Schnippenkoetter Wendelin, Hoque Mohammad, Maher Rebecca, Van de Wouw Angela, Hands Phillip, Rolland Vivien, Barrett Luke, Sprague Susan
CSIRO Agriculture and Food, 1 Clunies Ross Street, Canberra, ACT, 2601, Australia.
School of BioSciences, The University of Melbourne, Parkville, VIC, 3010, Australia.
Plant Methods. 2021 Dec 1;17(1):122. doi: 10.1186/s13007-021-00822-6.
Blackleg disease, caused by the fungal pathogen Leptosphaeria maculans, is a serious threat to canola (Brassica napus) production worldwide. Quantitative resistance to this disease is a highly desirable trait but is difficult to precisely phenotype. Visual scores can be subjective and are prone to assessor bias. Methods to assess variation in quantitative resistance more accurately were developed based on quantifying in planta fungal biomass, including the Wheat Germ Agglutinin Chitin Assay (WAC), qPCR and ddPCR assays.
Disease assays were conducted by inoculating a range of canola cultivars with L. maculans isolates in glasshouse experiments and assessing fungal biomass in cotyledons, petioles and stem tissue harvested at different timepoints post-inoculation. PCR and WAC assay results were well correlated, repeatable across experiments and host tissues, and able to differentiate fungal biomass in different host-isolate treatments. In addition, the ddPCR assay was shown to differentiate between L. maculans isolates.
The ddPCR assay is more sensitive in detecting pathogens and more adaptable to high-throughput methods by using robotic systems than the WAC assay. Overall, these methods proved accurate and non-subjective, providing alternatives to visual assessments to quantify the L. maculans-B. napus interaction in all plant tissues throughout the progression of the disease in seedlings and mature plants and have potential for fine-scale blackleg resistance phenotyping in canola.
由真菌病原体大斑壳二孢菌引起的黑胫病对全球油菜（甘蓝型油菜）生产构成严重威胁。对这种疾病的数量抗性是一个非常理想的性状，但难以精确地进行表型分析。视觉评分可能具有主观性，并且容易出现评估者偏差。基于对植物体内真菌生物量的量化，开发了更准确地评估数量抗性变异的方法，包括小麦胚凝集素几丁质测定法（WAC）、qPCR和ddPCR测定法。
在温室实验中，用大斑壳二孢菌分离株接种一系列油菜品种进行病害测定，并在接种后不同时间点收获的子叶、叶柄和茎组织中评估真菌生物量。PCR和WAC测定结果相关性良好，在不同实验和宿主组织中具有可重复性，并且能够区分不同宿主-分离株处理中的真菌生物量。此外，ddPCR测定法显示能够区分大斑壳二孢菌分离株。
与WAC测定法相比，ddPCR测定法在检测病原体方面更敏感，并且通过使用机器人系统更适合高通量方法。总体而言，这些方法被证明是准确且客观的，为视觉评估提供了替代方法，以量化在幼苗和成熟植物疾病发展过程中所有植物组织中大斑壳二孢菌与甘蓝型油菜的相互作用，并且在油菜精细尺度黑胫病抗性表型分析方面具有潜力。
