Computational protocol: A role for random, humidity-dependent epiphytic growth prior to invasion of wheat by Zymoseptoria tritici

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Protocol publication

[…] Confocal images were obtained using a Leica SP8 with LAS-X software and 40× oil immersion lens, 60× water immersion lens or 20× dry lens. An argon laser with emission at 500 nm was used to excite GFP fluorescence and chloroplast autofluorescence, detected at 510–530 and 600–630 nm, respectively. Samples of infected leaf material were mounted in water and fields of view selected randomly. Z-stacks were taken which encompassed fungi entirely. Fungi were scored for inside/outside leaves by looking (a) through stacks for any green fluorescent fungi inside leaves; (b) at 3D projections of stacks, tipping the projection to obtain views from various angles and (c) by use of the Leica LAS-X software’s depth coding function. Fungi were scored as internal if any part of an individual was so. In the context of the work presented here, the term ‘individual’ is taken to mean any contiguous fungal unit, which may be a single yeast-like cell (arising from budding growth), a branching macroconidium of any size, or a hyphal network. It may also be a mixture of macroconidial-type and hyphal-type cells. All of these types of individual can be observed on leaves initially inoculated with either filtered ‘yeast-like’ growth from a plate or with pycnidiospores raised in planta. Cells within an individual are interconnected and can be assumed to share nutrients and other signals. Time-courses of confocal microscopy were halted at 12 dpi, as after this point and individuals could not be easily distinguished. Hyphal growth was scored if any portion of an individual exhibited the hyphal growth form (characterised by long, narrow cells readily distinguished from the shorter, wider cells of both yeast-like growth and pycnidiospores). Five independent experiments were carried out to score fungal individuals for hyphal growth and for location at the inside/outside of the leaf. In each, a minimum of thirty fungal individuals were scored each day, from at least three randomly selected fields of view on each of three randomly selected leaf pieces. [...] For fungicide assays, sprays were carried out at 7 or 14 dpi, using an Iwata Revolution SAR airbrush. Fungicide solutions were adjusted to pH 6, mixed with 0.01% (v/v) Silwet L-77 (Momentive Specialty Chemicals, UK), and sprayed on both surfaces of the leaves at a rate of 1 ml per cell of a 24-cell tray (2 plants per cell, avoiding run off). Phosphite (prepared from phosphorous acid; Sigma, UK), an antifungal which causes plant defence elicitation (), was used at a concentration of 100 mM; carbendazim (Sigma, UK), a benzimidazole fungicide which is active on the leaf surface () was used at a concentration of 100 μM. Controls consisted of either no fungicide application or mock treatment with water only, again avoiding runoff. To quantify disease, leaves were removed from plants 28 days after inoculation, and taped, abaxial side up, onto 3mm white perspex sheets and weighed down between dry tissue papers for 24 h. Leaves were scanned at high resolution and pycnidia number and leaf area measured using a bespoke ImageJ () script. Two independent repeats of this experiment were carried out. […]

Pipeline specifications

Software tools LAS X, ImageJ
Databases PSP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Zymoseptoria tritici, Triticum aestivum, Fungi
Diseases Infection