Tracking Rye Pathogenesis with Calcofluor White Staining

The seedlings of susceptible rye cultivar Słowiańskie were inoculated with Prs single spore isolate No. 1.1.6. Leaf samples were collected at 4, 8, 12, 16, 20, 24, 36, 48, and 72 hpi and stained with calcofluor white as described by Orczyk et al. [46 (link)]. Briefly, samples were fixed for 24 h with an ethanol:dichloromethane (3:1) solution supplemented with 0.15% trichloroacetic acid, after which they were rinsed twice with 50% ethanol, twice with 0.05 M sodium hydroxide, three times with water, and once with 0.1 M Tris. They were then stained with calcofluor white (3.5 mg/ml).
The stained leaf fragments were examined with the Diaphot fluorescence microscope (Nikon) for the presence of germinating spores, HMCs, and micronecrosis symptoms. Additionally, the number of infection sites was calculated. Observations were made in 80 on average (but not less than 30) infection sites per leaf sample. For selecting time-points for additional analyses of plant–pathogen interactions, the germinating spores and appressoria at infection sites were counted. The following four infection site profiles were used to reflect plant–pathogen interactions: (i) appressoria; (ii) appressoria and HMCs; (iii) appressoria, HMCs, and micronecrosis; and (iv) appressoria and micronecrosis. The analysis of pathogenesis and the percentage of profiles in the preliminary experiment with cv. Słowiańskie (Fig 1) were calculated according to: Eqs 1, 2, 3 and 4. The analysis of pathogenesis and the percentage of profiles in the main experiment with lines L318, D33 and D39 (Fig 2) were calculated according to: Eqs 5, 6, 7 and 8.
$\text{Percentage}\text{of}\text{profile}\text{i}=\frac{n_i}{(n_{gs}+n_i+n_{ii}+n_{iii}+n_{iv})}\times 100\%$
$\text{Percentage}\text{of}\text{profile}\text{ii}=\frac{n_{ii}}{(n_{gs}+n_i+n_{ii}+n_{iii}+n_{iv})}\times 100\%$
$\text{Percentage}\text{of}\text{profile}\text{iii}=\frac{n_{iii}}{(n_{gs}+n_i+n_{ii}+n_{iii}+n_{iv})}\times 100\%$
$\text{Percentage}\text{of}\text{profile}\text{iv}=\frac{n_{iv}}{(n_{gs}+n_i+n_{ii}+n_{iii}+n_{iv})}\times 100\%$
$\text{Percentage}\text{of}\text{profile}\text{i}=\frac{n_i}{(n_i+n_{ii}+n_{iii}+n_{iv})}\times 100\%$
$\text{Percentage}\text{of}\text{profile}\text{ii}=\frac{n_{ii}}{(n_i+n_{ii}+n_{iii}+n_{iv})}\times 100\%$
$\text{Percentage}\text{of}\text{profile}\text{iii}=\frac{n_{iii}}{(n_i+n_{ii}+n_{iii}+n_{iv})}\times 100\%$
$\text{Percentage}\text{of}\text{profile}\text{iv}=\frac{n_{iv}}{(n_i+n_{ii}+n_{iii}+n_{iv})}\times 100\%$
where: n_gs−number of infection sites with germinating spores, n_i−number of infection sites with appressoria, n_ii−number of infection sites with appresoria and HMC, n_iii−number of infection sites with appresoria, HMC and micronecrosis, n_iv−number of infection sites with appresoria and micronecrosis.
The number of infection sites with germinating spores and the rates of the four profiles were used to select the following time-points for further analyses of plant–pathogen interactions: 8, 17, 24, and 48 hpi. The first time-point (8 hpi) was selected because of the considerable abundance of appressoria at established infection sites. The 17 and 24 hpi time-points were associated with HMC formation and intense pathogen growth, respectively. The final time-point (48 hpi) corresponded to the beginning of the resistance reaction. Leaf samples were collected at the selected time-points, stained, and analysed regarding plant–pathogen interaction profiles.