A breeding line and one genotype of the wild species Rosa wichuraiana were found to be resistant against all isolates tested. These data will be employed in further investigations on the genetics of blackspot resistance in roses and for resistance breeding programmes. [ABSTRACT FROM AUTHOR]

Blackspot, caused by the Ascomycete fungus Diplocarpon rosae, is the most widespread and pernicious disease of cultivated roses. While some species of rose possess resistance to D. rosae, none of the modern-day rose cultivars are fully resistant to the pathogen. In the current study, Biolistic gene delivery was used to introduce a rice gene, encoding a basic (Class I), chitinase into embryogenic callus of the blackspot-susceptible rose (Rosa hybrida L.) cv. Glad Tidings. The plasmid used for transformation carried the neomycin phosphotransferase (nptII) gene facilitating the selection and regeneration of transgenic plants on medium containing 250 mg/l kanamycin. Southern analysis confirmed integration of 2–6 copies of the chitinase gene into the rose genome; gene expression was confirmed by enzyme assay. Bioassays demonstrated that expression of the chitinase transgene reduced the severity of blackspot development by 13–43%. This degree of resistance to the pathogen correlated with the level of chitinase expression in the transgenic rose plants. The introduction of disease defence genes into rose provides a method of producing blackspot-resistant rose cultivars sought by breeders and growers. [ABSTRACT FROM AUTHOR]