Figure 1.
Appearance of Brassica napus resistant Charlton and susceptible RQ001-02M2 when inoculated with Sclerotinia sclerotiorum.
Samples were taken at 24, 48, 72, 96 hours post inoculation (hpi). “Control” represents the mock inoculated control comparison for resistant and susceptible genotypes.
Table 1.
Description of growth of Sclerotinia sclerotiorum isolate MBRS-5 on the cotyledon surface of resistant (Brassica napus Charlton) and susceptible (B. napus RQ001-02M2) genotypes over time (12 to 72 hours post inoculation).
Figure 2.
Histology of resistant and susceptible Brassica napus genotypes in response to Sclerotinia sclerotiorum.
Spring type B. napus resistant Charlton and susceptible RQ001-02M2 were inoculated with S. sclerotiorum isolate MBRS-5. (A)–(C), (F)–(H), (J)–(L) Samples were cleared in acetic acid: ethanol: water (2∶2∶1), stained with 1% cotton blue, and photographed using a Zeiss Axioplan 2 microscope photograph system. (D), (E), (I) 2 μm thick sections obtained and photographed using the same photograph system. (A) Impeded fungal growth on resistant Charlton at 24 hours post inoculation (hpi). Arrow indicates the presence of simple appresoria. (B) Hyphal growth on susceptible RQ001-02M2 at 24 hpi. Arrows indicate the presence of simple appresoria. (C) Increase in hyphal diameter of fungal cells on resistant Charlton at 24 hpi. (D) Cytoplasmic disorganization and necrotic cells (arrows) of palisade mesophyll cells in the susceptible RQ001-02M2 at 24 hpi. (E) Darkly-stained areas (arrows) around the dead cells of palisade mesophyll layer at 24 hpi in the resistant Charlton. (F) Hyphal growth on cotyledons of the susceptible RQ001-02M2. Arrows indicate the extension of hyphal growth beyond the periphery of the inoculum droplet area. (G) Repeated dichotomous branching of the terminal hyphae led to formation of appresoria (arrow) at 48 hpi on susceptible RQ001-02M2. (H) Hyphal growth on resistant Charlton at 48 hpi. (I) Fungal invasion up to palisade mesophyll cells and extensively damaged upper epidermis in the susceptible RQ001-02M2 at 48 hpi. (J) Hyphal growth on susceptible RQ001-02M2 extended across almost whole of the upper surface of the cotyledon at 72 hpi (K) Hyphal growth within the periphery of the inoculum droplet area (arrow) on resistant Charlton at 72 hpi. (L) Disintegration of hyphal cell wall (arrows) on resistant Charlton at 72 hpi.
Figure 3.
Representative image of resistant cv. Charlton cotyledon proteins separated by two-dimensional electrophoresis (2-DE).
The 2-DE was performed for both Brassica napus resistant Charlton and susceptible RQ001-02M2 at 12, 24, 48 and 72 hours post inoculation (hpi) by using 11 cm immobilized-pH-gradient (IPG) strips. Gels were stained with Coomassie Brilliant Blue (CCB) and their images were acquired by GS-800 imaging densitometer (Bio-Rad) with a red filter (wavelength 595–750 nm) and a resolution of 63.5×63.5 µm. The numbers shown correspond with the spot numbers mentioned in Table 2.
Table 2.
Details of the proteins identified in Brassica napus resistant Charlton and susceptible RQ001-02M2 at various times after inoculation with Sclerotinia sclerotiorum.