Gautier Angélique, Laval Valérie, Balesdent Marie-Hélène
INRAE, UR Bioger, Université Paris-Saclay, Place de L'Agronomie, Palaiseau Cedex, CS, 80022, 91120, France.
BMC Microbiol. 2025 Mar 21;25(1):159. doi: 10.1186/s12866-025-03855-2.
Leptosphaeria maculans is a fungal pathogen causing stem canker of oilseed rape (Brassica napus). The disease is mainly controlled by the deployment of varieties with major resistance genes (Rlm). Rlm genes can rapidly become ineffective following the selection of virulent isolates of the fungus, i.e. with mutations, including deletions, in the corresponding avirulence genes (AvrLm). Reasoned and durable management of Rlm genes relies on the detection and monitoring of virulent isolates in field populations. Based on previous knowledge of AvrLm gene polymorphism, we developed a tool combining multiplex PCR and Illumina sequencing to characterize allelic variants for eight AvrLm genes in field L. maculans populations.
We tested the method on DNA pools of 71 characterised L. maculans isolates and of leaf spots from 32 L. maculans isolates. After multiplex-PCR and sequencing with MiSeq technology, reads were mapped on an AvrLm sequence database. Data were filtered using thresholds defined from control samples included in each sequencing run. Proportions of each allelic variant per gene, including deletions, perfectly correlated with expected ones. The method was then applied to around 1300 symptoms (42 pools of mainly 32 leaf spots) from nine B. napus fields. The proportions of virulent isolates estimated by sequencing leaf spot pools perfectly correlated with those estimated by pathotyping single isolates. In addition, the proportions of allelic variants determined at the national scale also correlated with those previously determined following individual sequencing of AvrLm genes in a representative collection of isolates. Finally, the method also allowed us to detect still undescribed and rare allelic variants.
Despite the diversity of mechanisms generating virulent isolates and the gene-dependent diversity of AvrLm gene polymorphism, the method proved suitable for large-scale and regular monitoring of L. maculans populations, which will facilitate the deployment of effective Rlm genes and the early detection of resistance breakdowns.
十字花科黑斑病菌是一种引起油菜（甘蓝型油菜）茎溃疡病的真菌病原体。该病主要通过种植具有主要抗性基因（Rlm）的品种来控制。在选择该真菌的毒性分离株后，即相应无毒基因（AvrLm）发生包括缺失在内的突变后，Rlm基因会迅速失效。对Rlm基因进行合理且持久的管理依赖于对田间种群中毒性分离株的检测和监测。基于先前对AvrLm基因多态性的了解，我们开发了一种结合多重PCR和Illumina测序的工具，用于鉴定田间十字花科黑斑病菌种群中八个AvrLm基因的等位基因变体。
我们在71个已鉴定的十字花科黑斑病菌分离株的DNA池以及32个十字花科黑斑病菌分离株的叶斑上测试了该方法。在进行多重PCR并用MiSeq技术测序后，将读取结果映射到AvrLm序列数据库上。使用每次测序运行中包含的对照样品定义的阈值对数据进行过滤。每个基因的每个等位基因变体的比例，包括缺失，与预期比例完全相关。然后将该方法应用于来自九个甘蓝型油菜田的约1300个症状（42个池，主要为32个叶斑）。通过对叶斑池进行测序估计的毒性分离株比例与通过对单个分离株进行致病性鉴定估计的比例完全相关。此外，在全国范围内确定的等位基因变体比例也与先前在代表性分离株集合中对AvrLm基因进行个体测序后确定的比例相关。最后，该方法还使我们能够检测到尚未描述的罕见等位基因变体。
尽管产生毒性分离株的机制具有多样性，且AvrLm基因多态性存在基因依赖性差异，但该方法被证明适用于对十字花科黑斑病菌种群进行大规模和定期监测，这将有助于有效Rlm基因的部署以及抗性丧失的早期检测。
