Molecular characterization of bacterial leaf streak resistance in hard winter wheat

Planting and experimental design

The entire study consists of two greenhouse experiments (GH1 and GH2 in 2016 and 2017, respectively) and one field experiment (2017) constituting of two replications each. Both in the field and the greenhouse, spring wheat cv. Briggs (susceptible check: SC1) and germplasm accession SD1001 (pedigree: PFAU/MILAN//TROST, susceptible check: SC2); SD52 (pedigree: CNO79//PF70354/MUS/3/PASTOR/4/BAV92*2/5/HAR311, resistant check: RC1) were included in the study as susceptible and moderately resistant checks, respectively.

For greenhouse experiments, three seeds of each accession were planted in the cones which were 3.8 cm in diameter and 20 cm in height (Stuewe and Sons, Inc., Corvallis, OR). To the soil in each container, 2.5 g of multicote slow release commercial fertilizer with a 14–14–16 Nitrogen Phosphorous and Potassium composition (Sungro Horticulture Distribution Inc. Agawam, MA, USA) was applied at the time of planting. Each cone consisted of three plants per replication. Each cone was considered as an experimental unit, and the third leaf of each plant was regarded as a sampling unit. Both greenhouse experiments were performed under controlled temperature: 30/18 °C diurnal cycle (day/night) with a 16 h photoperiod and relative humidity (>85%) (Silva et al., 2010) in a randomized complete block design. In the field experiment, the 299 HWWAMP accessions were planted in ∼90 cm long single row plots (40 seeds per row) with a plot to plot distance of 19.5 cm. In total there were two blocks (1rep per block) along with resistant and susceptible checks. The experimental unit in the field was 10 flag leaves from each plot. Accessions were randomized in each block and for better representation, three plots of checks were planted in each block.

Inoculum, infiltration and disease assessment

A highly virulent strain isolate Xct-017 of X. campestris pv. translucens, was used in this study. A fresh culture of the isolate Xct-017 was initiated from −80° C freezer by streaking on a Kim-Goepfert (KG) agar media (agar = 20 g; magnesium sulphate = 1.5 g; proteose peptone = 20 g; potassium phosphate = 1.5 g) (HiMedia Labs Inc. Mumbai, India). The bacterial cells were obtained from a two day old culture by adding 20–25 ml of distilled water into each plate and scraping the surface using a flamed microscope slide. The inoculum density was adjusted to 3 × 10⁸ colonies forming unit ml⁻¹ using a turbidometer (Biolog Inc., Hayward, CA, USA). In the greenhouse, the infiltration was performed by injecting 10 to 15 µL of inoculum into a fully expanded third leaf using a needleless disposable syringe (Faris et al., 1996). The infiltrated areas were marked by a non-toxic sharpie, permanent marker and the plants were placed in trays with water. These plants were kept in a mist chamber for 24 h post-infiltration to enhance the infection process. The mist chamber was built in house and a humidifier (Vicks Filter-free cool mist humidifier-V4600) was installed for moisture. With the help of a timer, mist from the humidifier was released every 15 min for a time span of 4 min. After 24 h the plants were moved to a growth chamber (Conviron A1000 growth chamber). In the field, at third leaf stage, plants were inoculated using a powered backpack mist blower (Solo 451; Solo, Sindelfingen, Germany) with a fixed pressure. A 3 g of Carborundum (Fischer Scientific) was added to 1 liter of inoculum (1 × 10⁸ cfu/ml) to create non-lethal wounds on the leaves.

Disease rating

BLS is a very complex disease and requires stringent and highly accurate phenotyping. We rated the genotypes after 14 days of infiltration or inoculations. In the greenhouse environment (a controlled environment), we applied a disease rating scale of 1–5. The length of the initial infiltration area (L0) was recorded and the length of the final infected area (L14) was recorded. The difference in the length (L14-L0) was calculated and used to determine the rating level for each plant (Fig. S2) as following: category 1 (resistant, R): 0–0.49 cm increase; category 2 (moderately resistant, MR): 0.5–0.9 cm increase; category 3 (moderately susceptible, MS): 1–1.49 cm increase; category 4 (susceptible, S): 1.5–1.99 cm increase; and category 5 (highly susceptible, HS): ≥ 2 cm. In field evaluation, we rated the plants as percent leaf area necrotic ; 1: 0–20%, 2: 21–40%, 3: 41–50%, 4: 51–70% at the soft dough stage (Feeks 11.2) and 5: >70%. The statistical parameters including mean, frequency distribution, analysis of variance (ANOVA) were calculated using a statistical program R (De Mendiburu Delgado, 2009; Team, 2014). Broad sense heritability (H²) for field and greenhouse experiments was estimated separately using Eqs. (1) and (2) respectively. (1)${\displaystyle H^2=\frac{{\sigma }_G^2}{{\sigma }_G^2+{\sigma }_e^2∕r}}$ (2)${\displaystyle H^2=\frac{{\sigma }_G^2}{{\sigma }_G^2+{\sigma }_E^2∕n+{\sigma }_{G^{\ast }E}^2∕nr}}$ ${\displaystyle {\sigma }_G^2=genotype,{\sigma }_e^2=error,}$ ${\displaystyle {\sigma }_E^2=experiment,{\sigma }_{G^{\ast }E}^2=genotype\ast experiment,}$ ${\displaystyle r=numberofreplications,\mathrm{and}n=numberofexperiments}$

Combined best linear unbiased estimator (BLUE) values for GH and field data were calculated using Eq. (3) and employing META-R (Vargas et al., 2013). (3)${\displaystyle {\mathrm{Y}}_{ijk}=\mu +{\mathrm{Gen}}_{\mathrm{i}}+{\mathrm{Exp}}_{\mathrm{j}}+{\mathrm{Rep}}_{\mathrm{k}}\left({\mathrm{Exp}}_{\mathrm{j}}\right)+{\mathrm{Gen}}_{\mathrm{i}}\times {\mathrm{Exp}}_{\mathrm{j}}+{\mathrm{e}}_{\mathrm{ijk}}}$ where Y_ijk is the BLS score, µ is the mean effect, Gen_i is the effect of the ith genotype, Rep_k is the effect of the kth replicate, Exp_j effect of jth experiment, and e_ijk is the error.