Epidemiological aspects of blackspot disease of roses caused by Diplocarpon rosae Wolf

Conidia of Diplocarpon rosae Wolf are dispersed mainly by water-splash. Within a crop the spread of the disease can be traced from primary foci. Infection follows the ‘compound interest’ pattern, sensu van der Plank (1963), which is typical of most leafspot diseases. Accumulation of inoculum, together with certain conditions of rainfall and temperature in August, led to a characteristic, rapid development of the disease in September 1963 and 1964, after an incubation period of 2–4 weeks.     

Host/parasite interaction in blackspot disease of roses caused by Diplocarpon rosae Wolf

The utilization of amino acids by Diplocarpon rosae Wolf was investigated and compared with amino acid compositions of leaves of resistant and of tolerant roses. No amino acid appeared to determine resistance of roses to blackspot on a nutritional basis, but phenylalanine marginally increased resistance when taken up by wounded leaves of resistant or tolerant varieties. There is an accumulation of phenolic compounds in response to infection by D. rosae. Most phenolic compounds are toxic to D. rosae in vitro, particularly ellagic acid and p-coumaric acid, but their presence and quantity in healthy rose leaves could not be related to disease resistance. Inhibition of germination on rose leaves of conidia of the fungus was determined in a manner that suggested production of a phytoalexin. It is suggested that resistance of roses to D. rosae may be related to accumulation of polyphenols and possibly to the production of a phytoalexin.     

Genetic analysis of resistance to blackspot (Diplocarpon rosae) in tetraploid roses

  Diplocarpon rosae is the causal agent of rose blackspot, one of the most severe diseases of field-grown roses. The genetics of resistance to this pathogen was investigated in crosses between tetraploid rose genotypes. The hybrid breeding line 91/100-5, which exhibits a broad resistance to all isolates tested so far, was selfed to produce an F₂ population, backcrossed to the susceptible tetraploid variety ‘Caramba’ and crossed to the susceptible varieties ‘Heckenzauber’, ‘Pariser Charme’ and ‘Elina’. Infection experiments resulted in segregation ratios consistent with the presence of a single dominant resistance locus in the duplex configuration in the hybrid 91/100-5. This suggests, together with previous data on the race structure of the fungus, a “gene-for-gene” type of interaction in the pathosystem Diplocarpon/Rosa. We propose to designate this gene Rdr1, which is the first resistance gene described in the genus Rosa. The advantages and limitations of such an interaction type for future rose breeding programmes and for marker-assisted selection strategies are discussed. Received: 29 August 1997 / Accepted: 19 September 1997     

Resistance of roses to pathotypes of Diplocarpon rosae

Colonies of Diplocarpon rosae derived from single conidia were isolated on malt extract agar, multiplied (at 23°C) and stored (at ?20°C) on surface‐sterilised leaf discs of a universally susceptible rose, ‘Frensham’. The resistance of 16 species and cultivars of Rosa to different isolates of D. rosae was assessed using surface‐sterilised leaf discs. Four pathotypes of D. rosae were distinguished on the basis of host range. One species and one hybrid were resistant to all pathotypes. Two species and two cultivars were susceptible to all pathotypes. Four species and six cultivars were interpreted as having vertical resistance because they were strongly resistant to some but not all pathotypes. Only species and hybrids of the section Cinnamomeae were resistant to the pathotype identified as CW1 whereas only roses of other origins were resistant to the pathotype DA2.     

Microscopic and biochemical evidence of differentially virulent field isolates of Diplocarpon rosae causing black spot disease of roses

Black spot disease caused by Diplocarpon rosae is one of the most widespread diseases of roses that are very difficult to control due to the generative reproduction and complex genetic constitution of roses and up to now the control of black spot still requires intensive use of systemic fungicides. Here we report for the first time evidence of differentially virulent field isolates of D. rosae. Using a combination of fungal structures, disease symptoms and host cells protein expression pattern analysis we here provide direct biochemical evidence that tropical field isolates of D. rosae are more virulent and caused disease symptoms earlier than their temperate counterparts. The tropical fungal field isolates strongly induced an excessive accumulation of ROS and repressed activity of pathogenesis-related proteins such as peroxidases, chitinase and phenylalanine ammonia lyase compared to their temperate counterparts. These findings bring insights into a hidden pathogenic characteristic of tropical D. rosae field isolates compared to their temperate counterparts and open a novel dimension of parameters to be considered when controlling black spot disease of roses by fungicides in tropical versus temperate regions. Interestingly, we found that treatment of rose leaves with ROS (H₂O₂) prior to fungal inoculation promoted plant defense response regardless of the isolate virulence based on protein expression pattern analysis, suggesting that ROS (H₂O₂) can be efficiently incorporated into black spot disease management.     

Expression of a chitinase transgene in rose (Rosa hybrida L.) reduces development of blackspot disease (Diplocarpon rosae Wolf)

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.     

Efficacy of triazoles and strobilurins in controlling black spot disease of roses caused by Diplocarpon rosae

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.     

The TNL gene Rdr1 confers broad‐spectrum resistance to Diplocarpon rosae

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.     

Prediction of the Diplocarpon rosae secretome reveals candidate genes for effectors and virulence factors

Rose black spot is one of the most severe diseases of field-grown roses. Though R-genes have been characterised, little information is known about the molecular details of the interaction between pathogen and host. Based on the recently published genome sequence of the black spot fungus, we analysed gene models with various bioinformatic tools utilising the expression data of infected host tissues, which led to the prediction of 827 secreted proteins. A significant proportion of the predicted secretome comprises enzymes for the degradation of cell wall components, several of which were highly expressed during the first infection stages. As the secretome comprises major factors determining the ability of the fungus to colonise its host, we focused our further analyses on predicted effector candidates. In total, 52 sequences of 251 effector candidates matched several bioinformatic criteria of effectors, contained a Y/F/WxC motif, and did not match annotated proteins from other fungi. Additional sequences were identified based on their high expression levels during the penetration/haustorium formation phase and/or by matching known effectors from other fungi. Several host genotypes that are resistant to the sequenced isolate but differ in the R-genes responsible for this resistance are available. The combination of these genotypes with functional studies of the identified candidate effectors will allow the mechanisms of the rose black spot interaction to be dissected.     

Identification of molecular markers linked to Rdr1, a gene conferring resistance to blackspot in roses

Blackspot resistance in the tetraploid rose genotype 91/100–5 had been characterised previously as a single dominant gene in duplex configuration. In the present study a tetraploid progeny (95/3) segregating for the presence of the blackspot resistance gene Rdr1 were screened with 868 RAPD and 114 AFLP primers/primer combinations. Seven AFLP markers were found to be linked to Rdr1 at distances between 1.1 and 7.6 cM. The most closely linked AFLP marker was cloned and converted into a SCAR marker that could be screened in a larger population than the original AFLP and was linked at a distance of 0.76 cM. The cloned fragment was used as an RFLP probe to locate the marker on a chromosome map of diploid roses. This is the first report of markers linked to a resistance gene in roses, and the possibilities of using them for a marker-assisted selection for blackspot resistance as well as for map-based cloning approaches are discussed. Received: 23 December 1999 / Accepted: 25 March 2000     

Influence of phylloplane colonizing biocontrol agents on the black spot of rose caused by Diplocarpon rosae

Abstract

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.     

The sulphur: Nitrogen ratio of conifer foliage in relation to atmospheric pollution with sulphur dioxide

Summary The S : N ratio of the current foliage of two commonly planted conifers, Sitka spruce (Picea sitchensis (Bong.) Carr.) and Scots pine (Pinus sylvestris L.) was determined for areas known to have unpolluted atmospheres. The mean values of the ratio (0.034 and 0.028 respectively) were used to compare similar foliage from a) an industrialised area, and b) a series of transects radiating from a brickworks chimney. The established ratios were used to predict foliar S concentrations and the greater amounts found were ascribed to foliar absorption of atmospheric S. The ‘excess’ S was negatively related to distance from the supposed source, and also to needle weight which itself was positively related to distance. It is concluded that this measure derived from the S : N ratio is theoretically and practically a more sensitive indicator of the accumulation of S in conifer foliage exposed to atmospheric pollution than the analysis for elemental S or the SO₄-ion alone. re]19751115 Department of Forestry and Natural Resources University of Edinburgh     

The response of wheat straw varieties to mild sulphur dioxide treatment

Straws of five wheat cultivars, including soft and hard and dwarf and semi-dwarf varieties, were treated with sulphur dioxide (SO₂), and the effect of treatment on monosaccharide composition and cell wall (CW) degradation by rumen microorganisms was studied. Despite agrobotanical differences, the monosaccharide profiles of the straw hydrolysates were similar. Glucose, xylose, uronic acids and arabinose comprised about 98% of the total monosaccharides and the ratio of glucose to xylose was generally 2:1. The major and general effect of SO₂ treatment was expressed in solubilization of the matrix polysaccharides, cellulose being hardly affected. More than 20% of the permanganate lignin was solubilized by SO₂ in four of the cultivars. The ‘Lakhish’ cultivar had the highest lignin content (13.3%), but its lignin proved resistent to SO₂ treatment. Organic matter digestibility in vitro (IVD) was maximally increased in four varieties (excluding the ‘Lakhish’ straw) to 77–84%, and the IVD of monosaccharides to 88–93%. The treatment effect on the ‘Lakhish’ variety was weaker, the IVD of organic matter and monosaccharides was only increased to 67 and 77%, respectively. Other straw batches of the ‘Lakhish’ variety have shown a strong response to SO₂ treatment similar to that found in the straw batches of the other four cultivars. The present study provides support to previous conclusions about the major biodegradation obstacles in wheat straw. The results of this study imply that mild SO₂ treatment is generally effective in increasing the degradability of wheat straw by rumen microorganisms.     

The effects of sulphur dioxide pollution on bark epiphytes

Abstract

Sematophyllum substrumulosum (Hampe) Broth. was found in 1995 on a decaying trunk of Pinus in Tresco, Isles of Scilly, the first report of the species in the British Isles.     

The retting of flax after preservation with sulphur dioxide

Experiments were carried out to compare the retting of moist flax preserved with sulphur dioxide with that of green dried flax, using whole straw samples. When retted in water at either a constant 20°C or 28°C dried flax was fully retted after 15 and 10 days respectively whereas the sulphur dioxide treated flax (20 g sulphur dioxide kg“¹ flax DM) had undergone almost no retting after 20 days at 20dC or 10 days at 28°C. Pre-soaking the treated flax for 24 h in water and changing the acidified water, raised the pH of the retting liquor to a more normal value but did not significantly increase the rate of retting. Addition of the pectinase enzyme preparation ‘Flaxzyme’ to retting liquor at the rate of either 1.5 g kg^-1 or 3.0 g kg^-1 water, and at a constant temperature of 20°C, substantially increased the rate of retting of both sulphur dioxide treated and dried flax. Optimum degree of retting was achieved at 24 h with the treated flax and at 97 h with the dried flax. Pre-rinsing of the sulphur dioxide treated straw only served to reduce the rate of retting. It was concluded that natural water retting of sulphur dioxide treated flax is retarded by the presence of acidic residues of sulphur dioxide, while enzyme retting is enhanced by these. In further smaller scale experiments using bundles of cut flax straw Flaxzyme was added at concentrations ranging from 0–8.0 ml litre ¹ to containers containing flax in water at ratios from 1:10 to 1: 600 flax:water and the producion of galacturonic acid was used as an indicator of retting progress. Retting took place more rapidly at higher flax to water ratios for a given enzyme concentration. This effect was attributed to the lower pH of higher flax to water ratios which created pH conditions closer to the pH optimum for the retting enzymes. When enzyme retting was compared at a range of buffered pH's the optimum was pH 4.0. At a buffered pH of 4.0 and a temperature of 19°C, retting of sulphur dioxide treated moist flax (flax to water ratio of 1:10) was achieved with Flaxzyme concentrations as low as 0.5 ml litre”‘,much lower than the previously reported minimum of 3.0 ml litre’.     

Detailed description of developmental growth stages of Diplocarpon rosae in Rosa: a core building block for efficient disease management

Black spot disease of roses caused by the ascomycetous fungus Diplocarpon rosae teleomorph (Marssonina rosae anamorph) is a widespread and devastating disease. Despite considerable progress in the management of black spot disease in the recent years, it is still unclear by which mechanisms this fungus colonises and invades the host system, and without a good knowledge of such infection machineries, it not possible to fully overcome the challenges of D. rosae infection. By exploring research contributions up to date, we highlight in this review the ultrastructure of D. rosae infection cycle in the host cell by emphasising on several aspects related to its in vitro and in vivo germinations, the infection mechanisms of the fungus, the different fungal structures formed in the host cells and the optimum storage conditions for D. rosae to retain its viability and pathogenicity over time. Here, attention is particularly focused on the asexual life cycle of D. rosae, with the sexual cycle being briefly discussed. In addition, a new dimension of research approaches to effectively control black spot disease of roses, that is, how to accurately use the advanced biotechnology tools to speed up the current state of the disease management, is proposed here.