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Phoma stem canker is a disease of oilseed rape (Brassica napus) caused by closely related plant pathogens Leptosphaeria maculans and L. biglobosa. It is an economically important disease, causing annual yield losses of approximately £770 million worldwide. When colonising oilseed rape, L. maculans and L. biglobosa exist in close proximity on the leaf, competing for resources as they move through the main leaf vein and into the stem. Fungicides are commonly used to decrease severity of phoma stem canker on oilseed rape. However, the efficacy and longevity of active chemicals is under threat from evolution of resistance in pathogen populations and government legalisation. Moreover, it has been suggested that both L. maculans and L. biglobosa differ in their sensitivity to azoles, and important class of fungicides that are used to control the disease through the inhibition of lanosterol 14-α demethylase (erg11, CYP51). This project aims to further understand the role that fungicides have in controlling phoma stem canker by investigating their efficacy against L. maculans and L. biglobosa in crops, in vitro and in planta. In field experiments, established in Cambridgeshire across four cropping seasons, the fungicide mixture penthiopyrad (SDHI) plus picoxystrobin (QoI) was as effective at controlling phoma leaf spotting and phoma stem canker in winter oilseed rape as prothioconazole (DMI), suggesting that both fungicides could be used to reduce phoma stem canker symptoms. The two pathogens differed in their growth rates in vitro, with L. biglobosa growing faster than L. maculans when untreated or treated with lower fungicide concentrations. Fungicide sensitivity assays suggest that L. maculans and L. biglobosa are both sensitive to DMI, SDHI and QoI fungicides and that differences between the species are minor. Prothioconazole and penthiopyrad + picoxystrobin had a similar efficacy on oilseed rape cotyledons colonised with either L. maculans or L. biglobosa. There was no difference between species on prothioconazole treated plants, although there was a difference between L. maculans and L. biglobosa when treated with 20 μg/ml penthiopyrad + picoxystrobin. Heterologous yeast expression of LmCYP51B and LbCYP51B with fungicide sensitivity testing of the yeast transformants suggests that LmCYP51B and LbCYP51B are similarly sensitive to azole fungicides flusilazole, prothioconazole-desthio and tebuconazole. These findings are supported by homology protein modelling, which predicts that LmCYP51B and LbCYP51B are structurally very similar, specifically at the azole-binding site. In conclusion, fungicides are still an effective control method for reducing phoma stem canker symptoms caused by Leptosphaeria species in the UK, and a useful tool to in the sustainable production of oilseed rape.

Leptosphaeria maculans and L. biglobosa co-infect winter oilseed rape plants to cause phoma stem canker disease. The sexual spores of both species are produced in pseudothecia on infected winter oilseed rape stem debris after harvest and this is the most important source of inoculum for infection of newly-emerged plants in autumn. Field experiments investigated the effects of host resistance on proportions of pathogens Leptosphaeria maculans and L. biglobosa in most leaf and stem tissues during 2011/2012, 2012/2013 and 2013/2014 growing seasons and on the pseudothecial development on crop debris on nine winter oilseed rape cultivars; cvs Adriana (Rlm4 + quantitative resistance (QR)), Bilbao (Rlm4), Capitol (Rlm1), Drakkar (no R gene against L. maculans), DK Cabernet (Rlm1 + (QR), Es-Astrid (QR), Excel (Rlm7), Roxet (Rlm7) and NK Grandia (QR). Cultivars with a combination of R-gene resistance and QR [Adriana (Rlm4 + QR), DK Cabernet (Rlm1 + QR)] or cultivars with only QR [(Es-Astrid and NK Grandia)] had more numbers of L. maculans leaf spots than other cultivars in autumn but less stem canker damage. There was greater number of L. biglobosa leaf spots on leaves of cvs Roxet and Excel with resistance gene Rlm7 than those of other cultivars and later more L. biglobosa DNA was detected in their stems than in those of other cultivars. In all cultivars in the three growing seasons, there was a greater amount of L. biglobosa DNA than L. maculans DNA in basal stem canker and upper stem lesions. The cv. Drakkar (no R gene against L. maculans) was susceptible in all three growing seasons, with a great number of L. maculans and L. biglobosa leaf spots and severe stem cankers. There were four cultivars (Adriana, Bilbao, Drakkar and NK Grandia) selected for the study of pseudothecial development under natural conditions with different times of exposure and in controlled environment conditions (20oC, continuous wetness). The fastest development was on the susceptible cv. Drakkar (no R gene against L. maculans), followed by Bilbao (Rlm4), Adriana (Rlm4 + QR) and NK Grandia (QR) for stem base cankers and upper stem lesions in controlled conditions. Results for pseudothecial development on stems of the nine winter oilseed rape cultivars that were exposed in natural conditions at Bayfordbury support the controlled environment results, with pseudothecia on stems of cultivars with a combination R-gene and QR consistently maturing later than those on other cultivars, regardless of the weather conditions in three growing seasons. Ascospores produced in pseudothecia are the primary inoculum that initiate phoma stem canker epidemics in autumn. Ascospore release was later in autumn 2011 than in autumn/winter 2012/2013 or 2013/2014 because of dry weather. The pattern of ascospore release had a peak, or maximum in autumn/winter 2011/2012 (4958 spores/m-3 on 22 Jan 2012) and several maxima in autumn/winter 2012/2013 (1307 spores/m-3 on 5 Nov 2012, 1291 spores/m-3 on 15 Nov 2012, 1306 spores/m-3 on 25 Dec 2012) and 2013/2014 (4575 spores/m-3 on 27 Oct 2013, 4619 spores/m-3 on 3 Nov 2013, 3674 spores/m-3 on 9 Nov 2013, 3521 spores/m-3 on 12 Dec 2013). Results from the qPCR showed that ascospores of L. maculans were released earlier than ascospores of L. biglobosa at Bayfordbury in the 2013/2014 growing season. There were differences in phenotype of isolates amongst ninety-five isolates of L. maculans and forty-eight isolates of L. biglobosa obtained from different sources (phoma leaf spots, upper stem lesions or basal stem cankers) on different cultivars. Cotyledon tests showed that the resistance genes Rlm4, Rlm5, Rlm6 and Rlm7 are still effective in England. Most isolates from phoma leaf spots carried avirulent AvrLm4 (39 isolates; 97.5%), AvrLm5 (39 isolates; 97.5%) and AvrLm6 alleles (36 isolates; 90%) and all 40 isolates carried the avirulent allele AvrLm7 (100%). Fewer isolates from basal stem cankers carried avirulent AvrLm4 (4 isolates, 16.7%) or AvrLm6 alleles (16.7%) but all 24 isolates carried the avirulent AvrLm7 (100%). Fewer isolates from upper stem lesions carried the avirulent AvrLm4 allele (5 isolates; 16.1%), but 15 isolates carried avirulent AvrLm5 (48.4%), 21 isolates carried AvrLm6 (67.7%) and all 31 isolates carried AvrLm7 (100%). By contrast, all isolates were virulent against Rlm1, Rlm2, Rlm3 and Rlm9. This knowledge, together with knowledge about R genes present in current winter oilseed rape cultivars, should be useful to provide recommendations on cultivar selection to growers based on regional frequencies of avirulent alleles of Avr allele genes in the L. maculans populations (races) and improved understanding of the race structure of L. maculans. There is a need to further investigate any R genes that operate against L. biglobosa (possibly from wild brassicas) and to study if any R genes or QR can provide resistance against both L. maculans and L. biglobosa.

To improve understanding of the effectiveness of host resistance against Leptosphaeria spp., three aspects of effectiveness of resistance were investigated. With focus on the major Rlm-mediated resistance against L. maculans, changes in effectiveness of Rlm7-mediated resistance to prevent initiation of disease epidemics at the leaf spot stage were investigated in winter oilseed rape field experiments at five sites in the UK over the period with the cropping seasons 2009/2010 - 2013/2014. L. maculans isolates virulent against Rlm7 were identified in the UK. This may be associated with observed changes in lesion phenotypes on the Rlm7 cultivars in field conditions. However, despite increased severity of phoma leaf spotting on Rlm7 cultivars, there was no associated increase in phoma stem canker severity at the end of the cropping seasons. The effectiveness of winter oilseed rape cultivars for control of phoma stem canker (caused by L. maculans or L. biglobosa) was affected by the coexistence of the two Leptosphaeria species in oilseed rape crops. Weather conditions influenced ascospore release of both species and favoured L. biglobosa ascospore release in 2011, resulting in subsequent increased L. biglobosa phoma leaf spotting and stem canker severity. However, coexistence of Leptosphaeria spp. on oilseed rape crops was affected by the cultivar resistance against L. maculans. CE experiments showed that there were interactions between the two Leptosphaeria spp. in planta. Their coexistence on B. napus was influenced by the different host responses that they trigger during host colonisation. Effects of increased temperature on effectiveness of resistance against L. maculans and on severity of symptoms by Leptosphaeria spp. on B. napus were investigated. Increased temperature affected both Rlm4- and Rlm7-mediated resistance, when assessed by phenotypic and molecular techniques. Increased temperature was associated with increased symptom severity, for both L. maculans and L. biglobosa lesions on plants. Cultivar quantitative resistance background increased effectiveness of resistance against phoma stem canker pathogens at increased temperature and should be deployed in in strategies for adaptation to climate change to avoid increased phoma stem canker epidemics in the future.