Understanding R Gene-Mediated Resistance Against Leptosphaeria maculans for Effective Control of Phoma Stem Canker in Oilseed Rape

Abstract

This project aims to improve understanding of R gene-mediated resistance against Leptosphaeria maculans for effective control of phoma stem canker in oilseed rape. This was achieved through three objectives by (i) monitoring the UK regional distribution of races of L. maculans, (ii) investigating molecular mechanisms of mutation to virulence in L. maculans populations, (iii) understanding effects of environmental factors on Brassica napus resistance against L. maculans. The regional distributions of L. maculans races in the UK were monitored in air and crops in 2015/2016, 2016/2017 and 2017/2018 cropping seasons. The release of ascospores in the air was monitored using Burkard spore samplers at four different sites. The frequencies of avirulent alleles of effector genes AvrLm1 and AvrLm6 in the L. maculans ascospore populations were determined by qPCR. There were differences between the four sites and seasons in patterns of ascospore release and in dates of first major ascospore release. L. maculans ascospores with avirulent AvrLm1 alleles were mostly released earlier than ascospores with AvrLm6 alleles. AvrLm6 alleles were detected more frequently than AvrLm1 alleles in L. maculans ascospores sampled at all sites in all seasons. Changes in frequencies of avirulent alleles of different effector genes were investigated by inoculation of conidial suspensions onto the cotyledons of a set of Brassica napus differential cultivars/lines. The AvrLm7 allele was predominant in the UK L. maculans populations until 2015/2016; however, the frequency of isolates virulent towards the corresponding resistance gene Rlm7 has increased from 6.8% (2016/2017) to 16.3% (2017/2018), suggesting that there is a risk of breakdown of Rlm7-mediated resistance in the UK. There were differences in the frequencies of avirulent alleles of different effector genes in L. maculans populations from leaf samples (single pycnidial isolates) between sites during three cropping seasons. Overall, the frequencies of AvrLm5 (85.5%), AvrLm6 (97.2%) and AvrLm7 (94.7%) were greater than those of AvrLm1 (5.7%), AvrLm2 (3.6%), AvrLm3 (3.2%), AvrLm4 (17.7%) and AvrLm9 (1.2%) in L. maculans populations from leaf samples. Similarly, for L. maculans populations from stem samples (single ascospore isolates) from two different sites in 2016/2017, the frequencies of avirulent alleles of AvrLm5 (100%), AvrLm6 (94.1%) and AvrLm7 (75.9%) were greater than those of AvrLm1 (22.7%), AvrLm2 (11.9%), AvrLm3 (23.7%), AvrLm4 (15.6%) and AvrLm9 (16.5%). Seven races were identified in the L. maculans populations from leaf samples in 2015/2016. In 2016/2017, 15 and 19 races were identified in the L. maculans isolates from leaf and stem samples, respectively. A total of 17 races were observed in the UK L. maculans populations from leaf samples in 2017/2018, however Av5-6-7 remained the major race for the three cropping seasons. Molecular mechanisms of mutation in L. maculans populations leading to virulence towards Rlm1, Rlm4, Rlm6 or Rlm7 resistance genes were investigated. Sanger sequencing (55 L. maculans isolates) and whole genome sequencing (40 L. maculans isolates) were used in this study. For Sanger sequencing, whole gene deletion of the AvrLm1 gene was observed in 96% of the isolates. For the remaining isolates, partial deletion (1%) and single-base point mutation (70%) were identified as the cause of virulence towards the resistance gene Rlm1. For whole genome sequencing, whole gene deletion of the AvrLm1 gene was identified in all the isolates (100%). Molecular mechanisms of mutation leading to virulence towards Rlm4 were identified as whole gene deletion (6.6%) from Sanger sequencing. From whole genome sequences, whole gene deletion (18.2%), partial deletion (3.03%), single-nucleotide (non-synonymous) point mutation (84.8%) and single-nucleotide (synonymous) point mutation (6.1%) were identified. Eighteen different single-nucleotide polymorphisms were identified at thirty-four codon sites; that had led to amino-acid changes or created stop codons. From whole genome sequences, whole gene deletion of the AvrLm6 gene was identified in one L. maculans isolate virulent towards the Rlm6 resistance gene and no other molecular mechanisms of mutation were identified. From whole genome sequences, various molecular mechanisms of mutation leading to virulence towards the Rlm7 resistance gene were identified in this study, such as whole gene deletion (27.3%), partial deletion (13.6%), single-nucleotide (non- synonymous) point mutation (68.2%), single-nucleotide (synonymous) point mutation (9.1%), Nonsense point mutation (50%), single-nucleotide deletion (27.3%), repeat-induced point mutation (RIPs) (68.2%), transition (68.2%) and transversion mutation (13.6%). Effects of environmental factors on Brassica napus resistance against L. maculans were investigated in three cropping seasons. Severities of phoma leaf spot and phoma stem canker were assessed on twelve different oilseed rape cultivars at different environments/sites in the UK. There were differences in the phoma leaf spot and phoma stem canker severities between sites and between cropping seasons. Among all the sites, Morley site had the greatest phoma leaf spot and phoma stem canker severities; whereas, Trumpington and Risby sites had the least disease severity. Among the three cropping seasons, less phoma stem canker severity was observed among the sites in 2016/2017 than in other two seasons. Cultivars Drakkar, Incentive and Mentor (with no R gene against L. maculans) developed more severe phoma leaf spots and phoma stem cankers than cultivars with R genes and background quantitative resistance. Cultivars with the Rlm7 gene (DK Exalte, Whisky, Amalie, Harper and DK Extrovert) had less severe phoma leaf spotting and less severe phoma stem canker than cultivars DK Cabernet (with the Rlm1 gene) and Adriana (with the Rlm4 gene). Cultivar Angus (with the LepR3 gene) also had less phoma leaf spot and phoma stem canker severity. Cultivars with QR (Es Astrid, Adriana and DK Cabernet) had less severe phoma stem cankers in summer even though they had severe phoma leaf spots in autumn/winter. Analysis of the relationship between disease severity and weather data among the different sites in the three growing seasons showed that increased severity of phoma leaf spots was associated with increased rainfall and moderate mean temperatures during the phoma leaf spot development stage and increased severity of phoma stem canker was associated with increased temperature during the stem canker development stage.