Plants
Wheat (variety Galaxie) was sown on John Innes No. 2 compost in 24-cell trays with 2–5 plants per cell, and maintained on a 16:8 h light:dark cycle at 18 °C (day) and 15 °C(night), with 80% RH in a MLR-352H-PE climate chamber (Sanyo). All plants were used for experiments at 2 weeks old.
Bacteria and fungi
Pseudomonas syringae pv. syringae strains reported to exhibit ice nucleation activity (281, 3010) or not reported to exhibit ice nucleation activity (1902, 3012) were purchased from the National Collection of Plant Pathogenic Bacteria (FERA, UK). Strains selected were not originally isolated from wheat (281, 1902—isolated from Syringa vulgaris; 3010, 3012—isolated from Malus sylvestris) and are not known wheat pathogens. Ice-nucleation activity was confirmed by floating smooth foil squares on the surface of a water/methanol ice bath, cooled to − 10, − 8, − 6, − 4 or − 2 °C. 100 μL droplets of sterile, distilled water were pipetted onto the foil squares and ice crystals formed spontaneously, within 1 min, only at − 10 °C. Two microliters of overnight bacterial culture were added to water droplets and time taken for ice formation recorded. Strain 281 showed the strongest ice nucleation activity and 1902 showed no detectable effect on ice formation; 3010 and 3012 were intermediate (Supplementary Table S1). Bacteria were maintained in 50% glycerol at − 80 °C and were grown on LB agar at 28 °C for all applications. For all experiments involving Z. tritici, the model isolate IPO3237 was used.
Bacterial pathogenicity tests
Bacteria were streaked onto LB agar and grown for 3 days before resuspension in 10 mM MgCl2 at 107 cfu/mL. Bacterial suspensions were sprayed onto wheat plants using a hand held atomiser at a rate of approximately 0.5 mL per cell of 2–5 two week old plants, giving visible misting of both leaf surfaces, on all leaves, without runoff of inoculum. Six cells of plants were inoculated with each strain. Plants were then returned to growth chambers and monitored for symptom development for 28 days. Only strain 3010 induced clear symptoms within this timeframe, although some plants inoculated with strain 3012 also showed mild chlorosis of leaf tips after day 14 (Supplementary Table S2). In further experiments, only strains 281 (INA+) and 1902 (INA−) were used.
Ion leakage measurements
10 cm lengths of 6–9 treated leaves were excised and placed in 10 mL ddH2O for 12 h. Conductivity of the ddH2O was measured using a conductivity meter and then leaves were boiled for 1 h and measurement repeated. ddH2O controls, without leaves, were treated in the same fashion. Ion leakage was reported as a percentage of total conductivity after boiling; control values were subtracted.
Propidium iodide staining for cell death
1 cm leaf sections were immersed for 1 h, in the dark, in 0.05% (w/v) propidium iodide (PI), mounted in 0.1% (v/v) phosphate buffered saline (PBS, pH 7) and viewed using a Leica SP8 confocal microscope using argon laser emission at 500 nm with detection at 600–630 nm. Five leaf sections were viewed for each treatment and 3 fields of view visualised in each leaf section. Cell death was scored as number of cells showing internal (cytoplasmic or nuclear) red fluorescence / total number of cells in field of view. No cell death was recorded.
Wheat inoculation
14 day-old wheat plants were inoculated with either INA+ or INA− bacteria suspended in 10 mM MgCl2 at 107 cfu/mL by spraying with a handheld atomiser until leaves were visibly beaded with moisture on both surfaces, avoiding runoff. Controls were sprayed with 10 mM MgCl2 only.
For assays involving co-inoculation of plants with bacteria and Z. tritici, plants were allowed to dry for an hour before inoculation with Z. tritici blastospores. Blastospores were suspended in sterile distilled water at 105 cfu/mL, a low inoculum density preventing saturation of infection28. Inoculated plants were returned to growth chambers and kept under plastic cloches for 72 h, then maintained as usual.
Analysis of STB speed and severity
Plants were observed at 7, 10, 12, 14, 16, 18, 21, 24 and 28 days post inoculation (dpi) and the most severe symptom on each leaf recorded. At 28 dpi, all inoculated leaves were harvested, rehydrated for 1 h, then scanned at high resolution. Pycnidia were enumerated and leaf area measured in scanned images using ImageJ28.
Antibiotic and competitor application
INA+ bacterial inoculation was carried out as before. Plants were allowed to dry for 1 h at room temperature, then spray inoculated with INA− bacteria suspended in 10 mM MgCl2 at 107 cfu/mL, or sprayed with ampicillin solution (50 μg/mL). Following this second treatment, plants were returned to growth chambers for 24 h. Freezing treatment and subsequent Z. tritici inoculation was then carried out as above.
Estimation of bacterial populations
Two methods were used to estimate bacterial populations on leaves. Firstly, 1 cm leaf samples were harvested from 3 randomly selected leaves in each treatment. These samples were mounted on glass slides in phosphate buffered saline, to which BacLight Green bacterial stain and propidium iodide counterstain were added at 0.05% (w/v) each. After 10 min, leaf samples were imaged at 20× magnification using a Leica SP8 confocal microscope. 5 μM z-stacks were collected at three randomly selected fields of view for each leaf sample and maximum projections created from these. Laser power, gain, and other parameters were held constant between fields of view and samples. The number of green pixels in each projection was then counted using ImageJ software and summed across the three fields of view. The percentage of pixels in the three fields of view which were green was then calculated and used as a proxy for percentage leaf area covered by bacteria. Secondly, 1 cm leaf samples were harvested from 3 more randomly selected leaves in each treatment and homogenised in 10 mM MgCl2. Homogenate was diluted 1/10, 1/100 and 1/1000 in MgCl2 and five 10 μL samples of each dilution spotted onto King’s B29 agar with Pseudomonas selective supplement CFC (Oxoid). Colonies were counted after 24 h incubation at 28 °C. Both procedures were carried out at 1, 4, 7, 10 and 14 dpi.
Experimental design and statistical methods
Specific details of experimental design and analyses are presented in the figure legends alongside the relevant results. Some general principles were applied. Randomisation: where a set of pots of plants was divided between treatments, pots were numbered and randomly generated numbers used to select those assigned to each treatment. For selection of microscope fields of view within a leaf, the slide was placed so that the ‘bottom left’ part of the leaf was in view and a random distance (in mm, bounded by length and width of the leaf) moved in the x and y dimensions to select each field of view, returning to the 0,0 position before each selection.
Replication: technical replication was used in all experiments, with the number of such replicates given in each figure legend. Three complete repeat experiments were carried out in most cases, with figure legends stating where replication was different (min. 2 repeats) and all data presented represent the mean of such replicates, with error bars showing standard errors. Statistical analyses: data were analysed using ANOVA unless otherwise stated, with appropriate checks for homoscedasticity and other assumptions.
Source: Ecology - nature.com
