Net blotch (NB) is a major disease of barley caused by the fungus Pyrenophora teres f. teres (Ptt), and P. teres f. maculata (Ptm). Ptt and Ptm infect the cultivated crop (Hordeum vulgare) and its wild relatives (H. vulgare ssp. spontaneum and H. murinum ssp. glaucum). The main goal of this research was to study the NB-causing pathogen in the crop center of origin. To address this, we have constructed a Ptt (n = 15) and Ptm (n = 12) collection isolated from three barley species across Israel. Isolates were characterized genetically and phenotypically. Aggressiveness of the isolates was determined based on necrotrophic growth rate on detached leaves of barley. In addition, isolates were genetically characterized by the mating type, followed by phylogenetic analysis, clustering them into seven groups. The analysis showed no significant differentiation of isolates based on either geographic origin, host of origin or form (Ptt vs. Ptm). Nevertheless, there was a significant difference in aggressiveness among the isolates regardless of host species, geographic location or sampling site. Moreover, it was apparent that the isolates derived from wild hosts were more variable in their necrotrophic growth rate, compared to isolates sampled from cultivated hosts, thereby suggesting that NB plays a major role in epidemiology at the center of barley origin where most of the diversity lies. Ptm has significantly higher necrotrophic and saprotrophic growth rates than Ptt, and for both a significant negative correlation was found between light intensity exposure and growth rates.

Although cytoplasmic genetic variation influences agronomic traits, its potential for breeding applications remains unexplored. We validated cytoplasmic effects on grain traits in barley and tested their portability across nuclear backgrounds and environments. The cytonuclear multi-parent population validation panel (CMPPV) and Magal reciprocal validation panel (MRVP) were evaluated in replicated field trials across two thermally contrasting Israeli environments. The wild B1K-50-04 cytoplasm consistently increased thousand grain weight (TGW) by 6–11% and grain width (GW) by 2–3% relative to the corresponding cultivated cytoplasm within each reciprocal comparison, explaining up to 9.5% of TGW, and 8% of GW variance. These effects persisted in segregating populations derived from crosses with the elite cultivar Magal, demonstrating portability across nuclear backgrounds. Three-way analysis of marker × cytoplasm × environment interactions across a targeted set of loci identified a chromosome 5 H cluster of significant marker effects, including genotype × cytoplasm × environment signals near the ari-e locus; the hypothesized segregation of an HvDep1 mutant allele was confirmed by Sanger sequencing. Near-infrared spectroscopy analysis of grain composition revealed environment-dependent cytonuclear epistasis for the ari-e.GP allele, increasing protein content only in the B1K-50-04 cytoplasmic background at one field site. Image-based high-throughput phenomics under controlled conditions identified morphological and canopy spectral reflectance indices that diverged between cytoplasms and were correlated with final grain dimensions, suggesting that taking cytoplasmic variation into account can reveal novel trait relationships. Findings demonstrate that wild cytoplasms offer portable genetic resources for crop improvement, and that the utility of major nuclear loci in breeding can depend on cytoplasmic context.