Chinthani Shanika Karandeni Dewage, Kavithra Jayani Wijerathna, Henrik U. Stotz, and Bruce D. L. Fitt, 'Improved understanding of novel sources of resistance against the light leaf spot pathogen, Pyrenopeziza brassicae', paper presented at the Association of Applied Biologists Conference Crop Production in Southern Britain 2017, 15 - 16 February 2017, Peterborough, UK. Proceedings available online at: http://www.aab.org.uk/contentok.php?id=501.In this work, the endophytic growth phase of the light leaf spot pathogen Pyrenopeziza brassicae in selected lines from a doubled haploid (DH) population of oilseed rape, which is known to segregate for resistance against P. brassicae, was characterised using controlled environment (CE) experiments. Fungal staining techniques and pathogen-specific quantitative polymerase chain reactions (qPCR) were used to observe and quantify the pathogen biomass, respectively. The qPCR results showed that the resistant lines contained little P. brassicae DNA and there seemed to be little to no change in the amount of DNA over time. In contrast, there was a considerable increase in pathogen DNA in susceptible lines from 0 to 24 days post inoculation (dpi). These results were also reflected in observations made by a fungal staining method. In addition, leaf samples of these DH lines, collected at three different times from winter oilseed rape field experiments, were analysed using qPCR. The resistant lines had a considerably smaller amount of P. brassicae DNA in leaf samples collected later in the cropping season than that in susceptible lines

Fitt, Bruce D. L.

Karandeni Dewage, Chinthani Shanika

Stotz, Henrik U.

Wijerathna, Kavithra Jayani

University of Hertfordshire Research Archive

Research ArchiveCitation for published version:K. Dewage, K. W. Wijerathna, H. U. Stotz, and B. D. L. Fitt,‘Improved understanding of novel sources of resistanceagainst the light leaf spot pathogen, Pyrenopeziza brassicae’,Proceedings, Aspects 134: Crop Production in SouthernBritain 2017.DOI:Link to published proceedingsDocument Version:This is the Published Version.Copyright and Reuse: Reproduced with permission from Association of Applied Biologists.Content in the UH Research Archive is made available for personal research, educational, and non-commercial purposes only.  Unless otherwise stated, all content is protected by copyright, and in the absence of an open license, permissions for further re-use should be sought from the publisher, the author, or other copyright holder.EnquiriesIf you believe this document infringes copyright, please contact the Research & Scholarly Communications Team at rsc@herts.ac.ukAspects of Applied Biology 134, 2017Crop Production in Southern Britain221Improved understanding of novel sources of resistance againstthe light leaf spot pathogen, Pyrenopeziza brassicaeBy C S KARANDENI DEWAGE, K J WIJERATHNA, H U STOTZ and B D L FITTSchool of Life & Medical Sciences, University of Hertfordshire, Hatfield,Herts AL10 9AB, UKCorresponding Author Email: c.s.karadeni-dewage@herts.ac.ukSummaryIn this work, the endophytic growth phase of the light leaf spot pathogen Pyrenopeziza brassicae in selected lines from a doubled haploid (DH) population of oilseed rape, which is known to segregate for resistance against P. brassicae, was characterised using controlled environment (CE) experiments. Fungal staining techniques and pathogen-specific quantitative polymerase chain reactions (qPCR) were used to observe and quantify the pathogen biomass, respectively. The qPCR results showed that the resistant lines contained little P. brassicae DNA and there seemed to be little to no change in the amount of DNA over time. In contrast, there was a considerable increase in pathogen DNA in susceptible lines from 0 to 24 days post inoculation (dpi). These results were also reflected in observations made by a fungal staining method. In addition, leaf samples of these DH lines, collected at three different times from winter oilseed rape field experiments, were analysed using qPCR. The resistant lines had a considerably smaller amount of P. brassicae DNA in leaf samples collected later in the cropping season than that in susceptible linesKey words: Oilseed rape, Pyrenopeziza brassicae, yield loss, light leaf spot, host resistance, extracellular pathogensIntroductionThe hemibiotrophic fungus Pyrenopeziza brassicae is an economically important pathogen of winter oilseed rape in the UK, causing light leaf spot (Boys et al., 2007, 2012). Light leaf spot is a polycyclic disease and early infections during autumn and winter are able to kill seedlings, decrease plant vigour and increase susceptibility to frost damage (Fitt et al., 1998; Gilles et al., 2001). Light leaf spot on pods causes early maturation and pod shatter leading to further yield losses. Previously, severe disease epidemics have been recorded in Scotland and northern England, where the climatic conditions are favourable for disease development (Figueroa et al., 1995). However, according to recent disease survey data, the severity of epidemics has increased progressively across England since 2006 (CropMonitor, 2016). This frequent, widespread occurrence of light leaf spot has made its control a high priority for many oilseed rape growing areas in the UK. Light leaf spot disease control has mainly relied on the use of fungicides. Fungicide applications can be inefficient due to poor timing and reduced sensitivity to azole fungicides has also been reported (Carter et al., 2014). Therefore, deployment of cultivar resistance against P. brassicae is of a greater importance for successful management of this disease. However, there is only a limited amount of information available on the operation of resistance in oilseed rape against P. 222brassicae. Little is known about the mechanisms of genetic resistance in different commercial oilseed rape cultivars. With recent severe disease epidemics, breakdown of resistance of some cultivars with high resistance ratings has been reported. Therefore, there is a need to improve the understanding about this pathosystem and search for new sources of resistance in different genetic backgrounds. In this study, the endophytic growth phase of the light leaf spot pathogen in selected doubled haploid (DH) lines of oilseed rape derived from a cross between Brassica rapa and B. oleracea was characterised using in planta experiments. These DH lines had been observed to produce different amounts of P. brassicae sporulation under controlled environment conditions, three weeks after the conidial inoculation (K Downes, unpublished data) and in field experiments (C S Karandeni Dewage, unpublished data). In this study these DH lines were studied under controlled environment (CE) conditions after artificial inoculation in comparison to winter oilseed rape experiments with natural inoculum in order to assess the development of light leaf spot disease under the two experimental conditions. Materials and MethodsPlant growth and inoculation in controlled environment (CE) conditionsEight DH lines (four resistant lines labelled R1-R4 and four susceptible lines labelled S1-S4) and two commercial cultivars susceptible to P. brassicae (labelled cv1 and cv2) were grown in a glasshouse until the plants reached growth stage 1,04 (Sylvester-Bradley et al., 1985). Plants were point inoculated by placing c. 1 cm2 pieces of Whatman no. 1 filter paper soaked in a P. brassicae conidial suspension (105 spores mL) on four marked locations on the fourth true leaf. Inoculated plants were transferred into a controlled environment cabinet (A1000, Conviron, Cambridgeshire, UK) at 80% relative humidity and with a 12 h day length with a 210 μe m2 s-1 light intensity. Day and night temperatures were maintained at 16°C and 14°C, respectively. Plants were covered with a polyethylene sheet for 48 h after inoculation to maintain high humidity to facilitate spore germination and infection. Collection and preparation of leaf samples Leaf samples from randomly selected plants were collected at 0, 3, 7, 13, and 24 days post inoculation (dpi). Leaf discs of the same size (1.6 cm diameter) were taken from the inoculated fourth true leaf of each plant. The leaf discs used for DNA extraction were inserted into labelled sterile screw-cap tubes and stored at -20°C after freeze drying. The leaf discs used for staining were placed in labelled Petri dishes.DNA extraction and quantification of P. brassicae DNA in leaf samples Freeze-dried samples were processed in a FastPrep machine (MP Biomedicals, UK) with three sterile metal beads (3 mm diameter) until the leaf discs were ground into a fine powder. DNA was extracted with a DNAMITE DNA extraction kit according to the manufacturer’s protocol (DNAMITE Plant Kit, Microzone Ltd, UK). The DNA concentration was measured using a Nanodrop ND-1000 spectrophotometer (Labtech International, UK) and samples were adjusted to a final concentration of 20 ηg μL with sterile distilled water for qPCR analysis. The amount of P. brassicae in 50 ng of DNA from each leaf sample was quantified using P. brassicae diagnostic primers PbITSF and PbITSR (Karolewski et al., 2006) using an Mx3005 qPCR instrument (Agilent technologies, UK). qPCR reaction mixture was prepared by adding 10 μL Brilliant III ultra-fast SYBR Green qPCR mix, 6.3 μL sterile distilled water, 0.6 μL (10 μM) PbITSF, 0.6 μL (10 μM) PbITSR and 2.5 μL (20 ηg μL) template DNA. Five standards were prepared using P. brassicae DNA ranging from 10000 pg to 1 pg to plot a standard graph for P. brassicae DNA quantification. Template DNA replaced with sterile distilled water was the negative control and 1 ηg of P. brassicae DNA was the positive control.   No-template controls, five standards, positive control and plant sample DNA were added in duplicate for each run, in 96 mL × 0.2 mL PCR plates (ABgene) covered with cap strips and the 223PCR conditions were set for an initial denaturation temperature of 95°C for 2 min followed by 50 cycles of 15 s at 95°C, 45 s at 58°C, 45 s at 72°C and a data collection step of 15 s at 84°C.The final step of the qPCR consisted of a melting curve with cycling parameters of 95°C for 1 min, 58°C for 30 s and 95°C for 30 s. The amount of P. brassicae DNA in each sample was estimated using the standard curve. Results were expressed as amount (ρg) of P. brassicae DNA in 50 ηg total DNA from each plant sample. Assessment of  P. brassicae biomass in leaf samples with trypan blue stainingLeaf discs were decolourised by adding a solution of ethanol and chloroform (3:1, v/v) and stained with 0.025% trypan blue solution for a period of 4 h. Stained leaf discs were each mounted on a glass slide containing a drop of 70% glycerol and covered with a cover slip. The leaf discs were observed under a microscope at ×400 magnification and images were taken for analysis.Winter oilseed rape field experimentsIn the 2015/16 cropping season, a field experiment was established at Rothwell, Lincolnshire including the DH lines used in this study and two commercial oilseed rape cultivars labelled cv1 (susceptible to P. brassicae) and cv3 (resistant against P. brassicae). Leaves were sampled on 19 November 2015, 19 February 2016, and 4 April 2016 by randomly selecting 6–10 plants from each line and from control cultivars. Each leaf sample was put in a labelled 15 mL Falcon tube, freeze-dried and stored at -20°C for further analysis. Freeze dried leaf samples of the DH lines studied in the CE experiment and the two control cultivars were selected (Table 1) and leaf samples were ground separately with mortar and pestle until they became a fine powder. A sub-sample of 20 mg was used for DNA extraction. DNA extraction and the quantification of P. brassicae in these samples were according to the methods described.ResultsP. brassicae DNA in leaf samples at different timesAccording to the qPCR results for the average quantity of P. brassicae DNA in leaf samples collected from the controlled environment experiments at selected times, resistant lines had considerably less P. brassicae DNA than susceptible lines and the two susceptible cultivars. There was a considerable increase in the amount of P. brassicae DNA in susceptible lines and in the commercial cultivars between 0 and 24 dpi.  There was a considerable difference in the amount of pathogen DNA between resistant and susceptible lines at 24 dpi (Table 2). Table 1. Number of leaf samples of different lines/cultivars taken from the winter oilseed rape experiment for the quantification of  P. brassicae by qPCR. The field experiment was established at Rothwell, Lincolnshire in 2015/16 cropping season and leaf samples were collected at three different timesNumber of samplesSample date R1a R2 a R3 a R4 a S1b S2 b S3 b S4 b cv1c cv3 c19 Nov 2015 10 * * 6 8 6 6 10 6 619 Feb 2016 8 4 6 6 7 8 8 8 * 64 Apr 2016 6 4 4 8 8 7 6 6 10 8* No samples were taken; a - Lines resistant against P. brassicae; b - Lines susceptible to P. brassicaec - Commercial oilseed rape cultivars.224Table 2. Amount of  P. brassicae DNA (mean ± SEM) (ρg) in 50 ηg of total DNA extracted from different lines/cultivars from 0–24 days post inoculation. Plants were point inoculated in controlled environment conditions with a P. brassicae conidial suspension and leaf sampleswere taken at five different times (days post inoculation)P. brassicae DNA (ρg)Sample date (dpi)R1a R2a R3a R4a S1b S2b S3b S4b cv1c cv2cDay 0 1.4±0.1* 0.8±0.40.6±0.40.3±0.1* * 1.2±3.90.2±0.10.4±0.2Day 3 21.4±2.70.5±0.12.5±1.32.5±0.73.5±0.70.6±0.1* 26.1±2.730.4±3.74.6±0.5Day 7 34.4±19.7* 15.7±3.05.5±2.984.0±22.71.5±0.7* 60.3±18.426.3±6.051.6±0.7Day 13 20.7±3.4* 19.4±2.114.2±3.144.8±7.043.5±11.1* 42.9±4.6176.4±41.1173±20.6Day 24 32.6±19.329.3±9.414.7±2.117.4±1.460.7±5.775.2±18.7121.7±24.657.3±11.6105.4±24.6207.4±57.9* No data available for these sampling times; a - Lines resistant against P. brassicaeb - Lines susceptible to P. brassicae; c - Commercial oilseed rape cultivars.P. brassicae biomass in leaf samples assessed with trypan blue stainingThe microscopic images indicated that there was more P. brassicae colonisation in susceptible lines than in resistant lines. According to the observations made at different times, spore germination and penetration was observed by 3 dpi in most of the lines/cultivars and the P. brasscae colonisation had started by 7 dpi. There were differences between different lines/cultivars in the number of successful fungal penetrations as well as in the endophytic growth of P. brassicae. The resistant lines had little fungal penetration and colonisation. In contrast, the susceptible lines had substantial colonisation by 7 dpi and this increased progressively until 24 dpi. The two commercial cultivars, cv1 and cv2, were observed to have the most pathogen penetration and colonisation.Fig. 1. The amount of P. brassicae DNA in the leaf samples of different DH lines and cultivars collected from a winter oilseed rape field experiment in April 2016. (R-resistant lines, S-susceptible lines, cv1-susceptible cultivar and cv3- cultivar resistant against P. brassicae). 0204060R1 R2 R3 R4 S1 S2 S3 S4 cv1 cv3225Winter oilseed rape field experimentsThe qPCR results for the amount of P. brassicae DNA from the winter oilseed rape experiment samples collected later in the cropping season (4 April 2016) indicated a clear difference between the resistant and susceptible DH lines, cv1 (susceptible cultivar) and cv3 (resistant cultivar). There was more pathogen DNA in susceptible lines except for the line S1 than in resistant lines. The susceptible cultivar cv1 had a considerably greater amount of pathogen DNA compared to the resistant cultivar cv3 (Fig. 1). There was no clear difference between the resistant and susceptible lines in the amount of pathogen DNA in the leaf samples collected at the first two sampling times.DiscussionThe qPCR results for the average quantity of P. brassicae DNA in different lines at different sampling times suggested that P. brassicae grew less in resistant lines than in susceptible lines. The results were confirmed by the analysis of colonisation by P. brassicae using fungal staining of leaf samples at different times, where there was less pathogen biomass in the resistant lines than the susceptible lines. In resistant lines, the amount of pathogen DNA showed little increase from day 0 to day 24. In contrast, there was a considerable increase in pathogen DNA in susceptible lines from 0 to 24 dpi. The leaf samples collected from the winter oilseed rape field experiment in April 2016 also indicated the presence of less P. brassicae DNA in resistant lines than in susceptible lines. It was also observed that there were differences between different lines in number of successful infections. However, more research is needed to confirm this observation. It will also be useful to further study these DH lines to identify the effect on other stages of P. brassicae life cycle. There are previous studies on resistance against P. brassicae (Bradburne et al., 1999; Boys et al., 2012). Boys et al. (2012) reported a resistant phenotype that prevents asexual sporulation of P. brassicae. However, the pathogen was able to continue its growth and undergo sexual sporulation after leaf senescence.In a previous study, different amounts of P. brassicae sporulation were observed in different lines from this DH population (K Downes, unpublished data).  However, the resistant lines selected for the work described here produced no asexual sporulation. Combination of both visual and molecular assessment methods (such as qPCR) provide insights into the endophytic growth phase of P. brassicae and help to identify points at which the host resistance may operate in relation to the pathogen life cycle. Considering the fungal staining and the qPCR data from both controlled environment experiment and the field experiments, it can be suggested that asexual sporulation of P. brassicae is interfered with or prevented in the resistant lines by inhibiting the pathogen growth. This type of resistance may also contribute to reduce the amount of pathogen inoculum available. The resistant lines from this population can serve as pre-breeding material to provide a new source of resistance against the light leaf spot pathogen P. brassicae. Identification of markers linked to resistance loci can be used to improve the efficiency of breeding programmes. AcknowledgementsThe authors acknowledge the funding received from AHDB Cereals and Oilseeds (AHDB PhD studentship no. 2140010 and summer student bursary), the Gen Foundation and the University of Hertfordshire. We thank Mark Nightingale (Elsoms Seeds UK Ltd) and Dr Vasilis Gegas (Li-magrain UK Ltd) for their in-kind contributions to this work. ReferencesBoys E F, Roques S E, Ashby A M, Evans N, Latunde-Dada A O, Thomas J E, West J S, Fitt B D L. 2007. Resistance to infection by stealth: Brassica napus (winter oilseed rape) and Pyrenopeziza brassicae (light leaf spot). European Journal of Plant Pathology 118:307–321.226Boys E F, Roques S E, West J S, Werner C P, King G J, Dyer P S, Fitt B D L. 2012. Effects of R gene-mediated resistance in Brassica napus (oilseed rape) on asexual and sexual sporulation of Pyrenopeziza brassicae (light leaf spot). Plant Pathology 61:543–554.Bradburne R, Majer D, Magreth R, Werner C, Lewis B, Mithen R. 1999. Winter oilseed rape with high levels of resistance to Pyrenopeziza brassicae derived from wild Brassica species. Plant Pathology 48:550–558.Carter H E, Fraaije B A, West J S, Kelly S L, Mehl A, Shaw M W, Cools H J. 2014. Alterations in the predicted regulatory and coding regions of the sterol 14alpha-demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae. Molecular Plant Pathology 15:513–522.CropMonitor. 2016. Survey of commercially grown winter oilseed rape [Online]. Available: http://www.cropmonitor.co.uk/wosr/surveys/wosr.cfm [Accessed 1 Aug 2016].Figueroa L, Fitt B D, Welham S J, Shaw M W, McCartney H A. 1995. Early development of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) in relation to temperature and leaf wetness. Plant Pathology 44:641–654.Fitt B D L, Doughty K J, Gladders P, Steed J M, Sutherland K G. 1998. Diagnosis of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) in the UK. Annals of Applied Biology 133:155–166.Gilles T, Fitt B D L, McCartney H A, Papastamati K, Steed J M. 2001. The roles of ascospores and conidia of Pyrenopeziza brassicae in light leaf spot epidemics on winter oilseed rape (Brassica napus) in the UK. Annals of Applied Biology 138:141–152.Karolewski Z, Fitt B D L, Latunde-Dada A O, Foster S J, Todd A D, Downes K, Evans N. 2006. Visual and PCR assessment of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) cultivars. Plant Pathology 55:387–400.Sylvester-Bradley R. 1985. A revised code for stages of development in oilseed rape (Brassica napus L.). Aspects of Applied Biology 10, Field Trials Methods and Data Handling, pp. 395–400.

Improved understanding of novel sources of resistance against the light leaf spot pathogen, Pyrenopeziza brassicae

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Improved understanding of novel sources of resistance against the light leaf spot pathogen, Pyrenopeziza brassicae

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