Natural Variation in Elicitation of Defense-Signaling Associates to Field Resistance Against the Spot Blotch Disease in Bread Wheat (Triticum aestivum L.)

Abstract

Spot blotch, caused by the hemibiotropic fungus Bipolaris sorokiniana, is amongst the most damaging diseases of wheat. Still, natural variation in expression of biochemical traits that determine field resistance to spot blotch in wheat remain unaddressed. To understand how genotypic variations relate to metabolite profiles of the components of defense-signaling and the plant performance, as well as to discover novel sources of resistance against spot blotch, we have conducted field studies using 968 wheat genotypes at 5 geographical locations in South-Asia in 2 years. 46 genotypes were identified as resistant. Further, in independent confirmatory trials in subsequent 3 years, over 5 geographical locations, we re-characterized 55 genotypes for their resistance (above 46 along with Yangmai#6, a well characterized resistant genotype, and eight susceptible genotypes). We next determined time-dependent spot blotch-induced metabolite profiles of components of defense-signaling as well as levels of enzymatic components of defense pathway (such as salicylic acid (SA), phenolic acids, and redox components), and derived co-variation patterns with respect to resistance in these 55 genotypes. Spot blotch-induced SA accumulation was negatively correlated to disease progression. Amongst phenolic acids, syringic acid was most strongly inversely correlated to disease progression, indicating a defensive function, which was independently confirmed. Thus, exploring natural variation proved extremely useful in determining traits influencing phenotypic plasticity and adaptation to complex environments. Further, by overcoming environmental heterogeneity, our study identifies germplasm and biochemical traits that are deployable for spot blotch resistance in wheat along South-Asia.

Keywords: Bipolaris sorokiniana; Triticum aestivum; defense signaling; natural variation; salicylic acid; spot blotch; syringic acid; wheat.

Figure 1

Evaluation of wheat germplasm in South-Asia identifies promising sources of resistance against spot blotch. (A) Schematic summarizes the experimental design adapted in the present study. (B) A geographical map showing the locations across South-Asia (India, Nepal and Bangladesh) where field experiments were conducted. Thirty-one genotypes in the year 1, and 15 in year 2 trials were identified as resistant.

Figure 2

Variation in ROS signaling during spot blotch infection of wheat germplasm. Heatmaps show the H₂O₂ levels (μM g⁻¹ FW; A), SOD activity (Unit g⁻¹ FW; C) and MDA content (μmol g⁻¹ FW; E) in the wheat CRP lines, at various time intervals, upon spot blotch infection were analyzed. A strong negative correlation (p ≤ 0.05) of AUDPC to spot blotch-induced H₂O₂ (B) and SOD (D) contents was observed, whereas MDA (F) was positively correlated to disease progression. r is the correlation coefficient from Pearson correlation analysis. Susceptible genotypes are marked in red color whereas resistant genotypes are labeled in black.

Figure 3

Elicitation of salicylic acid (SA) is strongly related to resistance of wheat against spot blotch. (A) Variation in accumulation of SA (μg g⁻¹ FW) in 55 wheat genotypes before (0 hpi) and after spot blotch infection is presented with the help of a heatmap. Values on x-axis indicate time. (B) An inverse correlation between SA and the AUDPC of 55 CRP genotypes is shown. Red colored labels are susceptible genotypes.

Figure 4

Variation in accumulation of phenolic acids (μg g⁻¹ FW) in wheat CRP genotypes during the process of infection of spot blotch. Heat maps show accumulation patterns of (A) total free phenolic content, (B) 4-hydroxybenzoic acid, (C) syringic acid, (D) vanillic acid, (E) chlorogenic acid, (F) caffeic acid, (G) p-coumaric acid, (H) ferulic acid, as well as (I) total bound phenolic content, (J) p-coumaric acid, and (K) ferulic acid. In red are the names of susceptible genotypes.

Figure 5

Inverse-correlation between metabolite levels of phenolic acids and the AUDPC of 55 CRP genotypes. Levels of metabolites showing significant correlation (p ≤ 0.05) was plotted against AUDPC during spot-blotch infection. r (correlation coefficient) and p-values are from Pearson correlation analysis.

Figure 6

Syringic acid resists B. sorokiniana growth and disease progression. Pharmacological evaluations (A,B) and exogenous complementation assays (C,D) were conducted to determine anti-fungal role of syringic acid. (A) Representative pictures of B. sorokiniana growing on PDA plates containing different concentrations of syringic acid. (B) Area of plate covered by B. sorokiniana in presence of syringic acid as compared to the control (no syringic acid) is plotted. (C,D) The susceptible (Sonalika) plants were exogenously complemented with various concentrations of syringic acid, and disease severity (right panel; representative data of 0.5 mM vs. 0 mM control Sonalika plants) as well as AUDPC (left panel) were calculated over time course of infection. Values are expressed as mean ± SD for (C). (D) Is represented as % of disease severity. One-way ANOVA with Tukey's test was done on absolute values to show significant differences; values with the same letters are not significantly different (P ≤ 0.05) from each other, whereas ^* and ^*** indicate significant differences at P ≤ 0.05 and P ≤ 0.005, respectively.