Understanding interactions between phoma stem canker causal pathogens Leptosphaeria maculans and Leptosphaeria biglobosa

Abstract This project aimed to improve the understanding of interspecific interactions between Leptosphaeria maculans and Leptosphaeria biglobosa and to provide valuable insights for future integrated strategies for control of phoma stem canker on oilseed rape (Brassica napus) in the UK. These aims were achieved by investigating interspecific interactions between L. maculans and L. biglobosa in vitro, in planta and in natural conditions. In vitro experiments confirmed the ability of L. biglobosa to inhibit the production of sirodesmin PL by L. maculans under simultaneous co-inoculation. Further results showed diFerences between L. maculans and L. biglobosa in growth in liquid cultures; with L.biglobosa having a growth rate approximately three times greater than that of L.maculans. Growth of mycelia in simultaneously co-inoculated cultures showed a similar trend to that of L. biglobosa only, suggesting that the growth of L. maculans was inhibited by L. biglobosa. Sequential co-inoculation experiments showed that in liquid culture, L. biglobosa not only inhibits the production of sirodesmin PL by L. maculans but also inhibits the growth of L. maculans if inoculated before or up to one day after L.maculans, confirmed by quantitative polymerase chain reaction (qPCR) analyses. Interestingly, if L. biglobosa was co-inoculated three days after L. maculans, it was only able to partially inhibit the production of sirodesmin PL and the growth of L. maculans in liquid culture. Finally, if L. biglobosa was co-inoculated five days or later after L.maculans, enough time was provided for L. maculans to successfully produce sirodesmin PL, which in return led to inhibition of L. biglobosa growth by L. maculans. These results showed that the timing of interaction directly affects the outcome of interspecific interactions between L. maculans and L. biglobosa. Results from in planta experiments showed that L. biglobosa can inhibit the production of sirodesmin PL by L. maculans when simultaneously co-inoculated on cotyledons of oilseed rape. In addition, the phenotype of lesions observed in the simultaneous coinoculation treatment was similar to those of L. biglobosa only, suggesting that the growth of L. maculans was also inhibited by L. biglobosa in planta. Further results from in planta experiments with sequential co-inoculations showed similar results to those invitro, with a small difference in the timeline. When L. biglobosa was inoculated before or up to one day after L. maculans at the same site on cotyledons of oilseed rape, small, dark necrotic lesions appeared shortly after infection and expanded slowly under humid conditions, similar to those of L. biglobosa only. Furthermore, the growth of L. maculans was inhibited in those lesions, confirmed by qPCR. Conversely, when L. biglobosa was co-inoculated three days or later after L. maculans; small, grey lesions appeared by 7-10 days post inoculation (dpi) and rapidly expanded by 14-16 dpi, similar to those of L.maculans only and the growth of L. biglobosa was inhibited. Finally, the results for gene expression experiments in relation to plant defences indicated induction of systemic acquired resistance (SAR) through upregulation of PR-1 and defence responses against a necrotrophic pathogen through upregulation of WRKY33 but were otherwise inconclusive. These results showed that the timing of the interaction also plays a crucial role in determining the outcome of the competitive interactions between L. maculans and L. biglobosa in planta, which subsequently affects disease development and severity. Observations made by monitoring the patterns of ascospore release of L. maculans and L. biglobosa at Bayfordbury, Hertfordshire over four growing seasons (2020/2021 to 2023/2024) showed the release of ascospores of both Leptosphaeria species at similar times. The first ascospore release was generally observed at the end of September, with the maximum release of ascospores generally observed in late-October/early-November, dependent on environmental conditions such as temperature and rainfall. Next, results of field experiments at Harpenden, Hertfordshire and Norwich, Norfolk over two growing seasons (2023/2024 and 2024/2025) showed little to no difference in overall disease incidence (% of plants aFected) between different sites and different growing seasons for ten cultivars assessed. Interestingly, there were differences in the percentage of phoma leaf spot lesions caused by L. maculans and L. biglobosa. At both sites, a predominance of L. biglobosa in causing phoma leaf spot lesions was observed in the 2023/2024 growing season and L. maculans in the 2024/2025 season. These differences were most probably due to differences in weather conditions, stubble management practices and fungicide applications. Next, results of field experiments at Hereford, Herefordshire and Huntingdon, Cambridgeshire over two growing seasons (2021/2022 and 2022/2023) showed differences in overall phoma stem canker disease severity at different sites and in different growing seasons for two cultivars per assessment, despite showing little to no difference in percentage of Leptosphaeria species in stem cankers, where > 95 % of the DNA in stem basal cankers was of L. maculans, confirmed by qPCR. Finally, results of field experiments at Wisbech, Cambridgeshire over two growing seasons (2021/2022 and 2022/2023) showed an increase in phoma stem canker disease severity in the latter season for cultivars with only the Rlm7 resistance gene, with > 95 % of the DNA in stem basal cankers quantified as L. maculans, suggesting a breakdown of resistance event. Strikingly, for Line-A, which has both Rlm7 and RlmS, the percentage of L. biglobosa DNA in stem basal cankers was the greatest across both growing seasons and increased to > 50 % in the latter season, suggesting that combining multiple resistance genes against L. maculans in oilseed rape cultivars may increase their susceptibility to L. biglobosa. These results showed that environmental and human factors, such as weather conditions and various disease control practices, may influence the interspecific interactions between L.maculans and L. biglobosa, which may subsequently affect proportions of pathogen populations and the course of disease epidemics. This project improved the understanding of interspecific interactions between L.maculans and L. biglobosa by combining existing knowledge with novel findings from experiments and provided contemporary ideas to be incorporated into future integrated management strategies.