Phoma stem canker is a major disease of oilseed rape in the UK, leading to annual yield losses worth more than £100M. The disease is caused by two closely related species, Leptosphaeria maculans and L. biglobosa. L. maculans is generally considered more damaging, causing stem base canker; L. biglobosa is generally less damaging, causing upper stem lesions. Therefore, previous work has mainly focused on L. maculans and there has been little work on L. biglobosa. This work investigated the contribution of L. biglobosa to stem canker epidemics by assessing the amounts of DNA of L. maculans and L. biglobosa in upper stem lesions or stem base cankers on winter oilseed rape cultivars with different types of resistance against L. mac ulans. Diseased upper stem and stem base samples were collected from nine oilseed rape cultivars in a 2011/2012 field experiment at Rothamsted. The presence of L. maculans or L. biglobosa in each stem sample was detected by speciesspecific PCR. The abundance of L. maculans or L. biglobosa in each stem sample was measured by quantification of L. maculans DNA and L. biglobosa DNA using quantitative PCR (qPCR). The amounts of L. biglobosa DNA were greater than those of L. maculans DNA in both upper stem and stem base samples. These results suggest that the severe upper stem lesions and stem base cankers in the 2011/2012 cropping season were mainly caused by L. biglobosa, suggesting that L. biglobosa can sometimes cause considerable yield loss in the UK. There were differences between cultivars in the amounts of L. maculans DNA and L. biglobosa DNA, with the susceptible cultivar Drakkar having more L. maculans DNA than L. biglobosa DNA while resistant cultivars had less L. maculans DNA than L. biglobosa DNA. These results suggest that L. biglobosa can be an important cause of phoma stem canker on oilseed rape in the UK.Peer reviewedFinal Published versio

Fitt, Bruce

Huang, Yongju

Karandeni Dewage, Chinthani Shanika

English

Phoma stem canker is a major disease of oilseed rape in the UK, leading to annual yield losses worth more than £100M. The disease is caused by two closely related species, Leptosphaeria maculans and L. biglobosa. L. maculans is generally considered more damaging, causing stem base canker; L. biglobosa is generally less damaging, causing upper stem lesions. Therefore, previous work has mainly focused on L. maculans and there has been little work on L. biglobosa. This work investigated the contribution of L. biglobosa to stem canker epidemics by assessing the amounts of DNA of L. maculans and L. biglobosa in upper stem lesions or stem base cankers on winter oilseed rape cultivars with different types of resistance against L. mac ulans. Diseased upper stem and stem base samples were collected from nine oilseed rape cultivars in a 2011/2012 field experiment at Rothamsted. The presence of L. maculans or L. biglobosa in each stem sample was detected by speciesspecific PCR. The abundance of L. maculans or L. biglobosa in each stem sample was measured by quantification of L. maculans DNA and L. biglobosa DNA using quantitative PCR (qPCR). The amounts of L. biglobosa DNA were greater than those of L. maculans DNA in both upper stem and stem base samples. These results suggest that the severe upper stem lesions and stem base cankers in the 2011/2012 cropping season were mainly caused by L. biglobosa, suggesting that L. biglobosa can sometimes cause considerable yield loss in the UK. There were differences between cultivars in the amounts of L. maculans DNA and L. biglobosa DNA, with the susceptible cultivar Drakkar having more L. maculans DNA than L. biglobosa DNA while resistant cultivars had less L. maculans DNA than L. biglobosa DNA. These results suggest that L. biglobosa can be an important cause of phoma stem canker on oilseed rape in the UK.Peer reviewe

University of Hertfordshire Research Archive

Aspects of Applied Biology 127, 2014Crop Production in Southern Britain: Precision Decisions for Profitable Cropping 117Importance of Leptosphaeria biglobosa as a cause of phoma stem canker on winter oilseed rape in the UKBy Y J HUANG, C S KARANDENI-DEWAGE and B D L FITTSchool of Life and Medical Sciences, University of Hertfordshire, College Lane, Hatfield, Hertfordshire AL10 9AB, UKCorresponding Author Email: y.huang8@herts.ac.ukSummaryPhoma stem canker is a major disease of oilseed rape in the UK, leading to annual yield losses worth more than £100M. The disease is caused by two closely related species, Leptosphaeria maculans and L. biglobosa. L. maculans is generally considered more damaging, causing stem base canker; L. biglobosa is generally less damaging, causing upper stem lesions. Therefore, previous work has mainly focused on L. maculans and there has been little work on L. biglobosa. This work investigated the contribution of L. biglobosa to stem canker epidemics by assessing the amounts of DNA of L. maculans and L. biglobosa in upper stem lesions or stem base cankers on winter oilseed rape cultivars with different types of resistance against L. mac  ulans. Diseased upper stem and stem base samples were collected from nine oilseed rape cultivars in a 2011/2012 field experiment at Rothamsted. The presence of L. maculans or L. biglobosa in each stem sample was detected by species-specific PCR. The abundance of L. maculans or L. biglobosa in each stem sample was measured by quantification of L. maculans DNA and L. biglobosa DNA using quantitative PCR (qPCR). The amounts of L. biglobosa DNA were greater than those of L. maculans DNA in both upper stem and stem base samples. These results suggest that the severe upper stem lesions and stem base cankers in the 2011/2012 cropping season were mainly caused by L. biglobosa, suggesting that L. biglobosa can sometimes cause considerable yield loss in the UK. There were differences between cultivars in the amounts of L. maculans DNA and L. biglobosa DNA, with the susceptible cultivar Drakkar having more L. maculans DNA than L. biglobosa DNA while resistant cultivars had less L. maculans DNA than L. biglobosa DNA. These results suggest that L. biglobosa can be an important cause of phoma stem canker on oilseed rape in the UK. Key words: Leptosphaeria biglobosa, Leptosphaeria maculans, oilseed rape, phoma stem canker, yield loss IntroductionPhoma stem canker is a major disease problem on oilseed rape (Brassica napus) in the UK, causing losses worth more than £100M p.a. at price of £300 t-1, despite use of fungicides costing £20M p.a. (Fitt et al., 2008; Stonard et al., 2010). The disease is caused by two closely related species, L. maculans and L. biglobosa, which co-exist on their oilseed rape host. L. maculans is generally considered more damaging than L. biglobosa because L. maculans is often associated with damaging stem base cankers and L. biglobosa is often associated with less damaging upper 118stem lesions (Huang et al., 2005). Therefore, previous work has mainly focused on L. maculans and there has been little work on L. biglobosa. Although L. biglobosa can cause substantial yield losses in Poland (Huang et al., 2005) and China (Liu et al., 2014), there has been little work on yield losses caused by L. biglobosa in the UK.  Although there have been studies on the co-existence of L. maculans and L. biglobosa in the UK (Huang et al., 2005; Fitt et al., 2006a; Stonard et al., 2010), the potential yield losses that can be caused by L. biglobosa have not been evaluated. In the 2011/2012 oilseed rape growing season, the timing of the first major ascospore release was later than normal. When compared with the previous growing season (2010/2011), the first major release of ascospores was more than two months later. This was followed by severe upper stem lesions and raceme lesions before harvest in the 2011/2012 season. Severe upper stem lesions caused breakage of upper stems, which led to yield loss at harvest in 2012. Previous work has suggested that ascospores of L. biglobosa are released later than those of L. maculans (Huang et al., 2011). There is a need to investigate whether the severe upper stem lesions in 2012 were caused by L. maculans or L. biglobosa and to improve our understanding of the importance of L. biglobosa in relation to control of phoma stem canker on oilseed rape in the UK.  Materials and MethodsPreparation of stem samplesA field experiment with nine winter oilseed rape cultivars (with different combinations of R gene-mediated and quantitative resistance) was drilled on 30 August 2011 at Rothamsted, Harpenden. Before harvest, stems of the nine cultivars (Table 1) with upper stem lesions and/or stem base cankers were collected on 12 July 2012.  A small piece of diseased stem tissue was excised from each stem lesion or canker and placed in a 2 mL tube. The stem samples were freeze-dried and stored at -20°C for further analysis. A total of 102 stem samples collected from nine cultivars in the 2011/2012 growing season were used for this study. Table 1. Cultivars and numbers of upper stem and stem base samples taken on 12 July 2012 from a winter oilseed rape field experiment at Rothamsted in the 2011/2012 growing seasonCultivar QualitativeresistanceQuantitativeresistanceNumber of samplesUpper stem Stem base Adriana Yes (Rlm4) Yes 7 6Bilbao Yes (Rlm4) No 4 5Excel Yes (Rlm7) Yes 8 8Capitol Yes (Rlm1) No 6 4Roxet Yes (Rlm7) ? 5 6DK Cabernet Yes (Rlm1) Yes 6 5NK Grandia No Yes 6 5ES-Astrid No Yes 6 5Drakkar No No 5 5Total 53 49Extraction of DNAThe freeze-dried upper stem and stem base samples were ground separately into fine powder using a mortar and pestle. A 20 mg sub-sample from each ground sample was used for DNA extraction. The DNA was extracted using a DNA extraction kit (DNAMITE Plant Kit, Microzone Ltd, UK). 119DNA concentrations were measured using a Nanodrop ND-1000 spectrophotometer (Labtech International, UK) and adjusted to a final concentration of 20 ng µL-1.Detection of L. maculans and L. biglobosa by conventional PCRThe presence of L. maculans and/or L. biglobosa in each of the diseased stem samples was determined by conventional PCR using species-specific primers LmacF/LmacR (for L. maculans) and LbigF/LmacR (for L. biglobosa) (Liu et al., 2006). PCR reaction mixtures were prepared to a total volume of 20 µL, made up of 10 µL of Redtaq mix (Sigma Cat. No. R2532), 8.4 µL of sterile distilled water, 0.3 µL (10 µM) of forward primer and 0.3 µL (10 µM) of reverse primer and 1 µL (20 ng) of DNA sample. Positive and negative controls were also prepared by adding 1 µL (20 ng) of L. maculans (isolate ME24) DNA or L. biglobosa (isolate L. biglobosa 2003.2.8) DNA from pure cultures and 1 µL of sterile distilled water, respectively, for each set of PCR reaction mixtures prepared. PCR reactions were done in a thermal cycler TC-5000 (Billy Scientific, TECHNE) with the following PCR conditions; initial denaturation period of 95°C for 2 min followed by 30 cycles of 30 s at 95°C, 30 s at 65°C (for L. maculans) or at 63°C (for L. biglobosa) and 1 min at 72°C, then 10 min at 72˚C and a hold at 4˚C. PCR products were visualised on 1.5% agarose gel stained with ethidium bromide.Quantification of L. maculans and L. biglobosa DNA by quantitative PCRThe amounts of L. maculans or L. biglobosa DNA present in each of the stem samples were quantified by using SYBR Green qPCR with species-specific primers LmacF/LmacR (for L. maculans) and LbigF/LmacR (for L. biglobosa), as described by Huang et al. (2009).  PCR reaction mixtures were prepared to a total volume of 20 µL made up of 10 µL of supermix (Sigma, SYBR green jumpstart TAG ready mix), 6.22 µL of sterile distilled water, 0.6 µL (10 µM) of forward primer and 0.6 µL (10 µM) of reverse primer, 0.08 µL of Rox dye and 2.5 µL (50 ng total) of sample DNA.  All reactions were done in 96 × 0.2 mL PCR plates (ABgene) covered with cap strips, using a Stratagene Mx3000P quantitative PCR machine thermocycler. The thermocycling profile consisted of an initial cycle of 95°C for 2 min followed by 40 cycles of 30 s at 60°C, 45 s at 72°C and 15 s at 83°C. An additional melting curve was added to the end of the reactions, with a thermal profile consisting of 1 min at 95°C, 1 min at 60°C and 15 s at 95°C. In each qPCR run, a standard dilution series consisting of 10000, 1000, 100, 10 and 1 pg of DNA of L. maculans (isolate ME24) or L. biglobosa (isolate 2003.2.8) was used to produce a standard curve. The amount of L. maculans DNA or L. biglobosa DNA for each unknown sample was estimated using the standard curve. Results were expressed as amount (pg) of L. maculans or L. biglobosa DNA in 50 ng total DNA from diseased plant tissue. ResultsDetection of L. maculans and L. biglobosa DNA by conventional PCRThe conventional PCR results showed a clear difference between L. maculans and L. biglobosa. L. biglobosa was more frequently observed than L. maculans in most of the upper stem and stem base samples of the cultivars Capitol, DK Cabernet (DKC), Adriana, Excel, NK Grandia (NKG), Roxet and Bilbao.  In general, the percentage of stem samples in which L. biglobosa was detected was considerably greater than that in which L. maculans was detected (Fig. 1). For all the cultivars except Bilbao and Drakkar, the percentage of upper stem samples with L. biglobosa was greater than that with L. maculans (Fig.1a). For stem base samples, the percentage of samples with L. biglobosa was greater than percentage of samples with L. maculans, except for cultivars Drakkar and ES-Astrid (Fig. 1b).120Fig. 1.  Percentages of upper stem lesions (a) and stem base cankers (b) on different winter oilseed rape cultivars in which Leptosphaeria maculans (Lm) and/or L. biglobosa (Lb) were detected in samples collected on 12 July 2012 from a field experiment at Rothamsted in the 2011/2012 growing season. Quantification of L. maculans and L. biglobosa DNA by quantitative PCRIn the 2011/12 growing season, the amount of L. biglobosa DNA was greater than that of L. maculans DNA for both upper stem lesion (Fig. 2a, c) and stem base canker (Fig. 2b, d) samples for all cultivars except for Drakkar, for which the amount of L. maculans DNA was greater than that of L. biglobosa DNA. For upper stem samples, differences in the amount of L. biglobosa DNA between cultivars were also observed, with the greatest amount of L. biglobosa DNA observed in Drakkar. The smallest amount of L. biglobosa DNA was detected in both upper and base stem samples of Bilbao. In addition, there were differences between cultivars in the amount of L. maculans DNA, with the greatest amount in Drakkar.      Fig. 2. The amounts of Leptosphaeria biglobosa (a, b) or L. maculans (c, d) DNA detected by qPCR in diseased upper stem (a, c) and stem base (b, d) samples of different winter oilseed rape cultivars collected on 12 July 2012 from a field experiment at Rothamsted in the 2011/2012 growing season.DiscussionResults of this work suggest that in the UK L. biglobosa can cause severe upper stem lesions and stem base cankers that lead to serious yield losses. The results from both conventional PCR and quantitative PCR showed that L. biglobosa was dominant in both upper stem and stem base samples collected at Rothamsted in the 2011/12 growing season. The greater incidence of L. biglobosa than that of L. maculans in both upper stem and stem base samples suggests that both the upper stem lesions and stem base cankers were mainly caused by L. biglobosa. This conclusion is supported by the quantitative PCR results, indicating that the amount of L. biglobosa DNA was greater than   121that of L. maculans DNA in both upper stem and stem base samples. Previously, L. biglobosa has been considered less damaging in the UK and its contribution to yield loss has been ignored. There is a need for further research on L. biglobosa to investigate the importance of L. biglobosa in relation to control of phoma stem canker on oilseed rape in the UK.  There were differences between cultivars in the incidence of L. maculans and L. biglobosa and in the amounts of L. maculans DNA and L. biglobosa DNA. This suggests that host resistance affects the co-existence between L. maculans and L. biglobosa (Fitt et al., 2006b). The smallest amount of L. biglobosa DNA was detected in cultivar Bilbao and the greatest amount of L. biglobosa DNA was detected in cultivars Roxet and Capitol. The greatest amount of L. maculans DNA was in the susceptible cultivar Drakkar and smallest in the resistant cultivars Roxet and Excel. These results suggest that there are effects of cultivar resistance on interactions between L. maculans and L. biglobosa.   Previous work has shown that ascospores of L. biglobosa are generally released later than ascospores of L. maculans (Huang et al., 2011). The first major release of ascospores was more than two months later in 2011/2012 than in previous season (2010/2011). The large amount of L. biglobosa DNA detected in diseased stems in the 2011/2012 season may have been due to the presence of large numbers of L. biglobosa ascospores in the inoculum in the 2011/2012 season. This can be confirmed by examining the spore samples using species-specific qPCR. The results of this study suggest that L. biglobosa can cause severe phoma stem canker and thus produce subsequent yield losses in the UK. Further work is needed to determine the relative importance of L. maculans and L. biglobosa in relation to severity of phoma stem canker epidemics over a number of sites and growing seasons. AcknowledgementsThe authors thank the UK Biotechnology and Biological Sciences Research Council (BBSRC, BB/I017585/1), the Home Grown Cereals Authority (HGCA), Felix Thornley Trust and the Chadacre Agricultural Trust for funding the work. We also thank breeding companies DSV, Elsoms, Limagrain, Grainseed, LS Plant Breeding, Monsanto, Pioneer, Saaten-Union and Syngenta for in-kind contributions to the work. We thank Georgia Mitrousia, Siti N Mohamed-Sidique and Mohammed Rafi for help with field sampling.ReferencesFitt B D L, Brun H, Barbetti M J, Rimmer S R. 2006a. World-wide importance of phoma stem canker (Leptosphaeria maculans and L. biglobosa) on oilseed rape (Brassica napus). European Journal of Plant Pathology 114:3–15.Fitt B D L, Huang Y J, van den Bosch F, West J S. 2006b. Coexistence of related pathogen species on arable crops in space and time. Annual Review of Phytopathology 44:163–182.Fitt B D L, Hu B C, Li Z Q, Liu, S Y, Lange R M, Kharbanda P D, Butterworth M H, White R P. 2008. Strategies to prevent spread of Leptosphaeria maculans (phoma stem canker) onto oilseed rape crops in China; costs and benefits. 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Resistance to Leptosphaeria maculans (phoma stem canker) in Brassica napus (oilseed rape) induced by L. biglobosa and chemical defence activators in field and controlled environments. Plant Pathology  55:401–412.Liu  Z,  Latunde-Dada  A O, Hall A M, Fitt B D L. 2014. Phoma stem canker disease on oilseed rape (Brassica napus) in China is caused by Leptosphaeria biglobosa ‘brassicae’.  European Journal of Plant Pathology , DOI 10.1007/s10658-014-0513-7.Stonard J F, Latunde-Dada A O, Huang Y J, West J S, Evans N, Fitt B D L. 2010. Geographic variation in severity of phoma stem canker and Leptosphaeria maculans/L. biglobosa  populations on UK  winter oilseed rape (Brassica napus). European Journal of Plant Pathology 126:97–109.

Importance of Leptosphaeria biglobosa as a cause of phoma stem canker on winter oilseed rape in the UK

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Importance of Leptosphaeria biglobosa as a cause of phoma stem canker on winter oilseed rape in the UK

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