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DEFRA Final Report (CSG13) To investigate the biology and virulence of the rose blackspot fungus Diplocarpon rosae leading to improved methods of control. Aims, Objectives and Milestones: Milestone No. Aim 01 01/01 01/02 01/03 S01 01/04 S02 Title of Milestone Determine the pathogenicity, specificity and host/ cultivar distribution and frequency of D. rosae in the UK and obtain infected material from a range of UK sources. Collect at least 100 isolates from a diverse range of host cultivars from within the UK. Complete a literature survey to highlight conditions favourable for infection. Develop a standard pathogenicity test for evaluating pathotypes. Develop a standard pathogenicity test for evaluating new cultivars and fungicides. Assess pathogenicity of isolates on selected cultivars. Determine the trade pathways for roses entering the UK. Page No. 1-2 2-3 3-4 4-5 5-7 7-8 Aim 02 Investigate the genetic diversity and population biology of D. rosae by molecular and physiological analysis. 02/01 02/02 S03 S04 Collect at least 20 isolates from diverse geographical areas from outside the UK. Investigate the genetic diversity and population biology of the fungus. Investigate cultural morphological and physiological types of the fungus. Ascertain the level of fungicide resistance/tolerance of selected isolates. 8 8-11 11-12 12-13 Aim 03 Determine the resistance of a carefully selected range of host cultivars and species to different pathogen genotypes. Determine the resistance of a range of selected cultivars in vivo to pathotypes. 13-15 Define pathotypes (based on evidence from objectives 1-3) and develop molecular markers. Describe pathotypes based on genetic and physiological characters. Describe molecular markers for pathotypes. Assess the feasibility of in vivo identification of pathotypes with molecular diagnostic tools. Determine the prevalence of the sexual stage on fallen overwintering leaves. Ascertain if the sexual stage can be produced in vitro. 15 15 15 15 16 Determine the cultural and environmental factors favouring disease development to enable effective disease management and control. Determine inoculum sources. Ascertain the environmental factors favourable for disease development. Develop a strategy for improved disease management and control. 16 17-18 18-19 03/01 Aim 04 04/01 04/02 S05 S06 S07 Aim 05 05/01 05/02 05/03 Aim 01: Determine the pathogenicity, specificity and host/ cultivar distribution and frequency of D. rosae in the UK 01/01 Collect at least 100 isolates from a diverse range of host cultivars from within the UK 01/01 Methods a) Collection of infected leaves Infected leaves were gathered from a wide area of the UK e.g. Yorkshire, Hertfordshire, Devon, Staffordshire etc and also from a broad variety of rose species and varieties (climbers, teas, patio roses etc.). This stratified method of collection was used to ensure that a range of isolates were collected to represent different geographical locations and also from different rose types. Fresh young leaves were chosen that were showing signs of blackspot with acervili. 1 Depending on leaf size, between 2 and 5 compound leaves were removed close to the stem and placed in a labelled polythene bag. b) Isolation of D. rosae from infected rose leaves A range of methods were used to obtain the fungus in pure culture (Appendix 01/01b). All isolations were made from single acervuli. c) Storage of isolates Colonies of D. rosae were multiplied on the isolation media (1/4PDA) at 20C. The isolates were then stored in three ways:- as agar plugs in vitro, on frozen leaf discs and in liquid nitrogen. The methods are detailed in Appendix 01/01d. 01/01 Results a) Collection of infected leaves: Detail of material collected is listed in Appendix 01/01a. In total 211 rose blackspot samples were obtained from UK origin. b) Isolation of D. rosae from infected rose leaves: The most efficient isolation technique was method 1 (Appendix 01/01b). In brief, isolates were obtained from fresh acervuli using a 30m sterile dissecting needle and grown on 1/4 PDA under standard laboratory conditions (c. 18C, 12 hr day/night cycle). (c) Storage of isolates: Isolates obtained on media were placed into the culture collection at UH and CSL York (0.5 cm3 agar cubes placed in sterile water and stored at 4C). Isolates were maintained on frozen leaf discs both at UH and also at CSL Selected isolates were also stored in liquid nitrogen at CABI. Due to the slow growing nature of the fungus on solid media, the isolates were also maintained on the original agar plates at 4C at UH (Appendix 01/01d and 01/01e). 01/01 Discussion The fungus was difficult to obtain in pure culture, as it is extremely slow growing (c .0.5 cm increase in colony size per month) and readily becomes overrun by saprophytic fungi (mainly Penicillium, Cladosporium etc.) and bacteria. From the original 211 samples of infected rose leaves obtained (Appendix 01/01a), 104 were successfully grown in pure culture (Appendix 01/01c). Method 1 (see above) was selected and so all cultures used in the study originated from a single acervulus. 01/01 Outcome 104 isolates were obtained and stored for future use. 01/02 Complete a literature survey to highlight conditions favourable for infection 01/02 Methods A broad literature survey was undertaken to highlight conditions favourable for infection. Resources used included CABI Information databases and relevant journals. 01/02 Results The conditions favourable for infection as found in the literature are summarised below in Table 01/02. The full report on the conditions favourable for infection can be seen in Appendix 01/02. 2 Germination Table 01/02: Conditions favourable for infection Dispersal Overwintering Leaves are seen to be most susceptible to infection whilst they are still expanding (6-14 days old) (Horst, 1983). The fungus tolerates a wide range of temperatures (15-27°C), mainly through an inverse relation of humidity and moisture (Horst, 1983). To germinate, conidia must be wetted for at least 5 minutes. For infection to occur, conidia must be immersed in water and be continuously wet for at least 7 hours (Horst, 1983). Conidium germination is optimum at 18°C, at this temperature, germination begins in 9 hours and may reach 96% in 36 hours (Horst, 1983). Acervuli form in 11 days on the upper leaf surface. Conidia may be produced 10-18 days after infection. Production of acervuli decreases after about 1 week and may end in 10 days, but new acervuli are continually formed at the margins of spots (Horst, 1983). Some conidia adhere to ruptured cuticle fragments but most are dispersed in rainwater by rain-splash. Conidia are disseminated by splashing water, people during cultivation, or by contact with sticky body parts of insects. Fallen leaves blown by the wind may disperse the pathogen locally, but conidia are usually airborne only in water drops. Ascospores are found very rarely (Horst, 1983). Conidia have been reported to be splashed by rain to a height of 5-10 cms above infected fallen leaves in winter and early spring (Cook, 1981). The fungus does not survive in the soil and spores do not remain viable for more than a month (Horst, 1983). The fungus overwinters as mycelia in fallen leaves or in infected canes. Over-wintering in fallen leaves may be as conidia in existing acervuli, as acervuli in which new conidia are produced in spring, or as mycelia that in spring produce either new acervuli or apothecia in which conidia and ascospores form. Bud scale infection has been reported to be associated with stem infection. (Cook, 1981). Previous observations have indicated that D. rosae did not form apothecia on over-wintered rose leaves at Wisley (Cook, 1979), however they were seen at Silwood Park (Knight and Wheeler, 1977). 01/02 Discussion The results obtained from the literature search were used to inform the design of both the pathogenicity tests (Sections 01/03, 01/04 & S01) and the epidemiology study (Section 05/02). Particular attention was paid in epidemiological studies to leaf surface wetness (for germination), ambient temperature, humidity and height and frequency of rainsplash. 01/02 Outcome Conditions favourable for infection were defined, and used to design inoculation methods (01/03 & S04). The above results (Table 01/02) were used to inform the design of the epidemiology experiments. 01/03 Develop a standard pathogenicity test for evaluating pathotypes (a) Rose cultivar selection for use with pathogenicity studies 01/03a Method The results of the literature study and discussions with rose growers and breeders were used to determine the most suitable rose cultivars to use for pathogenicity studies. Rose cultivars were selected in accordance to their known historical and current susceptibility to D. rosae. These were grouped into three major categories:- very susceptible, susceptible and resistant. 01/03a Results A total of ten differential roses were selected for use with all pathogenicity studies (Table 01/03a). 3 Table 01/03a Ten differential roses selected for use with all pathogenicity studies Susceptibility Differential Reference VERY SUSCEPTIBLE Frensham Martin Frobisher SUSCEPTIBLE Baby love Mrs Doreen Pike or Pink Surprise (or Rosa bella) Climbing Allgold Knight & Wheeler (1978). Initially considered resistant but Svejda (1980) found black spot. Also see: www.mirror.org/group/crs/mfrobisher.html Recorded as recently becoming susceptible (Yokoya et al (1999 ) Yokoya et al. (2000) and discussions with David Austin and Michael Marriot Yokoya et al. (2000) and discussions with David Austin and Michael Marriot Noack - in D Austin catalogue recorded as ‘resists all diseases’ Described as ‘intermediate’ by Saunders (1970) Described as susceptible to race 2 by Debener (1998) but susceptible to all races by Yokoya (1999). Yokoya et al (2000) and discussions with David Austin and Michael Marriot. Described as resistant by Castledine (1981) and very resistant by Saunders (1970). Flower carpet Masquerade Rosa rugosa RESISTANT Rosa davidii Allgold (b) Development of standard pathogenicity test 01/03b Method The results of the literature survey were used to assess a range of methods. These were tested and developed in the laboratory under conditions suitable for disease development (Table 01/02) and a standard operating procedure was written (Appendix 01/03). The method chosen for the in vitro study on detached leaves was broadly based on that of Knight & Wheeler, 1978. The method was tested using a known very susceptible cultivar (cv. Frensham) and resistant rose cultivar (Allgold) as selected in section 01/03a. Inoculum was used from naturally infected leaves and pure fungal culture of isolate DR26. Sterile distilled water was used as a control. 01/03b Results Table 01/03b: standard method for testing the susceptibility of D. rosae on detached leaves Inoculum Source Culture Leaf Water control Time taken until blackspot formation (days) Susceptible host 7 7 No necrosis seen Time taken for acervuli to form (days) Resistant host No necrosis seen No necrosis seen No necrosis seen Susceptible host 14 10 No necrosis seen Resistant host No acervuli seen No acervuli seen No acervuli seen 01/03 Discussion The standard method developed was suitable for testing detached rose leaves for their susceptibility to rose blackspot. 01/03 Outcome 01/03a: 10 differential hosts selected 01/03b: An easily reproducible pathogenicity test developed. S01 Develop a standard pathogenicity test for evaluating new cultivars and fungicides. S01 Method The standard pathogenicity test developed in 01/03 was modified to enable growers/breeders to test new rose cultivars using attached leaves. This involved the use of a mini-inoculator that was affixed to the test leaf. A standard operating procedure was written (Appendix S01). An in planta test kit was produced and independently evaluated on selected cultivars very susceptible (Frensham), susceptible (R. rugosa) and resistant (R. davidii) cultivars) based on the knowledge obtained from 01/03 (a) growing in the field at RHS Wisley). Various methods of inoculating test plants with the mini-inoculator were trialled and a questionnaire completed. 4 The in planta test kit protocol and questionnaire is shown in Appendix S01. S01 Results Results from the in planta test questionnaire are shown in Appendix S01b. Table S01 shows the effect of different inoculation methods and rose blackspot infection. Table S01: Effect of different inoculation methods and infection Inoculation method No inoculum No inoculum No inoculum Agar plugs Agar plugs Rosa cultivar Agar plugs Leaf discs Leaf discs Leaf discs R. rugosa R. davidii Frensham R. rugosa ---- Spore suspension Spore suspension Spore suspension R. davidii Frensham R.rugosa Leaf lost --- R. davidii Frensham R. rugosa R. davidii Frensham Presence of acervuli ----- Key : --: No acervuli : Acervuli present S01 Discussion The feedback from the in planta evaluation sheet indicated that the mini-inoculators in general were easy to use and the leaf disc inoculator was regarded the most suitable method of inoculation. It is necessary to inoculate the leaflet nearest the petiole of the compound leaf in order to reduce the amount of damage to the junction between the petiole and the stem of the plant, particularly as the leaf blows in the wind. The tests were carried out late in the season, when there was already a high level of natural infection on the test plants. However, the mini-inoculators could be used by breeders as they provide a number of benefits over the present testing method namely mass spraying of bushes with the fungus. These advantages are as follows: A suitable humid environment for infection and development of rose blackspot fungus is maintained (see section 03/01). Only a few leaflets per bush need to be tested A knowledge of which leaves are challenged with the fungus By placing a known susceptible variety e.g. Frensham in the field, it would be possible for growers to collect fresh infected leaves to test new cultivars using the mini-inoculator system. The mini inoculators are suitable for use on container grown roses, where they are shielded from the wind, however they are less suitable for field grown roses. S01 Outcome Mini inoculators were developed and tested and found to be successful for container grown roses. 01/04 Assess pathogenicity of isolates on selected cultivars. 01/04 Method Rose cultivars selected in section 01/03 were grown in a clean glasshouse environment with no overhead watering. Roses were monitored regularly and any sign of rose blackspot was removed. Only known clean leaves were used for the in vivo testing. Twenty isolates grown on ¼ PDA (section 01/01) were selected and tested for pathogenicity using the standard operating procedure developed in section 01/03, sterile distilled water was used as a negative control. 01/04 Results & Discussion No infection was observed on any of the test roses in 1999 and so viability and germination tests were carried out on the fungus which indicated that the spores obtained from the cultures were still viable, but there was a marked reduction in their germination (from 80-90% when fresh to <10%). The fungus obtained from these cultures also appeared to have lost its pathogenicity as even the very susceptible rose varieties were not infected. Various methods were used to try and 5 regain pathogenicity (see Appendix 01/04a), but these were all unsuccessful. The cultures used for the tests were approximately six months old, and the results show that the fungus loses its pathogenicity when maintained on solid agar for this period. A number of tests were carried out from fresh cultures (approximately 1 month old) on a susceptible host and pathogenicity was maintained. An additional step was therefore added to the pathogenicity test protocol in order to overcome this problem and is described below: Additional method Initial isolation of the fungus from an acervulus was performed as previously described (Appendix 01/01b). When the culture was approximately one month old, a suitable concentration of rose blackspot spores was obtained and used to infect the very susceptible variety Frensham (using standard operating procedure developed in section 01/03). Pathogenicity was conserved by maintaining cultures on Frensham. After 14 days, spores were harvested and used for pathogenicity testing of selected rose cultivars (section 01/03). In order to reduce potential seasonal variability, all in vitro pathogenicity tests carried out in each year were set up at the same time, Results Fifteen isolates were tested in the year 2000, and a further 15 isolates tested in 2001. A summary of the results is shown in the following table 01/04a (raw data are given in Appendix 01/04b). Table 01/04a: Assessment of isolate pathogenicity on selected cultivars Isolate Ref. Year Location Rose type Climbing All gold Frensham Davidii All gold Martin Frobisher Baby love Mrs Doreen Pike Flower Carpet Masquera de R. rugosa Group DR C 2001 CH rugosa -- Hybrid Scots briar -- DR F 2000 CH Shrub -- CH Damask -- -- -- -- -- -- 2 -- -- 1 CH -- 2001 -- DR R -- -- -- -- 3 -- -- -- -- -- 4 -- -- -- -- -- -- -- -- -- -- -- 5 -- -- -- -- -- -- -- -- 6 --- -- -- -- 7 -- -- -- -- 7 -- -- -- -- 7 -- -- -- -- 7 -- -- -- -- -- 8 -- -- -- -- -- -- 9 -- -- -- -- -- -- 9 -- -- -- -- -- -- 9 -- -- -- -- -- -- 10 -- -- 12 -- -- -- 13 DR N 2000 DR O 2000 CH Shrub DR I 2001 W -- DR K 2001 York Hybridtea Climber -- -- DR D 2000 CH DR T 2001 CH Hybridmusk Shrub -- -- DR P 2001 CH Shrub DR A 2000 CH Shrub -- DR B 2000 CH -- DR C 2000 CH DR Q 2001 CH Hybrid tea Hybridmusk Shrub -- --- DR G 2000 CH Gallica DR K 2000 DR167 ? -- DR R 2000 CH Shrub -- DR Sl 2001 CH Shrub -- DR Sm 2001 CH Shrub -- DR Sh 2001 CH Shrub -- DR O 2001 CH Shrub -- CH Shrub --- DR E 2000 DR H 2000 CH HP DR E 2001 CH Alba -- DR216 2000 Dagenham HybridTea -- ----- 6 -- ------- -- ---- -- 1 1 1 1 4 6 6 11 DR M 2000 CH English DR Q 2000 CH English DR V 2001 York DR L 2001 CH Hybridtea Shrub -- DR M 2001 CH Shrub -- ---- --- -- -- -- -- -- -- -- -- -- 13 -- 14 -- 15 -- -- 16 -- -- 17 Key: CH W -- Year Castle Howard RHS Wisley Susceptible No infection 2000/ 2001 01/04 Discussion The modified method was suitable for observing differences in pathogenicity between the isolates collected. These results show that rose black spot is a highly variable fungus as out of 30 isolates tested over two years, 17 distinct infection groups were observed. These results may indicate some resistance with rose cultivars Climbing All-gold, Masquerade, Mrs Doreen Pike, All-Gold and Davidii as although they were challenged by 30 different rose blackspot isolates, less than a third of isolates produced a compatible reaction. Despite the claims in the literature that two cultivars were resistant (Table 01/03a), all the roses tested were susceptible to at least four of the isolates used. Rose cultivar Frensham was the most susceptible as it was infected with all of the isolates. Climbing All Gold appeared to be the most resistant of all the roses tested. With respect to the latest findings, the list shown in section 01/03 could be modified as follows in Table 01/04b. Table 01/04b: Differences in rose cultivar susceptibility between literature review and current studies Rose variety Climbing All Gold Frensham Davidii All Gold Martin Frobisher Baby love Mrs Doreen Pike Flower Carpet Masquerade Rosa rugosa Literature Susceptible Very susceptible Resistant Resistant Very susceptible Susceptible Susceptible Susceptible Susceptible Susceptible Current studies Susceptible Very susceptible Susceptible Susceptible Very susceptible Very Susceptible Susceptible Very Susceptible Susceptible Susceptible These results were obtained from in vitro tests and so it is likely that the fungus would be more stressed then in the wild. The results show that whatever was claimed by growers or in the literature, none of the rose cultivars tested maintained their resistance. The results also suggest that the fungus itself is highly variable with up to 17 different pathogenicity groups identified. The varieties which show resistance to more isolates of the fungus are All Gold climbing, susceptible to four isolates in three pathogenicity groups and R. rugosa, susceptible to 14 isolates in seven pathogenicity groups. Mrs Doreen Pike is susceptible to five isolates in one pathogenicity group and Masquerade is susceptible to five isolates in five pathogenicity groups. In contrast, Frensham, chosen as the universally susceptible host, was susceptible to all 30 isolates in all 17 pathogenicity groups. 01/04 Outcome Pathogenicity was assessed and it was found that the pathogen was more variable than expected with respect to pathogenicity and host. Contrary to the literature all the cultivars tested were susceptible to a proportion of the isolates. S02 Determine the trade pathways for roses entering the UK (plants, rootstocks, cut flowers) S02 Method 7 A literature survey was carried out to determine the trade pathways for roses entering the UK. S02 Results and Discussion According to the ‘The Plant Health Guide for Importers’ (MAFF, 1998) Rosa is prohibited from all non-European countries unless they are dormant plants free from leaves, flowers and fruit. A phytosanitary certificate issued by the country of origin must accompany material and may be subsequently inspected by MAFF PHSI as part of routine monitoring. Prohibited material may be imported with a licence issued by MAFF. A limited range of prohibited material may be imported under a ‘derogation’ of the EC legislation. We are not aware of either a licence or a specific derogation concerning Rosa being issued in the last three years. Rosa as cut flowers is not prohibited. Figures for the trade in cut flowers and live rose plants have been obtained from MAFF statistics for both import and export (details are given in Appendix S02). Based on the latest figures (1998), live rose plants are imported to the UK from primarily Netherlands (82% of total import values). There is some trade with Germany (10%), Denmark (3%), Poland (1.5%), and low levels with Uganda, Kenya, Italy, Belgium, France and ‘others’. Cut flowers are primarily imported from Netherlands (55%) and Kenya (24%). Further information has been obtained from the International Floriculture Trade Statistics 1998 (Appendix S02). These figures refer to cut flower imports only. It is interesting to note that in 1997 there was an increase of 28% from 1996 in the amount of cut roses imported. Several countries are listed as new:- Austria, Ethiopia, Saudi Arabia, and Thailand. S02 Outcome The main source of live rose plants is the Netherlands but a small proportion are source from other continents. Aim 02: Investigate the genetic diversity and population biology of D. rosae by molecular and physiological analysis 02/01 Collect at least 20 isolates from diverse geographical areas from outside the UK 02/01 Method Naturally infected leaf material was sent and collected from the Netherlands, Germany, Canada, Romania and India. Isolations onto ¼ PDA were made as described in section 01/01. The isolates obtained were tested using the standard pathogenicity test developed in section 01/03. Results + Discussion A total of 15 isolates were collected from Germany, India and Romania (Appendix 02/01). Leaf samples sent from Netherlands and Canada degraded during transit and so no isolates were obtained. Isolates obtained from Germany, India and Romania were grown in pure culture. These isolates were incorporated into the morphological, molecular and pathogenicity studies. See results for the molecular (02/02, 04/01 and 04/02) and morphological studies (S03). The problems described in milestone 01/04 mean that there were no results from these isolates in the main pathogenicity tests. However, the Indian isolate DR212 from Thora successfully infected Frensham and Rosa laxa in studies in UH. 02/01 Outcome 15 isolates were obtained from outside the UK and 10 added to the culture collection. 02/02 Investigate the genetic diversity and population biology of the fungus 02/02 Introduction The available strains of Diplocarpon rosae isolated in support of objectives 01/01 and 02/01 were subcultured at CABI Bioscience and DNA was extracted according to standard protocols (Appendix 02/02a). As for other project partners, the slow and uncertain growth in culture of the rose blackspot pathogen caused some difficulties, with a significant number of cultures failing or becoming contaminated, and problems were encountered in comparing our studies with those of other partners. Nevertheless, DNA was obtained from a total of 103 isolates of Diplocarpon rosae and a single strain of a related species, D. maculatum which is a pathogen of woody Rosaceous plants. 25 representative strains have been preserved in the CABI Culture Collection (Appendix 01/01e). Molecular analysis has not previously been attempted for Diplocarpon rosae, and none of its presumed close relatives have been studied either. This presented some problems as there was little or no indication of the processes by which the rose blackspot fungus populations evolved, necessitating a wide-ranging molecular study. 8 Four techniques were used to investigate the genetic diversity of the rose blackspot fungus: SS-REP PCR, AFLPs, restriction analysis of the ITS region and ITS sequencing. The protocols for each of these are available in Appendix 02/02a. In addition, the AFLP data were incorporated with morphological and cultural observations made by UH to investigate possible correlation between conidial size, colony colour and genetic profile. DNA extraction was also attempted directly from rose leaves, thereby bypassing the problematic cultural stage. Each of the four techniques are described individually below. Methods SS-REP PCR This method provides a rapid fingerprinting method for fungi which is particularly useful to detect individual genotypes, i.e. clonal populations. As it operates on the entire genome rather than the phylogenetically useful portions, it is of limited use in determining relationships, although there is often some correlation between major branches and phylogenetic position. The SS-REP sequence (GACA)4 was used for this study (Appendix 02/02). Results SS-REP PCR The results show a very large range of genotypes, with five major groupings (along with a few outliers) and at least 50 distinct genotypes (Figure 1 in Appendix 02/02b). Two of the clusters (DR 89 - DR 138 at the top of the dendrogram and DR 129 - IMI 381359 at the bottom) have fewer bands than the others, and it was at first suspected that their clustering might be due to inefficiencies in the amplification process. The sequencing work carried out at a later stage indicated that at least some of the strains involved are genuinely related. The remainder of the clusters show a complex branching pattern, with the outgroup species D. maculatum presented as distinct. Of the remaining strains, one genotype appeared to be present six times within the set of isolates amplified, three appeared five times, and one on four occasions. Of the genotype appearing six times, five strains were isolated from the Castle Howard rose collection. Of the genotypes isolated on five occasions, one was isolated exclusively from Castle Howard with the other two of mixed provenance, each from three separate locations. Most of the Castle Howard strains were strongly clustered, and represented a large proportion (68%) of one major branch. However, the significance of this may not be great due to the large proportion of the strains analysed which came from that location. Discussion SS-REP PCR This evidence suggests that genetically homogenous strains do occur within Diplocarpon rosae, but that variation due to recombination (not necessarily sexual, secondary milestone S06 and S07) is probably an important reproductive strategy. Not surprisingly, many genetically homogenous strains show a wide geographical distribution, presumably a reflection of the extensive movement of rose stock within the UK garden trade. Method AFLP analysis AFLPs provide a generally more robust fingerprinting product than SS-REP PCR, but are more complex technically and require larger amounts of DNA. AFLPs were therefore generated for a subset of the strains analysed with SS-REP PCR with three separate primer pairs CAT-GC, CTC-GC and ACC-GC, using the methodology described in Appendix 02/02a. Results and Discussion AFLP analysis The results are presented as a dendrogram (Figure 2, Appendix 02/02b). The dendrogram derived from the AFLP results shows a complex tree with two major branches, one of which has three major sub-branches, but as with the SS-REP results, no clear pattern which might indicate taxonomic separation emerges. AFLPs fingerprints use the entire genome, so this is not particularly surprising. Further evidence that the major branches are phylogenetically uninformative may be obtained from the fact that the major branches in the dendrograms for AFLP analysis and SS-REP PCR do not correlate significantly. The number of bands produced by AFLP analysis is larger than for SS-REP, so identifying clones is more problematic in some circumstances. In many cases, strains with identical SS-REP bands did not show identical or near-identical AFLP patterns strongly suggesting that while similarity of SS-REP banding pattern shows genetic homogeneity, this does not indicate clonality. If clonality does occur, it appears to occur rarely. Method ITS-RFLP analysis This molecular technique analyses sequences from the ITS region of the rDNA, a part of the genome which has been shown to be phylogenetically informative for other fungi at around the species level (i.e. which can detect infraspecific and infrageneric groups as well as species). The ITS region was amplified from DNA of 43 strains and cut using the two restriction enzymes HpaII and HaeIII, according to the methods detailed in Appendix 02/02a. For analysis of the resulting band patterns, we used two matching coefficients suitable for RE cleavage patterns such as the ITS-RFLP banding pattern. 9 Results and Discussion ITS-RFLP analysis The resulting dendrograms (Figures 3a and 3b in Appendix 02/02b) were very similar in branching pattern. The Different Bands coefficient is based on the number of different bands between two isolates and it is congruent with the distance between them. The Dice coefficient is based on the similarity between all possible pairs of fingerprints. Both band coefficients only take into account presence/absence of bands. The dendrogram analysis is therefore subject to the band definition and position on the gel as well as RE site positions, enzyme efficiency and digest conditions and the running conditions of the gels. Slight variations in these would cause differences in clustering, and are likely to be the reason why DR 159 and DR 91 were not clustered together with DR 53 / DR 100 / DR 103 / DR 150 as they were in the sequence dendrogram. The overall picture is of a surprisingly complex branching pattern, indicating a considerable degree of variation in ITS sequence. Our experience is that with the computer program used to analyse the data (GELCOMPAR) a similarity of more than about 90% is likely to indicate an identical sequence. RFLP analysis is a much cheaper process than sequencing, and can therefore be carried out on a much wider range of samples. However, the results are sometimes difficult to interpret, and minor sequence differences may result in large separations in the resulting dendrogram if they occur at restriction sites. Methods ITS sequencing The protocol for the ITS sequencing is described in Appendix 02/02a. Results ITS sequencing Full sequencing of the ITS region of the rDNA genome was carried out for nine strains of Diplocarpon rosae and one of D. maculatum, and the resulting dedrogram is shown below (Figure 02/02). It can be seen that the D. rosae strains fall into two clusters, with six of them (DR53 - DR 159) having identical ITS sequences. The remaining three (DR129, DR212 and DR168) are not identical with sequence divergence of less than 1%. The strain of D. maculatum was a clear outlier, diverging in ITS sequence from the D. rosae strains by 8.2%. In most cases, filamentous fungal species seem to differ in ITS sequence by between 1 and 4% (Cannon et al., 2000), so while the two groupings deserve further investigation, there is no clear justification at this stage to treat them as separate taxa. Interestingly, the six identical sequences all derived from northern England (Castle Howard, Yorkshire and the Liverpool Botanic Garden). Of the other three sequences, one derived from a collection from the Royal Horticultural Society Garden in Surrey (DR129), one from India (DR212) and the last was obtained from Prof. A. Roberts (University of East London) but is unlocalised (DR168). Bearing in mind the large difference in anamorph form between D. maculatum and D. rosae, a sequence divergence of only 8.2 % is smaller than expected, but the molecular data do suggest that the fungi are related and that placement in the same genus is justified. DR53 DR91 DR100 DR103 DR150 DR159 DR129 DR212 DR168 IMI381398 8.2 8 6 4 Nucleotide Substitutions (x100) 2 0 Fig. 02/02: Dendrogram derived from ITS 1 and 2 sequences of Diplocarpon species. IMI 381398 is D. maculatum, the others are all from strains of D. rosae. Discussion ITS sequencing The two sequence groupings in Figure 02/02 are not exactly reflected in the ITS-RFLP or the AFLP data. The RFLP results might be expected to correlate completely with the ITS sequencing, but small differences in sequence can sometimes lead to major differences in band pattern if the diverging base-pairs involve restriction sites. As explained above, AFLPs are derived from the entire genome and do not necessarily reflect phylogeny, so a lack of correlation with sequence data is not surprising. Interestingly, though, the sequence and SS-REP data do correlate well with the larger 10 cluster (DR53 - DR159 in the Figure 02/02) corresponding to the large SS-REP cluster described above in which the majority of the Castle Howard strains fell. BLAST searches were done for all the sequences to gain an insight into the taxonomic position of Diplocarpon. Very few of its possible relatives have been sequenced, so the results are not particularly helpful. However, the closest matches were two species of Phialophora (P. finlandica and P. melinii). Phialophora species are morphologically simple hyphomycetes of widely diverging phylogeny (Gams, 2000). Some have sexual morphs in the Helotiales, where Diplocarpon is thought to belong. P. finlandica is an mycorrhizal species associated with conifer roots, and there has been a suggestion that it belongs to the Dothideomycetidae rather than the Leotiomycetidae, but the phylogenetic study on which this assertion is based (Harney et al., 1997) needs further analysis. P. melinii is a poorly known weak wood pathogen of a range of broadleaved trees, and its phylogeny has not been fully investigated. Future research will be conducted into the phylogenetic position of Diplocarpon (by CABI Bioscience). DNA extraction ex situ ITS rDNA of Diplocarpon rosae was successfully extracted from 16 samples of infected rose leaves obtained from CSL used in their pathogenicity studies. A 600 bp band corresponding to the pathogen and a larger one from the host plant were obtained, and the former was purified and re-amplified using a nested PCR technique. This demonstrates the potential of detection and identification of the pathogen without culturing, and the system could be used to detect asymptomatic infections and infraspecific groupings. CABI were only able to culture the pathogen from four of the rose leaf samples received from CSL, making validation of the method uncertain without further research. The PCR method also proved complex and time would be needed to improve the specificity and yield of the PCR product. As these methods are unlikely to be of practical use bearing in mind the unclear picture of infraspecific variation and the conspicuousness of the pathogen in the field, it was felt that further development was not justified at this stage. 02/02 Overall Discussion: population structure Evidence from the suite of molecular analyses indicates that no clear infraspecific population structure appears to exist. There is little indication of clonality and no major robust infraspecific groupings, suggesting that recombination (possibly parasexual rather than sexual) is frequent and widespread. There was no clear correlation between the molecular profile and the data on conidium size and cultural characteristics contributed by UH, providing further evidence that the rose blackspot pathogen is a single polymorphic species. This is in contrast to the preliminary observations presented by Ali et al. (2000). There was also no discernible correlation between rose growth form and molecular profile. Although there is some indication that strains from particular locations are more similar than those from separate sites, there is no clear geographical pattern of variation. This is emphasized by the positioning of most of the non-UK strains within UK-dominated clusters rather than as separate branches of the dendrograms. Bearing in mind the great potential for the pathogen to spread through frequent and wide-ranging movement of host plants due to the nature of the rose industry, the lack of a clear geographical component to the genetic variability of the pathogen is not surprising. These factors when taken together emphasize the complex population structure of the pathogen, leading to major challenges for the horticultural industry in developing control strategies. Even if newly developed chemical control strategies are developed which are effective against a large proportion of the genotypes currently present in the UK, constant changes in the pathogen populations would be likely to lead to the rapid acquisition and spread of resistance. The variation pattern is indicative of a long period of coevolution with the host plant rather than rapid clonal colonization frequently found in agricultural pathogens, so control strategies borrowed from arable farming environments are unlikely to be successful. As no previous molecular studies had been carried out on D. rosae this molecular data has significantly increased the knowledge of the rose blackspot pathogen, and a paper is currently preparing a paper on this aspect of the research, which will be submitted to a leading peer-reviewed journal. 02/02 Outcome 1. 2. 3. The complex population structure of D. rosae is a major challenge for the control of this pathogen The variation pattern suggests a long period of co-evolution between host and pathogen which presents a further challenge to the control of this pathogen. The molecular data obtained here have significantly increased the knowledge base for D. rosae. S03 Investigate cultural morphological and physiological types of the fungus 11 S03 Methods The same isolates were used for both the molecular and morphological work. Colonies were grown using the method in Appendix 01/01b and the colony colour and hyphal characteristics of the 104 isolates were assessed using a standard colour chart. a) An investigation into the pigmentation of all 104 isolates The colony colour and hyphal characteristics of each isolate were assessed using a standard colour chart (Ridgewood, 1912). See table in Appendix S03a. b) Measurement of spore size A conidial suspension was prepared from each isolate and 50 conidia from each were measured (length and width) after 8 weeks of growth in culture. c) An investigation into the different hyphal types of D. rosae in culture Slide cultures (Appendix S03b) were prepared from conidial suspensions and the hyphal characteristics of the fungus observed and the presence or absence of microconidia noted. d) Storage Isolates were stored at all three sites (CSL, CABI & UH) in the methods described in section 01/01. Spore size and colony colour were assessed after storage in STW and in liquid nitrogen. S03 Results a) An investigation into the pigmentation of different colonies of D. rosae in culture As the colonies of D. rosae matured, three distinct variations in colony colour were observed: yellow, pale/dark brown and olive green. (Appendix S03a and Appendix S03 Figure 1). b) Measurement of spore size Conidial length between isolates varied from 11.1m to 30.7m. An apparent relationship was found between colony colour and conidial size (Appendix S03 Figure 1). The longer conidia were associated with lighter yellow colonies and smaller conidia associated with darker colonies. .The range of spore size found in this study was greater then that previously reported in the literature for this fungus (Sivanesan & Gibson, 1976). These spore sizes for each isolate were very consistent (Appendix S03 Figure 1). c) An investigation into the different hyphal types of D. rosae in culture In preliminary observations, 3 hyphal types were recorded Type 1 was seen to be sterile and produced no conidia (Plate a). Type 2 was seen to produce macro conidia (Plate b) and a 3rd hyphal type produced both micro and macro conidia. (See Appendix S03c). d) Storage Results suggest that the morphology of isolates stored in STW and in liquid nitrogen change after storage. Spore size and also colony colour were found to be different when compared to pre-stored isolates (Appendix S03 Table 1 S03 and Appendix 01/01e). S03 Outcome The isolates examined showed a range of colour and conidial size. However the fungus also undergoes marked changes as a result of storage. S04 Ascertain the level of fungicide resistance/tolerance of selected S04 Methods This study used fungicides that are recommended for growers and amateurs for rose blackspot control. The fungicides used within the study are listed in Tables 1 & 2 in Appendix S04. The isolates used within the study were chosen from the stratified sample that took into account different geographical locations and also varying molecular results. A range of methods were used to assess the level of fungicide resistance/tolerance of selected isolates. a) Poisoned disk method A fungal lawn was prepared on a plate of ¼ PDA using a suspension of D. rosae spores at concentration of 10-4 spores. The plates were incubated for 24 hours prior to the biocide treatment. A single sterile disc (0.5mm in diameter) was 12 placed in the centre of the plate. The disc was then saturated with the active ingredient (0.04mls) at a field rate concentration. The zone of inhibition was then measured after seven or more days. (five replicates). The controls used were sterile water and 25% copper sulphate solution. b) Poisoned plate method The active ingredient at field rate concentration was added to molten 1/4 PDA and poured into plates (Table 3 in Appendix S04). A 0.5 ml suspension of spores at a concentration of 104 was spread on the plates using a flamed glass spreader. The plates were then incubated and spore germination was recorded after 72 hours, (10 replicates). The controls used were spores spread onto plates of 1/4PDA. c) Detached leaf method Detached rose leaves were sprayed with the field rate concentration of the active ingredient. This method was used to test the systemic compound Azoxystrobin as this only active in a living leaf. The leaves were placed into a plate containing 0.6% agar in order to keep the leaf hydrated and were left for 7 days. The leaves were then inoculated with an isolate of D. rosae and lesion development measured after 10-11 days, (10 replicates). S04 Results 10 Total inhibition Zone of inhibition (cms) 99 8 7 8 7 6 6 5 5 4 4 3 3 2 2 1 1 0 STW CuSo4 Myclobutanil Dichlofluanid Penconazole No inhibition DR137 DR212 DR156 DR60 DR130 DR216 Isolates Figure S04: Average zone of inhibition of D. rosae isolates in the presence of fungicide active ingredients (Poison disk method) The results show that Penconazole had a consistent repeatable inhibitory effect on D. rosae. A more varied response was seen with Myclobutanil and Dichlofluanid see Figure S04..The poison plate method using Captan (Appendix S04 Figure b and Table b) showed a total inhibitory effect on the isolates tested (DR212 & DR156). Detached leaves inoculated with DR212 and then sprayed with the Azoxystrobin showed no symptoms after 14 days (Appendix S04 Figure c). S04 Discussion Growers report that it is hard to get repeatable consistent control of D. rosae. This may be due to the variability of the pathogen (02/02) or it may be due to variation in environmental conditions (05/02). These results for fungicide sensitivity suggested that there also can be a varied response to fungicides. This would be worth future investigation as it suggests that there is variation in sensitivity to these two fungicides in the population of D. rosae used in this study. Growers therefore need to pay particular attention to the active ingredient of the fungicide and aim to alternate mode of action, not just manufacturer brand. S04 Outcome 13 There is a variation in response of isolates to the standard fungicides used to control D. rosae. Growers need to be encouraged to use these fungicides wisely. Aim 03: Determine the resistance of a carefully selected range of host cultivars and species to different pathogen genotypes. 03/01 Determine the resistance of a range of selected cultivars in vivo to pathotypes 03/01 Method Three replicates of each rose cultivar selected in section 01/03 were grown in a clean environment with no overhead watering. Roses were monitored regularly and any sign of rose blackspot was removed. Ten of the 15 isolates tested in 2001 were used for this. Control tests were set up using distilled water. Inoculations were carried out at the same time as the in vitro pathogenicity work so comparisons could be made between the results. The standard operating procedure (Appendix 01/03) was used for all inoculations. 03/01 Results All ten isolates tested in vivo produced a compatible reaction on at least three rose cultivars. A summary table that also includes the in vitro results for comparison is shown in Table 03/01 below. Table 03/01: Resistance of a range of selected cultivars to the isolates Isolate. No. DR C Climbing All gold Frensham Davidii All gold --- DR R -- DR R -- -- DR C DR N -- -- -- DR N -- -- -- DR O -- -- -- DR O -- -- -- DR T -- -- -- DR T -- -- -- DR P -- -- -- DR P -- -- -- DR Q -- -- -- DR Q -- -- -- DR Sl -- -- -- DR Sl -- -- -- DR L -- -- DR L -- DR M -- DR M -- Key: In vivo (attached) In vitro (detached) ---- ---- Mrs Doreen Pike Flower Carpet Masquerade R. rugosa -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- Martin Baby love Frobisher -- -- ---- -- -- -- --- -- -- -- -- -- -- Susceptible (production of acervuli) -- Resistant (no acervuli development) No infection on any of the test cultivars was seen with the negative control. A comparison was made between wounded and unwounded leaves, both in vitro and in vitro. These are shown in Figures 03/01a and 03/01b in Appendix 03/01. 11 out of the 16 isolates showed more disease development on wounded 14 detached leaves. The results also showed that 9 out of 10 isolates showed more disease on wounded than unwounded attached leaves. 03/01 Discussion The in vivo method developed was suitable for observing differences in pathogenicity between isolates. In vitro and in vivo pathogenicity patterns were identical for 50% of the isolates tested, namely isolates DRC, DRR, DRO, DRT and DRSl. Four of the isolates, namely DRN, DRP, DRQ, DRR showed a susceptible reaction on more cultivars in vivo than in vitro. One isolate, L, showed one different reaction on one cultivar in vitro and one difference in vivo. These results indicate that the attached leaf test is the most sensitive method for detecting susceptible cultivars to the rose blackspot fungus, and the in planta tests indicated it was suitable for use with growers/breeders. The attached leaf assay is also more closely related to the field grown roses. Results from the wounded vs. unwounded tests showed no significant difference in the number of leaf discs infected with rose blackspot fungus on detached leaves, but a significant difference (p=0.01) was found between wounded and unwounded leaf discs from the in vivo tests. Wounding leaves significantly increased the number of rose blackspot infections. This effect is most probably due to the removal of the protective cuticle during wounding, allowing the fungus to enter the leaf more readily. It is unclear why no significant difference was found when detached leaves were wounded. It is possible that the removal of the leaf and the cutting of leaf discs triggers a defence response in the leaves. This could account for a) the reduced amount of susceptible reactions compared to those seen in vivo as the defence responses are already active in the detached leaves, reducing the amount of infection and b) the similarity in number of leaf discs affected when wounded compared to unwounded in the in vitro test. 03/01 Outcome This test can be used to test cultivars for resistance to D. rosae. Climbing Allgold, Allgold, and Masqurade appeared ‘resistant’ in these tests. However, other tests carried out in this study (03/01) showed that they are susceptible. So this test shows that it is unwise for growers to rely on a single test for resistance. Aim 04: Define pathotypes (based on evidence from objectives 1-3) and develop molecular markers 04/01 Describe pathotypes based on genetic and physiological characters 04/01 Methods & Results The results and outcomes described in 02/02 and 03/01 resulted in a reconsideration of this milestone. However, molecular analysis was performed on a number of isolates supplied by Prof. A. Roberts which were reported as distinct pathotypes (Yokoya et al, 2000). Some of these showed clearly different molecular profiles (e.g. DR216 was in group 1 and DR168, DR169, DR166a, DR217 and DR167a were all in group 2 (Appendix 02/02 Figure 2). However, those in group 2 though published as different pathotypes had similar patterns suggesting close genetic relatedness. 04/01 Discussion At the outset of the project, recent findings based on susceptibility of cultivars to the rose blackspot pathogen (Debener et al., 1998; Yokoya et al., 2000) indicated the likely existence of a number of pathotypes. It was anticipated that these might be correlated with molecular profile, rose type (cultivar or growth form), differing susceptibility to chemical control measures, variation in environmental requirements, etc. However, the complex patterns of genetic variability which have been uncovered during these studies of the pathogen does not suggest that well-developed race specific pathotypes are likely to exist within the species. Molecular analyses were performed on a number of isolates supplied by Prof. A. Roberts (University of East London) which were reported as distinct pathotypes (Yokoya et al, 2000). Some of these showed clearly different molecular profiles, but others had closely similar patterns suggesting close genetic relatedness. It would have been highly desirable to correlate molecular data with the information on pathogenicity supplied by CSL. However, the extreme difficulties they encountered in isolating and maintaining the pathogenicity of the fungus in culture, and in obtaining infection from the cultured pathogen, meant that this was not possible. A much more detailed analysis of the genetics of Diplocarpon rosae (including a search for pathogenicity genes) would be advisable, but this lay outside of the remit of our project. 04/01 Outcome The pathogen has a very complex population structure and it is considered that recombination is frequent (02/02). There are also at least 17 pathogenic groups in the isolates tested. This means that it was not practical (or even useful) to 15 describe pathotypes as originally intended. The molecular analysis of the isolates supplied as pathotypes suggest that most of these are exhibiting close genetic relatedness. 04/02 Describe molecular markers for pathotypes This objective proved to be inappropriate in the light of molecular and physiological data acquired during the early part of the project. As explained in the sections 02/02 and 04/01 above, the results suggest that distinct pathotypes are not identifiable using molecular methods alone, and that further research is needed on the genetics of pathogenicity and the environmental factors which influence its expression. If this were completed, it would be possible to identify molecular markers from the DNA fragments that have been generated from the strains analysed, probably using SCAR technology (Hermosa et al., 2000). In the light of the results described above, it is also clear that development of a molecular marker would not be of benefit to growers and breeders in the immediate future. S05 Assess the feasibility of in vivo identification of pathotypes with molecular diagnostic tools In the light of the discussion above under 04/01 and 04/02 it was not feasible or useful to the growers to develop molecular diagnostic tools. S06 Determine the prevalence of the sexual stage on fallen overwintering leaves Fallen leaves, thorns and stems were examined for the presence of the sexual stage of the fungus throughout the three years of the project. Minute examination of stems, thorns, buds and fallen leaves etc. was routine throughout the studies at the Garden of the Rose and RHS Wisley. No evidence of the sexual stage was found. This is in accordance with the literature as the sexual stage of D. rosae has only been reported once in the UK (Knight & Wheeler, 1977). S06 Outcome No sexual stage has been found in vivo and so it is doubtful that the sexual stage plays a major part in the epidemiology and genetic variability of the fungus. This confirms the comments of Sivanesan & Gibson (1976). S07 Ascertain if the sexual stage can be produced in vitro S07 Method Rose blackspot cultures were tested under different temperatures (18C, 4C and 0C), cultures were also examined following treatments outlined in Appendix 01/04a. Cultures were regularly examined for the presence of the sexual structure (perithecium). In the course of the study 700 petri dishes were examined. S07 Results No evidence of the sexual stage of rose blackspot was observed from any of the treatments throughout the duration of the project. S07 Outcome No sexual reproduction was observed in vitro Aim 05: Determine the cultural and environmental factors favouring disease development to enable effective disease management and control 05/01 Determine inoculum sources 05/01 Methods The presence or absence of acervuli was monitored throughout the year on a 100 leaves that were being studied in 05/02b. This was done using a hand lens. A comparable number of thorns, stems and bud scales were also examined weekly throughout the study. 16 05/01 Results Acervuli in large numbers were recorded on infected leaves on the bush and on stems and thorns. However, acervuli were not detected on bud scales throughout the study. 05/01 Discussion The presence of active acervuli on stems and thorns early in the season provides an ample source of new inoculum even in the absence of fallen infected leaves. This has consequences for how growers handle roses at the root pruning stage. 05/01 Outcome Active acervuli on stems and thorns early in the year are an important inoculum source for the initiation of epidemics. 05/02 Ascertain the environmental factors favourable for disease development. 05/02 Methods a) Environmental monitoring Rainfall, air temperature (C) hourly, relative humidity (%RH) hourly (Gemini Tinytag Plus (-30—50C & 0-100% RH) and also leaf wetness (Delta-T Data Logger) were all measured throughout the years 2000 and 2001 in and around rose bushes (Appendix 05/02 Figure 2). In 2001 surface wetness was recorded every hour using the Gemini Tinytag leaf wetness recorder. b) Lesion and acervuli development Lesion development was monitored each week from April to October 2001 by tracing occurrence and growth of lesions on leaflets. Lesion development was assessed by calculating the percentage of infection on a leaflet. Acervuli, both immature and mature were also examined on the leaf surface at weekly intervals. Disease development was also mapped on the bush. A total of 80 leaves were studied in this study in 2001. The inoculum source for the disease was reported to be from diseased leaves at the bottom of the bush (Cook, 1981) and spread initially by upward splash by rain. The height of rain splashes were measured using a splashmeter (Appendix 05/02 Figure 1 & 2) and correlated to the appearance of disease development on the rose bush. c) Disease transport Upward splash was measured using a splashmeter, adapted from Shaw (1987) (Appendix 05/02 Figure 1). The device was placed within a rose bush (Appendix 05/02 Figure 2) in May 2001 and evaluated at intervals. Raindrops were allowed to fall into a small trough containing glass fibre, saturated with a cellulose binding dye, UVITEX NFW (CibaGeigy) diluted at a rate of 1:100 with distilled water. 3 ml. of the dye was placed in each trough. The splash drops were caught on a cylinder of chromatography paper placed in the centre of the troughs. The frequency and height of raindrops and the percentage of rain-splash were measured. 05/02 Results a) Environmental conditions and lesion development Observations within the bush showed that initial disease symptoms developed on leaves at the base of the bush. It was noticed that symptoms developed at different times vertically on the bush. Rainfall was most frequent between the months of August-December 2001 (Appendix 05/02 Figure 5). This also correlates with high leaf surface wetness and relative humidity. Disease development on a leaf surface was influenced by leaf wetness and relative humidity. Results showed that as leaf wetness and % relative humidity increased so did the rate of lesion development Figure 02/05a. Leaf lesions were increased rapidly in the wetter months of September and October 2001 when the overall relative humidity and leaf wetness readings were higher, (Figure 05/02a also Appendix 05/02 Figure 1) than in June and July there was an overall reduced incidence of leaf wetness and humidity and the rate of lesion development was slower, (Figure 05/02b see also Appendix 02/05 Figure 2). Key: 17 Temperature Humidity Leaf wetness Lesion development on a single leaf Temperature (C), Humidity (%) Leaf wetness (%) Lesion development (mm3) Figure 02/05a Example of printout for temperature, humidity, leaf wetness and lesion development in October 2001 showing a rapid rate of lesion development linked to high leaf wetness and humidity. Figure 02/05b Example of printout for temperature, humidity, leaf wetness and lesion development for June 2001 showing a slow rate of lesion development linked to low leaf wetness and humidity. (Key as Figure 02/05a). b) Rain-splash and disease transport The splashmeter allowed the height and density of rain splashes within the bush to be quantified (Figure 3 and figure 4 Appendix 05/02 ). The greatest concentration of rain splashes was at the base of the splashmeter with less frequent rain splashes being recorded high on the splashmeter. The disease symptoms were mapped throughout the bush. Symptoms occurred first at the bottom of the bush (0-30cms) which coincided with the greatest frequency of rain splash on the splashmeter (5-15cms). Symptoms slowly progressed to the middle of the bush (30-60cms) where the frequency of rainsplash ranged from 30-40% and then to the top of the bush where the frequency of rain-splash ranged from 0-5% (Figure 3 Appendix 05/02). At this height there were few rain splashes to initiate infection (Figure 4 Appendix 05/02). By the end of the season (October 2001), rose blackspot was evenly distributed throughout the bush. 05/02 Discussion Initial infection occurs as a result of rain-splash within and into the lowest part of the bush. The source of spores carried by splashes is either from acervuli on thorns and branches or from fallen leaves. Initiation of infection above 60cms is 18 from occasional rain-splashes. However, once lesions occur at height in the bush, infection spreads both up the bush by splash and down the bush by drip. These results also emphasise the importance of surface wetness both for initial infection and lesion development. The rate of disease development is clearly corralated with surface wetness. 05/02 Outcome Two additional environmental parameters (surface wetness and rain splash) have been quantified and can be used in an overall disease control strategy. The progress of an epidemic within a rose bush has been quantified and followed. 05/03 Overall Discussion and Conclusion The aims and objectives of this project were formulated making some assumptions. In particular, it was assumed from the literature (Yokoya et al, 2000, Deberner,) at the start of the project that D. rosae and its hosts might behave in a manner parallel to a ‘typical’ pathogen of arable crops. It was assumed that there might be race specific pathotypes and that these could be effectively characterised by using molecular techniques, and that this knowledge would inform the development of a more coherent control strategy for this pathogen. However, the results reported here suggest that this is not how D. rosae behaves. The morphological and physiological studies (S03) show the fungus to be highly variable with respect to colony colour and spore size. The work on the storage of the fungus (milestone 01/01) shows that not only does it lose pathogenicity very easily but also that storage has profound effects on the growth and behaviour of D. rosae with respect to e.g. spore size (on going work, not reported here). The molecular work (milestone 02/02 & 04/01) shows quite clearly that the fungus has a very complex population structure which is not dependant on the detectable presence of teleomorph (S06 & S07). It is suggested that this observed variability is probably due to parasexual recombination. In spite of the highly variable nature of this fungus, few clones of D. rosae were detected (milestone 02/02) and there are no clearly defined pathotypes (milestone 04/01). The pathotypes as published by others (Yokoya et al, 2000) also proved to not be clearly defined at the molecular level (milestone 02/02). Furthermore, there is some indication of a variable response of different isolates to the commonly used fungicides. Taken together these factors indicate that the horticultural industry faces a major challenge in controlling this pathogen. Assumptions that there are a few clearly defined pathotypes are not supported by the results reported here. There is no indication that the variability detected is linked to geographical location or the type of rose from which the isolates were obtained (milestone 02/02). Furthermore, there is little indication that the mainstream commercial breeders have any lasting inheritable resistance to rose blackspot in the ‘gene pool’ of the parentage of present cultivars. Overall, the results of this study indicate that there has been a long period of co-evolution between roses and D. rosae (milestone 02/02). This means that the underlying assumption of this study that, this host and pathogen would mimic a conventional arable pathosystem, was incorrect. A more satisfactory paradigm for this host pathogen complex is that of the pathosystem of a semi-natural or natural host community where pathogen and host are both genetically heterogeneous and have had a long period of co-evolution and are in dynamic equilibrium. Or to express it differently, Diplocarpon rosae is so variable, and so widespread geographically that, at present, provided the environmental conditions are favourable, a ‘strain’ of the fungus can attack any new cultivar the breeders produce. This is exacerbated by the fact that most rose breeders are utilising a relatively narrow gene pool for their breeding programme and are far more interested in other commercially important traits then disease resistance. Furthermore, ‘resistance testing’ is carried out late in the breeding programme, often only with local ‘strains’ of the isolate, and if the commercially desirable traits are particularly good, then the cultivar will probably be marketed anyway. Finally, the life of most commercial cultivars is quite short. In essence, a new approach is needed to manage and control this pathogen. The epidemiological studies demonstrate that sources of inoculum within the bush (acervuli on stems and thorns) are as important as inoculum from fallen leaves initiating epidemics (milestone 05/02). These studies also demonstrate that the initiation of the epidemic at the base of the bush is linked to the greatest concentration of rain splash. The quantification of leaf surface wetness confirms the vital importance of this parameter in disease development. This new epidemiologocal prospective should be utilised in the control strategy particularly with respect to the inoculum source within buses. 19 05/03 Develop a strategy for improved disease management and control. At present when growers uproot roses prior to root pruning and potting on for sale they stack the roses in 2-3 meter high trolleys and trickle water over them. This must be distributing conidia throughout the column of roses from the acervuli on the stems and thorns. Growers should either consider using a fungicide prior to this stage or stop trickling water through the column of roses. Growers should consider fungicide use before bud break to kill conidia in acervuli in thorns and on stems, thus lowering inoculum potential. The old advice to remove dead leaves and to prune hard and remove prunings is vital and should be used in conjunction with pre bud break fungicide. Breeders should consider using a far broader gene pool to seek for resistance. There is some indication that there is some resistance in species roses. Breeders should look for inherited race non specific resistance as this would give a measure of resistance to all strains of the fungus. Breeders should challenge candidate cultivars as early as possible with a wide range of isolates of the pathogen as possible (race non specific resistance will only be found when using as many isolates as possible of the fungus when challenging candidate cultivars). Growers should consider a universal susceptible variety (Frensham) as a bait plant within their breeding programme. This should assist in providing the range of strains needed for screening for race non specific resistance in the host. Growers should understand that the mode of action of a fungicide is of vital importance and should be prepared to change mode of action not just manufacturers brand (similar advice to that given to arable farmers by the FUNGICIDE RESISTANCE ACTION COMMITTEE FRAC should be given). Future strategy There is clearly a need for roses to have long term disease resistance not only to rose blackspot but also to powdery mildew, downy mildew and rust. At present, growers would like this to be achieved without the use of genetic manipulation or excessive pesticide use (the present researchers feel that genetic manipulation could be a long term solution to this problem). It is possible that this long term disease reduction could be achieved using Systemic Acquired Resistance (SAR). UH proposes to carry out preliminary studies on the use of elicitors (including salicylic acid) as a spray and chitin incorporated into the growing medium in summer 2002. 20
