Figure 1.
Stages of Leptosphaeria maculans growth in oilseed rape.
Assessment of quantitative resistance against growth of L. maculans in oilseed rape was considered in two stages; stage 1, resistance against growth from phoma leaf lesion along the main leaf vein and petiole to the stem; stage 2, resistance against growth in stem to produce stem canker; both stages were investigated in controlled environment experiments.
Table 1.
Inoculation methods, type of Leptosphaeria maculans inoculum, number of plants inoculated, design of experiments and assessment methods used in each of the controlled environment experiments with the doubled haploid (DH) lines A30 or C119.
Figure 2.
Leaf lamina and leaf petiole inoculation.
Inoculation of oilseed rape leaf lamina (A) or leaf petiole (B) with conidia or ascospores of Leptosphaeria maculans. The arrows indicate the droplets of spore suspension (either conidia or ascospores) placed on the lamina (A) or petiole (B) of leaves of doubled haploid lines A30 (susceptible) or C119 (with quantitative resistance). Plants were inoculated at 21 days after sowing when they had three fully expanded leaves.
Figure 3.
Phoma leaf spot symptoms produced in controlled conditions.
Phoma leaf spot symptoms on leaves of doubled haploid lines A30 (susceptible) or C119 (with quantitative resistance) inoculated with conidia or ascospores of Leptosphaeria maculans. Lesions on leaves of A30 at 15 days post inoculation (dpi) with conidia (A) or 10 dpi with ascospores (B); lesions on leaves of A30 (C) and C119 (D) at 22 dpi; symptomless growth of GFP-expressing L. maculans (isolate ME24/3.13) along a leaf vein towards the petiole of A30 viewed with brightfield illumination (E-1) or a GFP filter (E-2) at 18 dpi.
Figure 4.
Distance grown along leaf petioles and Leptosphaeria maculans DNA in petioles and stems.
Leaf laminas or leaf petioles of doubled haploid lines A30 (susceptible) or C119 (with quantitative resistance) were inoculated with conidia of GFP-expressing L. maculans isolate ME24/3.13 or ascospores from a natural population. The distance grown by L. maculans along the leaf vein/petiole towards the stem (leaf lamina inoculation, Fig. 2A) was measured by assessing extent of GFP fluorescence at 22–25 days post inoculation (dpi) for conidial inoculation (A) or by assessing extent of the visible necrosis (A) and by quantification of amount of L. maculans DNA (B) using qPCR at 18–20 dpi for ascospore inoculation. The growth of L. maculans in stem tissue (petiole inoculation, Fig. 2B) was assessed by quantification of amount of L. maculans DNA using qPCR (B) at 46 dpi inoculation with ascospores. Bars show standard errors (A, 28 d.f. for conidia and 54 d.f. for ascospores; B, 54 d.f. for leaf petiole and 50 d.f. for stem).
Figure 5.
Relationships between different methods for assessment of quantitative resistance.
Different methods were used to assess growth of Leptosphaeria maculans in leaf petioles or in stems of doubled haploid lines A30 (susceptible) or C119 (with quantitative resistance). Relationships between leaf lesion area and distance grown (DG) by L. maculans in the leaf petiole viewed by GFP in experiment LExpt 1 (A; R2 = 0.64) or amount of L. maculans DNA in leaf petiole in experiment LExpt 5 (B; R2 = 0.59); or between amount of L. maculans DNA and DG in leaf petiole viewed by extent of necrosis in experiment LExpt 4 (C; R2 = 0.78); or between amount of L. maculans DNA in the stem and stem canker severity score in experiment PExpt 3 (D; R2 = 0.56). The amount of L. maculans DNA in the petiole or stem was log10-transformed. Details of these experiments are presented in Table 1.
Figure 6.
Phoma stem canker symptoms produced in controlled conditions.
Leaf petioles of oilseed rape doubled haploid lines A30 (susceptible) or C119 (with quantitative resistance) were inoculated with conidia of GFP-expressing Leptosphaeria maculans isolate ME24/3.13 (A, B, C) or ascospores produced on stem debris under natural conditions (E, F). No visible stem canker symptom at the leaf scar (A-1) of A30 after the inoculated leaf had abscised at 25 days post inoculation (dpi) but growth of the pathogen was observed using a GFP2 filter (A-2). Stem canker was visible at the leaf scar of A30 at 36 dpi (B-1) and growth of L. maculans was visualised by GFP fluorescence (B-2) (the selected region in B-1 was viewed using a GFP2 filter). Symptoms at leaf scars of A30 (C, E) or C119 (D, F) at 87 dpi with conidia (C, D) or at 31 dpi with ascospores (E, F) (arrows indicate the leaf scars of inoculated leaves).
Figure 7.
Severity of phoma stem canker symptoms.
Leaf petioles of oilseed rape doubled haploid lines A30 (susceptible) or C119 (with quantitative resistance) were inoculated with conidia of isolate ME24/3.13 or ascospores of Leptosphaeria maculans; severity of stem canker was assessed on a 0–4 scale at 46 days post inoculation (dpi) (ascospore inoculum) or 87 dpi (conidial inoculum). Bars show standard errors (17 d.f. for conidial inoculation; 50 d.f. for ascospore inoculation).
Figure 8.
Growth of Leptosphaeria maculans in the stem cortex or pith.
Leaf petioles of oilseed rape doubled haploid lines A30 (susceptible) or C119 (with quantitative resistance) were inoculated with conidia of GFP-expressing L. maculans isolate ME24/3.13, stems of A30 (A, C) and C119 (B, D) were cut horizontally at the leaf scar (A, B) of the inoculated leaf or at the hypocotyl (C, D) 2–3 cm below the leaf scar at 87 days post inoculation (dpi). Stem cross-sections were viewed with brightfield illumination (A-1, B-1, C-1, D-1) or a GFP2 filter (A-2-3, B-2-3, C-2-3, D-2-3) immediately after cutting (0 h) or after incubation for 22 hours in darkness at 20°C (22 h).
Table 2.
Comparison of different methods for assessing Brassica napus quantitative resistance against Leptosphaeria maculans.