Abstract
BackgroundBlackleg 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.ResultsDisease 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.ConclusionsThe 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.
Highlights
Precise and accurate quantification of disease severity is required to determine the genetic basis of resistance, breed cultivars resistant to disease, assess the efficacy of fungicides, and calculate relationships between cropSchnippenkoetter et al Plant Methods (2021) 17:122 potential for specific, rapid and objective quantification of quantitative disease resistance (Oliver et al [3]).Blackleg disease of canola is caused by the hemibiotrophic fungal pathogen Leptosphaeria maculans (Howlett et al [4])
L. maculans was detected in inoculated cotyledons at 12 dpi using Wheat Germ Agglutinin Chitin Assay (WAC) and PCR methods
Compared to quantitative polymerase chain reaction (qPCR), droplet digital polymerase chain reaction (ddPCR) reagents are more expensive, but these costs are offset by lower labour costs, reduced variability as technical replication is not required and improved data handling
Summary
Precise and accurate quantification of disease severity is required to determine the genetic basis of resistance, breed cultivars resistant to disease, assess the efficacy of fungicides, and calculate relationships between cropSchnippenkoetter et al Plant Methods (2021) 17:122 potential for specific, rapid and objective quantification of quantitative disease resistance (Oliver et al [3]).Blackleg disease of canola is caused by the hemibiotrophic fungal pathogen Leptosphaeria maculans (Howlett et al [4]). Quantitative resistance (QR) is thought to be a polygenic trait and is expressed as partial resistance (Raman et al [9,10,11,12]; Delourme et al [8]), in that it does not prevent infection but decreases canker severity While both forms of resistance are useful, QR is desirable because it is considered race non-specific and appears to be more durable and so provide longer-term protection compared to MGR (Delourme et al [13], Sprague et al [14, 15], Brun et al [16]). 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. 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
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