Fig 1.
Light micrographs illustrating hyphae and crystal shapes observed on Brassica carinata infected with Sclerotinia sclerotiorum at 72 hours post inoculation: A-D: Four crystal shapes on the surface of stems. For these, the epidermis and the first few layers of cortical cells were cut from stems decolorized in acetic acid: ethanol: water (2:2:1), stained in 0.05% aniline blue and observed on glass slides. Photographed using an Olympus BX51 microscope and DP71 digital photographic system. A: bipyramidal crystal B: short rod crystals C: long rod crystals D: irregular—crystal sand. E, F: The presence of crystals in the stem cortex was revealed when transverse sections 200 μm thick were cut with a Vibratome 3000 sectioning system from stems fixed in glutaraldehyde, then observed under bright field illumination on glass slides. Photographed using Zeiss Axioplan microscope and Zeiss Axiocam digital photographic system. E: Tetragonal short rod crystal (arrow) within the stem cortex. F: Jagged crystals formed on the surface of a cortical cell. A, B, C, D, E, F, Scale bars = 50 μm.
Fig 2.
Classification of tetragonal crystals according to their aspect ratio (AR).
AR was assessed as the ratio of width to height. A and B: bipyramidal with AR > 5. C: short rod with AR between 5 and 0.5. D: long rod, AR < 0.5.
Fig 3.
Occurrence of calcium oxalate crystals.
Percentage of samples containing calcium oxalate crystals at 6, 12, 24, 48 and 72 hours post inoculation (n = 134. P = 0.002, l.s.d. = 25%).
Fig 4.
Changes in crystal shape over time.
Percentage of calcium oxalate crystal-containing samples in which each of four differently shaped crystal forms were observed at 6, 12, 24, 48 and 72 hours post inoculation (n = 96, P = 0.001, l.s.d. = 23.4%).
Fig 5.
Crystal shapes and arrangements.
Scanning electron micrographs illustrating hyphae and crystal shapes and arrangements observed on the surface of stems of Brassica carinata 72 hours post inoculation with Sclerotinia sclerotiorum A: Bipyramidal. B: Short rod. C: Oval plates. D: Crystal sand. E: Mixture of bipyramidal and oval plates. F: Bipyramidal and druse crystals (arrow). A, B, C, F, Scale bars = 5 μm. D, E, Scale bars = 50 μm.
Fig 6.
Crystal locations and arrangements.
Scanning electron micrographs illustrating crystal locations and arrangements observed on stems of Brassica carinata infected with Sclerotinia sclerotiorum. Unless otherwise noted, images were taken 72 hours post inoculation. A: Bipyramidal crystals on stem surface loosely associated with S. sclerotiorum hyphae at 24 hours post inoculation. B: Flower-like druse. C: Bipyramidal crystal cluster (arrow) at 48 hours post inoculation. D: Complex of rectangular crystals. B, D, Scale bars = 5 μm A, C, Scale bars = 50 μm.
Fig 7.
Scanning electron micrographs illustrating crystal locations, arrangements, hyphal masses and crystal conglomerates as observed on stems of Brassica carinata infected with Sclerotinia sclerotiorum. Unless otherwise noted, images were taken 72 hours post inoculation. A: Large crystal incorporated into an infection cushion at 48 hours post inoculation showing location of the detail shown in B (arrow). B: Small tetragonal crystals formed along the edges of a larger crystal (arrows). C: 200 μm thick transverse section showing the hyphal mass and the location of the detail illustrated in D (arrow). D: CaOx conglomerate. E: Conglomerate containing short rod crystals. F: Abundant short/long rod and bipyramidal crystals forming within the hyphal mass. B, D, E, F, Scale bars = 5 μm. A, C, Scale bars = 50 μm.
Fig 8.
Qualitative energy dispersive X-ray (EDS) spectrum showing typical elemental composition of crystals. Samples were coated with carbon and platinum.