Climate change intensifies drought frequency and severity, significantly reducing crop yields. Increasing global food production by 70% by 2050 necessitates improving crop production under stress. Manipulating plant-associated microorganisms offers a promising approach to enhance plant resilience. Beneficial microbes improve nutrient uptake, inhibit pathogens, and protect against stress, potentially modulating plant responses to water stress. However, understanding how microbial communities respond to water limitations, considering initial communities and plant genotype, is crucial before manipulation. Plant microbial communities are assembled vertically (parent to offspring) and horizontally (from the environment). Soil and seed-borne microorganisms significantly contribute to community assembly. Previous water stress exposure of these colonists directly impacts plant responses to environmental changes. Water-stressed microbes produce solutes and polysaccharides increasing resistance to water limitations. Long-term water stress shifts soil microbial profiles toward stress-resistant taxa. A previous lab experiment showed that wheat plants had higher root biomass when grown in historically water-stressed soil, suggesting that soil microbes previously subjected to water stress aid in coping with subsequent water stresses. However, this lab study lacked seed production to assess the impact on seed-associated microbial communities. Plant genotypes influence microbial recruitment via rhizodeposition, potentially shifting community composition toward drought-tolerant species that enhance plant drought responses. Plant-associated microbes also change through plant developmental stages. This study aimed to investigate the interactive effects of microbial stress history and plant genotypes on microbial community assembly across plant compartments and life stages under contemporary water stress. The hypothesis was that wheat plants in fields with contrasting soil water stress history would be colonized by different microbial communities, differentially affected by contemporary water stress, plant genotype, and growth stages.

The literature review supports the hypothesis that both historical and contemporary water stress conditions influence the composition and function of plant microbiomes. Studies have shown that prior exposure to drought can alter soil microbial communities, leading to increased resistance to future drought events. The role of plant genotype in shaping the rhizosphere microbiome is well-established, with different genotypes attracting different microbial communities. The dynamic nature of plant-associated microbial communities across developmental stages is also highlighted in previous research. These studies provide a foundation for understanding the complex interactions between plants, microbes, and water availability.

The experiment utilized two adjacent agricultural fields with contrasting water stress histories (37 years): one irrigated (without WSH), the other non-irrigated (with WSH). Four wheat genotypes (two drought-resistant and two drought-sensitive) were planted in plots within each field, with half irrigated and half non-irrigated. Soil properties were analyzed. Irrigation was applied at specific intervals. Soil water content and leaf relative water content (LRWC) were measured. Plant compartments (soil, rhizosphere, roots, leaves, seed epiphytes, and seed endophytes) were sampled at early stem elongation and early dough development stages. DNA was extracted, amplicon libraries prepared, and 16S rRNA gene sequencing performed. Statistical analyses (linear mixed-effects models, LMMs, ANOVA, and paired t-tests) were used to analyze bacterial and fungal α-diversity (Shannon index), community structure (PCoA), and relative abundance of dominant taxa. Yield, kernel weight, and protein content were also determined.

Soil water content was significantly higher in irrigated plots after 6 weeks. LRWC was higher in irrigated plots in the field without WSH. Yields were generally higher in the field without WSH, decreased significantly under non-irrigation, and varied by genotype. Kernel weight and seed protein content were mainly influenced by genotype. Seed-associated epiphytic bacteria from drought-sensitive genotypes had higher diversity in the field with WSH. Drought-tolerant genotypes showed higher epiphytic fungal diversity. Development stage significantly impacted bacterial α-diversity in roots, rhizosphere, and leaves. Fungal α-diversity was also affected by developmental stage in most compartments. PCoA revealed distinct microbial communities in each plant compartment; seed communities were similar to leaf communities. Field water stress history and development stage significantly shaped bacterial and fungal communities. Irrigation effects were more pronounced in the field without WSH, especially on leaf-associated communities. Gammaproteobacteria were more abundant in roots, leaves, and seeds than in rhizosphere and soil. Actinobacteria abundance was higher in the field with WSH. Firmicutes (linked to *Bacillales*) were more abundant in seeds and rhizosphere from the field with WSH. Seed epiphytic Ascomycota were more abundant in the field without WSH; the opposite was true for Basidiomycota. Developmental stage significantly impacted Firmicutes abundance in the rhizosphere, roots, and leaves. Actinobacteria abundance in the rhizosphere of the field with WSH was higher under non-irrigation, especially at the dough stage.

The field experiment confirmed and extended findings from a previous growth chamber study. Plant development stage and field water stress history were major drivers of microbiome composition across plant compartments. The study demonstrates that, within a single generation, seeds can become colonized by diverse microbial communities influenced by historical contingencies and contemporary conditions and plant genotype. Differences in seed and plant microbial communities between fields reflect the impact of soil microorganisms. The interaction between irrigation and water stress history affected microbial parameters, consistent with previous research showing environmental impacts on seed microbiomes. Drought-sensitive genotypes had more diverse seed endophytic bacteria, while drought-tolerant genotypes showed higher epiphytic fungal diversity. Higher leaf relative water content and yields under well-watered conditions in the field without WSH correlated with higher fungal α-diversity. However, differences in fertilization rates and soil properties between the fields may have influenced the results. Different microbial taxa exhibit varying responses to water limitation. Plant genotype and stage influence belowground carbon input and consequently microbiome composition. The study underscores the interactive effects of historical and contemporary water stress, genotype, and developmental stage on plant microbiomes.

This study shows that historical and contemporary soil water stress, plant genotype, and developmental stage interact to shape bacterial and fungal communities in all major plant compartments, including the seed microbiome. Historical environmental conditions alter seed microbiome composition. Future research should investigate the transfer of these effects through seeds to influence the next plant generation's growth under water stress. This research contributes to understanding stress ecology of microbiomes and informs strategies for improving plant stress tolerance.

The field water stress history treatment was based on two unique fields, limiting replicability. Different fertilization rates between the fields may have influenced microbial and plant responses. The seed endophyte data was limited due to issues with sequencing, therefore conclusions on seed endophytes are mainly based on seed epiphytes. Other plant traits besides drought tolerance might influence the observed interactions between genotype and other variables.