Strawberry Yield Improvement by Hydrogen-Based Irrigation Is Functionally Linked to Altered Rhizosphere Microbial Communities

Abstract

Molecular hydrogen (H₂) is crucial for agricultural microbial systems. However, the mechanisms underlying its influence on crop yields is yet to be fully elucidated. This study observed that H₂-based irrigation significantly increased strawberry (Fragaria × ananassa Duch.) yield with/without nutrient fertilization. The reduction in soil available nitrogen (N), phosphorus (P), potassium (K), and organic matter was consistent with the increased expression levels of N/P/K-absorption-related genes in root tissues at the fruiting stage. Metagenomics profiling showed the alterations in rhizosphere microbial community composition achieved by H₂, particularly under the conditions without fertilizers. These included the enrichment of plant-growth-promoting rhizobacteria, such as Burkholderia, Pseudomonas, and Cupriavidus genera. Rhizobacteria with the capability to oxidize H₂ (group 2a [NiFe] hydrogenase) were also enriched. Consistently, genes related to soil carbon (C) fixation (i.e., rbcL, porD, frdAB, etc.), dissimilar nitrate reduction (i.e., napAB and nrfAH), and P solublization, mineralization, and transportation (i.e., ppx-gppA, appA, and ugpABCE) exhibited higher abundance. Contrary tendencies were observed in the soil C degradation and N denitrification genes. Together, these results clearly indicate that microbe-mediated soil C, N, and P cycles might be functionally altered by H₂, thus increasing plant nutrient uptake capacity and horticultural crop yield.

Keywords: microbial functional gene; plant nutrient uptake; rhizophere microbiome; strawberry; yield.

Figure 1

Hydrogen-based irrigation positively improves strawberry yield. Photographs of strawberry fields were taken on 28 December 2020 (A). Three replicates of 9 m² plot were set up, and all soils in 12 plots were mixed and refilled from a 1.5 m depth in all plots. Corresponding total yield, yield per plant, and single fruit weight of strawberry (B) were then determined and calculated from 18 December 2020 to 6 April 2021. SW: surface water; HNW: hydrogen nanobubble water; SW + F: surface water plus fertilizers; HNW + F, hydrogen nanobubble water plus fertilizers. Data presented as mean ± SD (n = 3). Different letters indicate significant differences (p < 0.05) according to Tukey’s test. ** and *** indicate p < 0.01 and 0.001, respectively (t-test).

Figure 2

Changes in soil nutrients, diversity and composition of rhizosphere microbial communities in response to hydrogen-based irrigation. Reductions of soil available nitrogen (SAN), soil available phosphorus (SAP), soil available potassium (SAK), and soil organic matter (SOM) contents were calculated at the fruiting stage (10 November 2020) compared to that before planting (24 August 2020), respectively (A). Data presented as mean ± SD (n = 3). Boxplot showing Chao1, Shannon, and Simpson indexes of microbial communities (B). Principal coordinates analysis (PCoA) of the bacterial community composition based on Bray–Curtis dissimilarity at the genus level (C). Bacterial community composition on phylum level (D). Different letters indicate significant differences (p < 0.05) according to Tukey’s test. *, ** and *** indicate p < 0.05, 0.01, and 0.001, respectively (t-test).

Figure 3

Relative abundances of genes involved in soil carbon fixation (A) and degradation (B) in response to hydrogen-based irrigation. Bars indicate the total relative abundances across all treatments. The middle heatmap represents the relative abundances in each sample, and the right represents the relative abundances of genes in each dominant phylum.

Figure 4

Relative abundances of genes involved in soil nitrogen (A) and phosphorus cycling (B) in response to hydrogen-based irrigation. Bars indicate the total relative abundances across all treatments, and the middle heatmap represents the relative abundances in each sample, and the right represents the relative abundances of genes in each dominant phylum.

Figure 5

Correlations between key genes related to carbon, nitrogen, and phosphorus cycling in networks and strawberry yield in response to hydrogen-based irrigation. Yellow, green, and pink nodes represent genes involved in C, N, and P cycling, respectively (A). The size of each node is proportional to the number of connections. Red and blue lines, respectively, represent positive and negative linear relationships (|R| > 0.8, and p < 0.01). Pearson correlation coefficients between key genes in networks and yield or soil properties at the fruiting stage (10 November 2020; |R| > 0.8, and p < 0.01; (B)). SAK: soil available potassium content; SAN: soil available nitrogen content; SOM: soil organic matter content; SAP: soil available phosphorus content; SFW: single fruit weight; TY: total yield; YPP: yield per plant.

Figure 6

Correlation analysis underlying hydrogen-based irrigation improvement in strawberry yield was conducted by targeting the genes related to carbon, nitrogen, and phosphorus uptake in strawberries and rhizosphere microbes. Transcriptional profiles of genes related to N, P, and K absorption in strawberry roots at the fruiting stage (A), including nitrate transporter genes NRT1.1 and NRT2.1, nitrate reductase gene (NIA), phosphate transporter genes PT4 and PT8, and K transporter genes KUP4 and KUP8. Pearson correlation analysis among strawberry yield, soil nutrients reduction, and related gene expression in strawberry (B). Pearson correlation analysis between rhizosphere microbial abundances and strawberry yield, or soil nutrients reduction, or related strawberry genes expression (C). Different letters denote significant differences (p < 0.05) according to Tukey’s test. *, **, and *** indicate p < 0.05, 0.01, and 0.001, respectively. RSAK: reduction of soil available potassium content; RSAN: reduction of soil available nitrogen content; RSOM: reduction of soil organic matter content; RSAP: reduction of soil available phosphorus content; SFW: single fruit weight; TY: total yield; YPP: yield per plant.

Figure 7

An overall schema illustrating the contribution of microbe-mediated soil carbon, nitrogen, and phosphorus cycles and plant nutrient uptake capacity in strawberry yield improvement by hydrogen-based irrigation. The colors of genes denote alterations in abundance or expression in rhizosphere microbial communities or strawberry roots achieved by hydrogen nanobubble water (HNW), compared to the corresponding control, respectively. Red indicates a significant increase; blue indicates a significant decrease; and gray indicates no significant difference.