Characterization of Leptosphaeria maculans races in Germany and studies on resistance of Brassica napus to blackleg disease

Leptosphaeria maculans, a phytopathogen causes blackleg disease, is an economically significant biotic stress that affects oilseed rape (B. napus) production worldwide. Blackleg disease can be controlled by stubble management, crop rotation, fungicides, biocontrol agents, and resistant cultivars. However, until now the latter has been the most effective and sustainable method applied. Therefore, we focused in this study on different aspects of B. napus resistance to blackleg disease. Resistance to L. maculans infection in oilseed rape can be qualitative or quantitative. Qualitative resistance is mainly favoured for its complete effect while quantitative resistance is preferred for its durability and wide-spread effects against new emerged races. The first objective of this study was to characterise the races of L. maculans in Germany to evaluate the efficacy of blackleg race-specific major resistance (R) genes in B. napus. Due to the natural evolution of the pathogen, major R genes typically go through a “boom and bust” cycle and might lose efficacy a few years after their commercial release. Hence, races of L. maculans should be monitored on a regular basis. This provides the farmers with the necessary information that enable them to effectively control blackleg disease in the field. In this study, 574 isolates were collected in different regions in central and northern Germany. The avirulence profile of L. maculans population was defined either by pathogenicity tests on cotyledons of a differential set of oilseed rape or by amplifying avirulence gene-specific PCR markers. Ten avirulence genes were characterised in the isolates, namely, Avrlm1, Avlm2, Avrlm3, Avrlm4, Avrlm6, Avrlm7, Avrlm11, LepR1, LepR2 and LepR3. Results showed that the Avrlm6, -7, -11, AvrlepR1, -R2 was the most dominant race, and thus, the corresponding major R genes Rlm6, Rlm7, Rlm11, LepR1 and LepR2 were the most effective R genes in the German experimental field. In contrast, Avrlm2 was absent in all races, highlighting that the major R gene Rlm2 is 100% ineffective in Germany. The frequencies of Avrlm1, Avrlm3, Avrlm4 and AvrlepR3 were less than 42%. This demonstrated that the corresponding major R genes were partially effective in the fields explored. The ratio of L. maculans mating types MAT1-1 and MAT2-1 in the collection was also investigated. For that, a multiplex PCR assay was used to show that the mating types did not deviate from the ratio 1:1 in any of the regions examined. It was concluded, according to the random mating hypothesis, that the sexual outcrossing occured randomly and was dominant in the L. maculans populations studied. This is a factor that enhances the emergence speed of new L. maculans races. As major R genes might lose their efficacy a few years after commercialisation, a further aim of our study was to seek new sources of quantitative resistance in B. napus or its cruciferous relatives. For that, this project aimed to establish a reliable and repeatable inoculation method that enables a fast but effective greenhouse screening of sources of quantitative resistance. Four inoculation methods were compared in the greenhouse. Results showed that inoculation with a mycelial agar plug on an injured stem base was more reproducible and more accurately differentiated the variances in quantitative resistance to blackleg disease than inoculation with spore suspension on lamina, or inoculation with a mycelial agar plug on an injured petiole. Through stem-based inoculation, 354 DH lines were assessed for quantitative resistance to blackleg disease. The plant material investigated provided a wide genetic background, as it was produced from an interconnected multiparent mapping population generated from six elite varieties with quantitative resistance and one susceptible parent. By monitoring the volume of diseased tissues (VDT), it was clear that quantitative resistance of the DH lines distinctly differed. These results were used for a genome-wide association study that revealed eight significant QTLs which contributed towards 3 to 6% of the phenotypic variation in the mapping population. Although the greenhouse experiments were conducted in parallel to field trials, abiotic and biotic stresses severely hampered the success of the experiments in the field. Drought, insect damage and field raiding by wild boar were the main problems in our phenotyping studies for quantitative resistance against blackleg disease in the field. Furthermore, this study aimed to evaluate quantitative resistance to blackleg disease in new allohexaploid hybrids (2n= 6X= AABBCC). These hybrids were produced in Giessen University through embryo rescuing followed by chromosome doubling of a triploid (2n=3x=ABC) produced from crossing B. napus (2n=2x= AACC) X B. nigra (2n=2x= BB). By comparing the volume of diseased tissue at the stem bases of the parental genotypes and the hybrids, we concluded that an inherited resistance originated from the B genome of B. nigra was shown in the hybrids. Further breeding studies should aim to solve the instability problems of the genome combination in the crosses to allow this new resistance source to be used. An additional objective of the study was to investigate plant chemical defences in the pathosystem L. maculans- B. napus. For this experiment, phytoalexin accumulation was checked in a genotype with quantitative resistance and another without. Our analysis, using an HPLC-system combined with high-resolution Q-TOF mass spectrometry, revealed that caulilexin C existed in B. napus as a phytoanticipin before the infection with L. maculans. On the contrary, rutalexin, spirobrassinin, cyclobrassinin, and brassilexin were induced de novo upon the infection as phytoalexins. The resistant genotype clearly accumulated more phytoalexins than the susceptible one at 7 dpi while the differences were less at 14 and 21 dpi. However, spirobrassinin, which cannot be detoxified by L. maculans, was the highest accumulated phytoalexin at all time points in both genotypes. The experimental results showed that there is a correlation between resistance and the accumulation of the above-mentioned chemical defences. Further studies are needed to assure whether a causative connection exists. All in all, the results of this study pave the way for agricultural consultants to establishment strategies that preserve the commercialised major R gene efficacies in German regions. However, the study emphasized the importance of pyramiding major R genes with quantitative resistance to expand their efficacy. This work also provides breeders with potential new sources of quantitative resistance that either have been found in the interconnected multiparent mapping population investigated or from the B- genome of B. nigra. Additionally, this research opens the door for more studies on chemical defences induced in the L. maculans- B. napus pathosystem that can be used in breeding programmes or the agrichemical industry.

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